B i o c h i m i c a et B i o p h y s i c a A c t a , 515 (1978) 209-237 © Elsevier/North-Holland Biomedical Press
BBA 85185
REGULATION
OF MEMBRANE
ENZYMES
BY LIPIDS
H E I N R I C H S A N D E R M A N N , Jr. l n s t i t u t f i i r B i o l o g i e 1L B i o c h e m i e der P f l a n z e n , Universitiit Freiburg, D- 7800 F r e i b u r g i. Br. (G. F. R.)
(Received October 21st, 1977)
CONTENTS I.
Introduction
....................................................................
11.
Basic properties of m e m b r a n e proteins and phospholipids
210
210
.............................
A. M e m b r a n e proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2.
210 212
Thermal phase transition .................................................. Phase separations .........................................................
213
II1.
Arrhenius plots of some m e m b r a n e - b o u n d enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
213
IV.
Response of m e m b r a n e enzymes to lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
214
A. B. C. D.
214 215 216 218
V.
Obligatory lipid requirement versus lipid modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solubilization of substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solubilization of enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetic effects oflipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of lipid structure
..........................................................
A. Lipid specificity, and possible experimental pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Viscotropic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Interfacial regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI.
Summary and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII. Appendix: Highly purified m e m b r a n e enzymes
......................................
219
..
219 223 225 228 229
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
231
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
231
210 1. INTRODUCTION
"Allein sehr viel sch6ner sieht man ihre Eigenschaften, wenn man die StOcke der Haut mit Wasser zusammenbringt.., so dringt es auf allen Seiten in Tropfen und F~iden heraus.., zu langen nervenartigen Gebilden." These observations were made by Virchow in 1854 [1], and probably represent the first demonstration of the (macroscopic) fluidity of membrane phospholipids ('Haut'). Membrane fluidity now appears to be of general importance for processes ranging from passive permeability and lateral diffusion to cell recognition, differentiation and malignant transformation. The fluid-mosaic membrane model regards biological membranes as two-dimensional solutions of oriented globular proteins in a fluid lipid bilayer phase [2]. It has also been recognized that the two-dimensional organisation of most biological membranes is heterogeneous. There appear to be areas of restricted mobility, caused by special protein-protein, lipid-protein or lipid-lipid interactions [2-9]. For a study of these complex interactions at the level of molecular parameters one may follow the traditional way of biochemistry, that is, isolation of the protein species involved, followed by their reconstitution with lipids. Reconstituted enzyme activities may then be taken as sensitive probes of lipid-protein interaction and protein conformation. The feasibility of this approach was probably first demonstrated in the case of the mitochondrial membrane enzyme, /3-hydroxybutyrate dehydrogenase. This enzyme activity was abolished upon removal of membrane lipids by detergent extraction, but it was restored by addition of lecithins containing at least one unsaturated fatty acid [10]. The lipid dependence of this and numerous other membrane enzymes has been reviewed [11-14], but previous studies were mostly performed at the cellular level or with particulate membrane fractions. The present review will concentrate on some general aspects which have emerged from recent biochemical and biophysical studies employing purified enzymes and chemically defined lipids. This review is incomplete in that a number of relevant studies, for example, on the use of phospholipases, are not covered. The following related topics have been covered in recent reviews; techniques for lipidprotein reconstitution [15-20], reconstitution of vectorial enzyme and transport activities [18-20], protein-lipid interactions [21-23] and properties of selected membrane enzymes [24]. II. BASIC PROPERTIES OF MEMBRANE PROTEINS AND PHOSPHOLIPIDS
1114. Membrane proteins Quite a number of tightly membrane-bound proteins have been highly purified in recent years, using detergents [22,25], chaotropic agents [26] or organic solvents [27] as solubilizing agents. These proteins are listed in the Appendix (Section VII).
211
Cyt. b5 Red~,~se
) ©TN 1111111Jll 111111111 Fig. 1. Proposed structures of amphipathic membrane proteins, a. Cytochromebs (left) and cytochromebs reductase (right). The highly schematic arrangement of the polar and non-polar domains of these proteins is based on indirect data [22,28,29,169].Redrawn from ref. 185.b. Bacteriorhodopsin. The structure shown was derived from X-ray and electron microscopicstudies, and was redrawn from ref. 36. There are seven trans-membrane a-helices per polypeptide chain of bacteriorhodopsin (molecularweight 26 000; ref. 36).
Primary structure determinations have in several cases indicated that the purified proteins consist of polar and non-polar domains, and by this amphipathic structure resemble lipids or detergents. The first such examples have been cytochrome b s [22,28] and cytochrome b 5 reductase [29]. The presumed membrane localisation of these proteins is depicted schematically in Fig. la. Non-polar domains have also been detected in intestinal aminopeptidase and maltases [30], glycophorin [31] and cytochrome P-450 [32]. These proteins will not bind to lipid membranes when the non-polar domains are removed, e.g. by partial proteolysis. Some membrane proteins have an exceptionally high overall hydrophobicity, e.g., C55-isoprenoid alcohol kinase [33], rhodopsin [34] or bacteriorhodopsin [35]. The latter tightly membrane-bound protein is the only one for which a tertiary structure has been derived with a resolution of about 7 A [36]. Seven transmembrane a-helices were detected per molecule of bacteriorhodopsin, as depicted schematically in Fig. lb. An amphipathic structure may also arise from the presence of non-polar subunits in membrane enzymes with quaternary structure. This situation is known, for example, for the mitochondrial, chloroplast and bacterial ATPases [37-39], mitochondrial cytochrome oxidase [40] and microsomal fl-glucuronidase [41 ]. Still other proteins contain covalently bound fatty acid residues, e.g., the bacterial Braun lipoprotein [42] and penicillinase [43], and the proteolipids of myelin [44] and of sarcoplasmic Ca2+-ATPase [45]. In principle, of course, a non-polar domain could also be generated by some special folding of a polypeptide of 'normal' amino acid composition. Lipid-embedded proteins can undergo rotational and translational diffusion [4]. It is also known that amphipathic as well as peripheral proteins of biological membranes are asymmetrically distributed, and similar observations have been made for the lipids [3,9,46].
212
liB. Phospholipids Phospholipids, like detergents, are typical amphipathic compounds. When placed in excess water above a certain critical temperature [47], phospholipids will spontaneously form bilayer structures. This process is driven energetically by nonpolar interactions of the fatty acid chains. In contrast, the interactions of the phospholipid polar head groups have been characterized as being repulsive [21,48,49]. It is important to realize that both types of interaction are essential for maintenance of an intact bilayer structure. Recent years have seen a dramatic increase in knowledge about the physical chemistry of phospholipids, mainly due to the development of sensitive calorimetric, X-ray and neutron diffraction and a variety of spectroscopic methods. A number of reviews have appeared [4-6,19-24,50-53]. The effects of temperature on bilayer structure are of special importance, since the sensitivity of membrane enzymes to structural changes of the surrounding lipid phase has often been demonstrated by temperature studies. IIB-1. Thermal#hase transition. Artificial as well as certain biological bilayer membranes undergo reversible transitions from an ordered, quasi-crystalline to a disordered, liquid-crystalline state at characteristic critical temperatures, To. Changes in the packing arrangement (Fig. 2a) are accompanied by increases in the mobility of the lipid fatty acid chains, in bilayer permeability and in the number of 'kinks' (packing defects arising from trans --+ gauche isomerization of C-C bonds). The hydration of the polar groups and the capacity for the incorporation of foreign compounds are also increased above To. Tc values increase with the length and decrease with the degree of unsaturation or methyl-substitution of the fatty acid chains. These values also depend on the state of ionization and hydration of the phospholipid polar head groups. Tc values are modified by incorporation of foreign compounds, e.g. cholesterol, or by binding of divalent cations or proteins, or by changes in pH or ionic strength. It is therefore possible to induce the phospholipid order --+ disorder transition at constant temperature by changes in these environmental parameters. Even above T~, phospholipid bilayers contain a motional gradient. Mobility appears to be highest at the ends of the fatty acid chains, then decreases to a minimum near the glycerol backbone and increases again somewhat in the polar head group. 0
0
0
0
0 Oeeee
0
0@0000 0000 000 o o
000 • • 000 000 00 log oooo oo oooo •
oo'0
0
•
0
o
o o
•
•
Oo o
oo
o
o
°
o o
o
" 0
0
0
o 0
o
o
•
•
e O o e Oe •
Oo o
o o e
0
Fig. 2. Schematic illustration of phospholipid phase changes, a. Crystalline----, liquid-crystalline thermal phase transition of a lecithin bilayer, b. Lateral phase separation. In this top view of a bilayer of phospholipid (O), the second molecular species (O) could be another lipid or a protein.
213
IIB-2. Phase separations. When the physical state of lipid bilayers is measured with the aid of solubilized probe molecules, a lateral movement and aggregation of these molecules is usually induced below T~ (Fig. 2b). This type of lateral phase separation has, for example, been demonstrated in model membranes containing an electron-spin labeled steroid derivative [54]. A 'vertical' phase separation may also occur below T~, by preferential partitioning back into the water phase. This phenomenon has been demonstrated with another electron-spin labeled compound, TEMPO [55]. When binary phospholipid mixtures are studied, e.g., by calorimetry or electron-spin resonance spectroscopy, a co-crystallisation is usually observed belowT~. However, when the phospholipids are structurally so different that packing problems arise, lateral phase separation and crystallization of the individual phospholipid species will occur (solid phase immiscibility). The construction of phase diagrams for binary and more complex lipid systems is discussed in a number of recent reports [5,56-59]. From the biological point of view, phase separation phenomena occurring above T~ (fluid/fluid phase separations) are most interesting. Limited miscibility above T~is typically observed for mixtures of phospholipids and non-hydrating lipids, like triglycerides or cholesterol esters. A useful classification of hydrating and non-hydrating lipids is given by Small [60]. A lateral phase separation above T~ has also been described for the binary mixture of dipalmitoyl phosphatydylethanolamine and dielaidoyl phosphatidylcholine [57]. The formation of short-lived 'clusters' up to a temperature some 50 °C above T~ (-- 22 °C) has been reported for dioleoyl phosphatidylcholine [61]. Freeze-fracture electron microscopy [52,62] and electron-spin resonance spectroscopy [56,62] indicate that membranebound proteins can also undergo lateral phase separations below T~ of the lipid phase. However, some proteins (e.g. bleached rhodopsin, glycophorin, proteins of Staphylococcus membranes) appeared not to aggregate below Tc [52,56,62]. III. A R R H E N I U S
PLOTS OF SOME MEMBRANE-BOUND
ENZYMES
A correlation between the activity of membrane-bound enzymes and motional parameters of the surrounding lipid phase has frequently been sought by comparison of their temperature dependencies. The effects of temperature on the rate of enzyme reactions are extremely complex [63,64]. A number of auxiliary experiments have therefore been suggested in order to recognize some of the more trivial effects of temperature [63,64]. In most temperature studies of membrane functions this complexity has been ignored, and the logarithm of the experimental reaction rate has been plotted against the reciprocal of the absolute temperature. This treatment is based on the Arrhenius equation which may be written as dlogk = -
2.3 R
"d
where k is the reaction constant, E is the empirical activation energy and T is the
214 absolute temperature (K) [63]. Most soluble enzymes will yield a straight line, and in these cases the activation energy can be readily calculated from the slope of the curve obtained. Some soluble and many membrane-bound enzymes, however, will yield bi- or triphasic curves. Some examples for this behaviour will be discussed in Section VB (cf. Fig. 4). Usually these sudden changes in activation energy are not accompanied by a significant change in reaction rate. The change thus is in d V / d T and not in V. This behaviour is difficult to understand since it requires that the change in activation enthalpy be exactly compensated by a change in activation entropy [65,66]. It has been proposed, and has actually been demonstrated for the lactose permease system, that the discontinuities of biphasic Arrhenius plots may really extend over a broad temperature range and that a second discontinuity may be present at low temperature. The latter discontinuity may have remained undetected in may temperature studies [66]. The molecular mechanisms underlying bi- or triphasic Arrhenius plots have so far in no case been elucidated. It has, however, been shown that an explanation may become possible in terms of protein solubility in the lipid phase [6,67], so that enzyme/lipid phase diagrams should be constructed. An interesting attempt in this direction has been made for the lactose permease system ofEscherichia coli [66]. IV. RESPONSE OF MEMBRANE ENZYMES TO LIPIDS
1VA. Obligatory lipid requirement versus lipid modulation The classical way to demonstrate the lipid dependence of an enzyme has been the observation of a loss of activity on removal of lipid, followed by the restoration of activity upon re-addition of lipid [10-13]. It now appears that these criteria are not always sufficient proof for an obligatory lipid requirement. For some enzymes, a lipid requirement is only observed for reactions involving non-polar substrates. The mitochondrial succinate dehydrogenase may serve as an example. The highly purified and lipid-free enzyme is active with water-soluble substrates [68]. A lipid dependence exists, however, when the purified enzyme is used for reconstitution of partial reactions of oxidative phosphorylation, where water-insoluble ubiquinone and cytochromes act as electron acceptors [68,69]. Further examples for the role oflipids in the 'activation' of non-polar substrates are discussed below (Section IVB). A few membrane-bound enzymes will respond to phase changes of the surrounding lipid phase, but will remain active with water-soluble substrates in lipidand detergent-free form. This has, for example, been reported for cytochrome b 5 reductase [29]. In addition this enzyme and several other amphipathic membrane enzymes will remain active when the hydrophobic domain of these proteins is removed by partial proteolysis (e.g. cytochrome P-450 reductase, aminopeptidase, sucrase, isomaltase, bacterial penicillinase). Still other enzyme reactions initially appeared to require the presence of lipid, but more detailed studies have then indicated that only partial reactions of the
215 overall reaction pathway were lipid dependent. Such kinetic effects oflipids will be discussed in Section IVD. A special situation is offered by the mitochondrial, chloroplast and bacterial ATPases. The lipid-free 5-subunit enzymes (FI) are water-soluble and catalytically active [38,39,70]. The complete 8-11 subunit ATPases (F1-Fo) are water-insoluble and have amphipathic characteristics. These enzymes require lipid for full activity [38,39,70-73]. They are also sensitive to certain specific inhibitors and able to catalyze energy-dependent reactions. The above examples show that in some cases lipids may act as modulators of enzyme reactions, in addition to their role as obligatory lipid 'cofactors' for many other amphipathic membrane enzymes. IVB. Solubilization of substrate As noted above for succinate dehydrogenase, a lipid 'cofactor' may be required for the solubilization of water-insoluble substrates. These occur, for example, in reactions of lipid and glycolipid biosynthesis, in redox reactions involving long-chain ubiquinones or plastoquinones, or in glycosyl transfer reactions involving polyisoprenyl carrier lipids. In many of these reactions, a lipid dependence has been noted [12-14,74], and often certain detergents were able to substitute for phospholipid. The solubilizing effect of phospholipids has already been recognized in early studies, and the preparation of mixed micelles of phospholipids and ubiquinone or cholesterol was described in 1961 [75]. It must be emphasized that the term 'solubilization' is used here not in the sense of 'making water-soluble' or 'not sedimenting in the ultracentrifuge'. In the present context, solubilization means the incorporation of a non-polar foreign substance into a lipid bilayer or detergent micelle by a process analogous to mixed-micelle formation [21,25]. This definition also extends to the incorporation of amphipathic protein substrates (e.g., cytochromes) or the amphipathic enzyme proteins themselves (see Section IVC). The solubilizing lipid must fulfil the same structural requirements that are known for micelle or bilayer formation. The lipid concentration must be above the critical micellar concentration. The lipid must contain a suitable polar group so that lipid hydration can occur [21,60,76]. The temperature must be above the critical micellar temperature (Krafft point; refs. 15,25). In the case of phospholipids, this temperature is identical to Tc [47,50]. Below To, solubilization will be reversed, and a lateral or 'vertical' phase separation is likely to occur(see Section liB). The solvent properties of phospholipid bilayers have recently been studied in some detail. The partitioning ofn-hexane into lecithin bilayers or micelles of sodium dodecylsulphate appeared to be energetically somewhat less favorable than partitioning into bulk hydrocarbon [77]. Cholesterol linoleate [78] and squalene [79] did not appear to partition to a significant degree into lecithin bilayers. These bulky conpounds lack a polar group, and their incorporation into the bilayer interior would probably expose hydrocarbon to water. Cholesterol is similar in molecular
216 weight to squalene. It is, however, quite well soluble in various phospholipids, apparently, because its hydroxyl group can be packed into the bilayer/water interface [80]. One may conclude that a phospholipid bilayer cannot be treated as a microscopic 'bulk' hydrocarbon phase. For substances approaching or exceeding the size of lipid fatty acid chains the presence of hydrophilic polar groups and the packing geometry appear to be crucial for solubilization. The role of a phospholipid 'cofactor' as a solubilizing agent has been clearly demonstrated for sugar transfer reactions of lipopolysaccharide biosynthesis in Salmonella [81,82]. The galactosyl- and glucosyl-transferases involved have properties of peripheral [2] proteins. The lipopolysaccharide substrate, however, had only acceptor activity when presented as a complex with phospholipids like phosphatidylethanolamine or phosphatidic acid. The mobility of the fatty acid chains was shown to be essential [81]. It should be noted that membrane lipids can also activate by the solubilization of lipophilic products. This is, for example, known for fatty acid synthetase systems, where inhibition by the long-chain fatty acid-CoA esters is relieved after their solubilization into liposomes [83,84]. I VC. Solubilization of enzyme The use of short-chain lecithins has in some cases allowed the demonstration that enzyme activation occurred near the critical micellar concentration of the phospholipid. Such results have been obtained for mitochondrial ATPase [85] and fl-hydroxybutyrate dehydrogenase [86,87] Amphipathic proteins will quite generally bind mild detergents near their critical micellar concentrations. It has also been shown that this cooperative binding process is related to the exposed non-polar surface of amphipathic proteins [22,23,88]. A positive cooperativity in the binding of fluorescent phospholipids to Ca2+-ATPase has also been demonstrated [89]. Optimal activation by long-chain phospholipids is, however, usually obtained in the concentration range of 0.1-2 mM, which is far above their critical micellar concentrations (10 -1° M for dipalmitoyl phosphatidylcholine; ref. 21). The use of short-chain phosphatidylcholines or detergents will lead to micellar protein complexes which have often been suitable for the demonstration of nonvectorial enzyme activities. Reconstitution with normal long-chain phospholipids will yield vesicular preparations which, of course, are required for the study of vectorial enzyme and transport reactions [15-20]. Amphipathic proteins may either be partially embedded in the vesicular bilayer phase, or they may traverse the entire bilayer once or more than once (see Fig. 1). Vesicular lipoprotein complexes have been. characterized by a number of physical methods, as shown in Table I. The structural requirements for solubilization by phospholipids have been discussed in Section IVB, and they are schematically summarized in Fig. 3 for the case of enzyme proteins. The solubilization of proteins may be particularly favored in a phospholipid phase containing phase boundaries. It has been suggested that such systems will display strong density fluctations and high lateral compressibility
217 TABLE i EXAMPLES FOR RECONSTITUTED PHYSICALLY CHARACTERIZED
LIPOPROTEIN
SYSTEMS
WHICH
Protein
Methods of characterization
Refs.
Cytochrome br,
Gel filtration, ultracentrifugation, electron microscopy. ESR and 'H-NMR spectroscopy, fluorescence polarization, intrinsic fluorescence Gel filtration, electron microscopy, ultracentrifugation Surface pressure (monolayer) Gel filtration, electron miroscopy, calorimetry, ESR and '3C-NMR spectroscopy Gel filtration, ultracentrifugation, electron microscopy Ultracentrifugation, electron microscopy. ESR and '3C-NMR spectroscopy, fluorescence transfer Electron microscopy, ESR spectroscopy, X-ray diffraction Electron microscopy, ESR spectroscopy
99,169. 186-191
Cytochrome b.~ reductase Galactosyltransferase Glycophorin Band 3 proteins Cae+-ATPase Cytochrome oxidase Rhodopsin
HAVE
BEEN
169,191 192 104. 193-195 195-197 103,111.124, 198-200 97,98,102 139,201 62
[5,51,67,90]. When visualized as a process analogous to mixed-micelle formation, enzymatically active lipoprotein complexes may be formed by hydrophobic interactions of non-polar amino acid residues with mobile fatty acid chains, while the appropriately hydrated lipid head groups line up with the hydrated polar amino acid side chains to form a lipoprotein/water interface [91]. The degree of dependence on the lipid physical parameters may well differ for various proteins and may, for example, depend on the depth of penetration of the protein into the lipid phase. The motional state of the lipid phase will then determine the rotational and lateral diffusion rates of the solubilized protein, and probably its ability to undergo conformational changes. Unfortunately, exact data on the conformational behaviour of functional amphipathic membrane proteins are not yet available. FUNCTIONAL LIPOPROTEIN SYSTEM SOLUB ILIZATION MOBILITY ~ OF LIPID FATTY ACID CHAINS" I
HYDRATION [ " OF LIPID POLAR GROUPS
4 ,
PHASE SEPARATION NON - FUNCTIONAL SYSTEM Fig. 3. Schematic relationship between the physical state of a lipid phase and lipoprotein function. Redrawn from ref. 93.
218 Sometimes (e.g. ref. 92) polypeptides are depicted as being completely buried in the hydrophobic interior of the bilayer so that lipid and protein polar groups cannot line up. This arrangement appears to be very unlikely (see Section IVB). There is strong evidence that solubilization by itself is not sufficient for the activation of lipid-dependent enzymes. For example, the detergent Triton X-100 is a good solubilizing agent, but it failed to activate the C55-isoprenoid alcohol kinase. Addition of small amounts of phospholipid to the detergent micelles, however, resulted in full activation [93,94]. Similar observations have been made for other enzymes. For example, cytochrome oxidase is activated only by certain detergents [95], and only a single detergent is known as an activator of Ca2+-ATPase [96]. Such observations indicate that in addition to the physical requirements for solubilization (Fig. 3), there also exist certain structural requirements for proper lipid-protein interaction (see Section VA). In this context, it is of great interest that spectroscopic and lipid titration studies have shown that solubilized proteins will surround themselves with a distinct layer of 'boundary' lipid [5,22,97-102]. These studies have been extended to cytochrome oxidase [97,98,102], cytochrome b~ [99], CaZ+-ATPase [100] and the cytochrome P-450 oxygenase system [101]. The boundary lipid layers (also called 'annulus'; ref. 100) appeared to be immobilized relative to the bulk lipid phase when studied by electron spin resonance spectroscopy [97-102]. An exchange between boundary and bulk lipid, which was slow on the electron spin resonance time scale, has been demonstrated for cytochrome oxidase [102] and Ca2+-ATPase [103]. A boundary lipid fraction has also been demonstrated for glycophorin [104] and rhodopsin [105] by 13C- and 1H-nuclear magnetic resonance spectroscopy, respectively. It should be noted that preferential solvation is a rather general phenomenon which is often encountered when the role of the solvent in kinetics is studied [106]. The vast excess of lipid or detergent usually employed in solubilization or reconstitution studies will in general lead to a disaggregation of amphipathic proteins. Many such proteins, however, will maintain strong protein-protein interactions. This is obviously the case for membrane enzymes with quaternary structure and for proteins forming lattice structures, like bacteriorhodopsin [36] or the acetylcholine receptor [107]. Oligomeric protein structures which persist in a fluid lipid or detergent environment have been demonstrated for other proteins, like (Na+-K+)-ATPase [108,109], CaZ+-ATPase [110,111], cytochrome oxidase [95], band 3 proteins of erythrocytes [112] or glycophorin [113]. An oligomeric structure may be essential for the function of these proteins. It should be added that the physical state of lipid-embedded proteins may be modified by the binding of peripheral proteins. The important functional consequences of such interactions have been reviewed [3,7,8]
IVD. Kinetic effects oflipids Most membrane-bound enzyme and transport systems utilize water-soluble substrates and take place near the polyelectrolyte membrane/water interface. Due,
219 for example, to local concentration and charge effects, these reactions will be influenced by microenvironmental parameters. Such effects and their kinetic treatment have been reviewed for purely micellar systems [114] and for model membrane and lipid systems [115-117]. The effect of lipids on the apparent cooperativity of membrane enzymes has also been reviewed recently [118]. Kinetic effects of lipids have been thoroughly studied for the peripheral enzyme, pyruvate oxidase of Escherichia coli [ 119-121 ]. This water-soluble enzyme behaved rather differently from amphipathic membrane proteins. Lipid was bound to high-affinity sites in monomeric form in a way reminiscent of serum albumin [25,122]. The kinetic effects induced by lipids may therefore have arisen by some allosteric effect rather than the mechanism ofmixed-micelle formation discussed in Section IVC. In a few cases, the overall reaction pathway of lipid-dependent amphipathic enzymes has been kineticaUy resolved, and the lipid 'cofactor' has been found to be involved in only partial reactions (Table II). The underlying molecular mechanisms have not been elucidated. The phenomenon by itself, however, may indicate that lipids can exert a subtle control of enzyme reactions. V. EFFECTS OF LIPID STRUCTURE
VA. Lipid specificity, and possible experimental pitfalls There appears to be no example of a lipid-dependent enzyme possessing a strict specificity for the chemical structure of the fatty acid chains of its lipid 'cofactor'. In contrast, much emphasis has been placed on the specific recognition of chemically defined polar head groups [11-14], and these results have also been incorporated into the fluid-mosaic membrane model [2]. However, a number of reported lipid specificities have been refuted or modified in recent years (Table III). It appears appropriate therefore, to discuss possible experimental pitfalls of lipid specificity studies in some detail. TABLE II KINETIC EFFECTS OF LIPID ACTIVATORS Enzyme reaction
Proposed lipid-dependent step
Refs.
/3-Hydroxybutyrate dehydrogenase Cytochrome P-450 oxygenase system Cytochrome oxidase
Binding of NADH (and NAD)
202
Electron transfer between cytochrome P-450 ajnd reductase Electron transfer between cytochromes a and a 3 K+-Phosphatase regulated differently from ATPase Dephosphorylation of enzyme (Contrary reports:
203
(Na+-K +)-ATPase Ca2+-ATPase
204 205,206 207-209 129.210)
220 The aqueous dispersion of certain phospholipids, e.g., phosphatidylethanolamine, may be difficult and, in any case, it must be performed above T~..Reconstitution often requires certain optimal lipid concentrations which can vary considerably for different lipids. Specificity studies should therefore be done at a range of lipid concentrations. Some lipids, e.g., dioleoyl phosphatidylcholine, decompose easily, and decomposition products, like lysolipids or free fatty acids, are highly surface active. Peroxidation may occur with lipids containing highly unsaturated fatty acids. The enzyme protein itself may be unstable under some conditions. Protein-protein interactions of enzymes with quaternary structure may be affected during isolation or reconstitution. The enzyme preparation may contain residual lipid or detergent or foreign proteins. For example, the proteolipid contents of Ca2+-ATPase is usually ignored in reconstitution studies although this polypeptide may act as a Ca2+ATPase is usually ignored in reconstitution studies although this polypeptide may act as a Ca z+ ionophore [96,123]. Lipid specificity profiles will often greatly differ between assays performed in the absence or in the additional presence of detergent. Detergent may, for example, relieve lipid dispersion problems or latency of a portion of the enzyme. MichaelisMenten kinetics may not be applicable to particulate enzyme preparations, and microenvironmental effects may have to be taken into account (see Section IVD). An apparent polar group specificity or fatty acid effect may depend on the particular method used for reconstitution. This has, for example, been observed for Ca2+-ATPase [124], (Na+-K+)-ATPase [125] and fl-hydroxybutyrate dehydrogenase [126]. An extreme case has been reported for the intestinal sucrase/isomaltase. Only the amphipathic form of this sugar translocating protein would bind to liposomes, without catalyzing sugar transfer [127], but the nonamphipathic form of the enzyme was claimed to translocate sugars in black lipid film experiments [ 127,128]. Different specificity patterns are usually obtained in non-vectorial and vectorial assay systems. The requirement for unidirectional incorporation and the variable leakiness of lipid bilayers will complicate vectorial assay systems. These effects are, for example, well documented for the Ca2+-translocating ATPase of sarcoplasmic reticulum [129,130]. A largely unidirectional incorporation of mitochondrial enzymes has recently been achieved by use of mixed liposomes containing acidic lipids [ 131 ]. A specific requirement for phosphatidylglycerol has been reported for enzyme II of the phosphoenolpyruvate transport system ofE. coli [ 132]. However, only experiments performed at single lipid concentrations were reported, and sodium dodecylsulphate could partially (about 20%) substitute for phosphatidylglycerol [ 132]. The experiments in which a specific requirement for phosphatidylglycerol was derived from the inhibitory effect of a phospholipase D preparation [133] have been refuted [ 134]. Previous reviews [11,23] have indicated a specific requirement of the galactosyl-transferase of lipopolysaccharide biosynthesis for phosphatidylethanolamine.
Phosphatidylserine, for review see Cholesterol Various proposals, for review see Phosphatidylethanolamine Cardiolipin, phosphatidylglycerol
(Na+-K+)-ATPase
Rhodopsin Cs.~-isoprenoid alcohol kinase
Cytochrome oxidase
Proposed specificity
Catalytic protein
40,95 34,211 215
20,125,143 219,220
Refs.
REFUTED OR MODIFIED LIPID SPECIFICITIES OF MEMBRANE ENZYMES
TABLE Ill
Various lipids or detergents (+ endogeneous cardiolipin) Various lipids, certain detergents Various lipids or detergents
Various negatively charged lipids or mixtures
Recent result
212-214 93,216-218
125,153,154, 176 95,204
Refs.
t-,J
222 However, the original report on the lipid specificity of this enzyme reaction clearly indicates that phosphatidic acid, phosphatidylglycerol, cardiolipin and bovine brain cerebroside were also active [81]. Lecithins appeared to be inactive [81]. A more recent study [82] indicates that the related glucosyl-transferase enzyme is activated by phosphatidylcholine. Previous reviews [13,14] have also emphasized the report [135] that malate dehydrogenase, a peripheral enzyme from Mycobacterium avium, was specifically activated by a single molecule ofcardiolipin. However, the original report [135] also stated that full activation was possible with lecithin or a neutral detergent, when another assay procedure was used. In recent work with highly purified enzyme from Mycobacteriumphlei, the specificity for cardiolipin could not be reproduced [136]. A strict polar group specificity for activation by lecithin has repeatedly been confirmed for the mitochondrial fl-hydroxybutyrate dehydrogenase. However, even this enzyme was not specific for the bilayer structure, ester linkages or detailed head group structure of phosphatidylcholine [86,87,137]. The chemically similar detergent, stearoyl phosphorylcholine [86], and the phosphatidylcholine analogue, N,N,N-trimethylphosphatidylbutanolamine [137], could substitute for phosphatidylcholine. In addition, it has been observed for this enzyme [87,138], for a number of reactions of oxidative phosphorylation [ 17,18,69,131,95,139-142] and for Ca 2+ translocation [130] that lipid mixtures will activate more effectively than any single lipid species tested. This may be of interest with regard to the lipid heterogeneity of biological membranes (see below). When presented in mixed micelles with inactive lipids, only 2.5 to 4 molecules of phosphatidylcholine per polypeptide chain were required for activation [87]. Possibly, fl-hydroxybutyrate dehydrogenase is activated by a combination of non-specific solubilization (see Section IVC) and specific binding to high-affinity sites (cf. pyruvate oxidase, Section IVD). Several enzymes have been found to retain phospholipids even after treatment with excess detergent. In the cases of (Na+-K+)-ATPase [143] and Ca 2+ATPase [144] the retained phospholipid was very similar in composition to the bulk phospholipid. It is remarkable that detergent-treated (Na+K+)-ATPase [145] and mitochondrial ATPase [146] still gave X-ray reflections typical for lipid bilayer structure. Other proteins will retain specific phospholipids: sphingomyelin in the case of 5'-nucleotidase (ref. 147, but see also ref. 148 for a contrary report), cardiolipin in the cases of cytochrome oxidase [40,95], NADH-ubiquinone reductase [142] and acetylcholinesterase [149], and phosphatidylserine [150] or diphosphoinositide [151] in the case of glycophorin. These preferential lipid-protein associations may be related to the 'boundary' lipid concept (see Section IVC). Certain lipids may be bound preferentially in order to minimize a mismatch in the packing of boundary lipid around the irregular surface of the protein. The heterogeneous lipid composition of biological membranes may be necessary to satisfy the different packing requirements of the embedded proteins, and this may also explain the efficient reconstitution by mixed lipid phases (see above). At
223 present, however, there is insufficient data to substantiate these explanations. A recent example may illustrate the present uncertainty about the packing requirements of membrane proteins. It has been suggested that the activity of the sarcoplasmic CaZ+-ATPase is determined by an 'annulus' of about 30 phospholipid molecules, and that the enzyme activity responds sensitively to structural changes of these phospholipid molecules [100,103,130]. Recent results indicate, however, that most of the phospholipid 'annulus' can be replaced by the neutral detergent, dodecyloctaoxyethyleneglycol-monoether[96]. It should finally be noted that a preferential association with certain phospholipids is also shown by simple compounds. For example, cholesterol will preferentially associate with sphingomyelin in mixtures ofsphingomyelin and other phospholipids [ 152].
VB. Viscotropic regulation Functional activities of biological or reconstituted membranes often show bior triphasic Arrhenius plots (see Section III). The Arrhenius plots for activation of (Na+-K+)-ATPase with various defined species of phosphatidylglycerol are shown as an example in Fig. 4a. The temperature values of the discontinuities differed for the various lipid species, and correlated with their T,. values [153]. The term 'viscotropic' has been introduced by Kimelberg and Papahadjopoulos to designate the "influence of fatty acyl chain fluidity on enzyme functions" [153,154]. The term 'fluidity' is rather vague and only loosely defined. Associated physical parameters include spectral order parameters, relaxation times, partition coefficients of probe molecules, lateral or rotational diffusion coefficients, T,. values, and so on. These parameters differ greatly in their physical meanings, yet they have been used to establish a general correlation between lipid motional parameters and the funcz.
3 ¥
2 ¥
T
;>-
~I,,
~-C>: ~-4' _J00 - , ,
(.9 O ....I
i
3.1
1 xlO' 3,6 "T
3,3
I_x10 ~ +
i
3.6
T
Fig. 4. Viscotropic regulation, a. Arrhenius plots for the activation of(Na+-K+)-ATPase by various species of phosphatidylglycerol. 1, dioleoyl phosphatidylglycerol; 2, dimyristoyl phosphatidylglycerol; 3, dipalmitoyl phosphatidylglycerol; 4, distearoyl phosphatidylglycerol. The calorimetric Tc values of three of these lipids were within the temperature range studied, and are indicated in the upper part of the graph, Redrawn from ref. 153. b. Arrhenius plots for the cytochrome b 5 reductase reaction in liposomes of dimyristoyl phosphatidylcholine. The molar ratio of cytochrome b 5 to cytochrome b 5 reductase was approx. 2 in curve 1, and 45 in curve 2. The Tc value of dimyristoyl phosphatidylcholine (23.7 °C) is indicated in the upper part of the graph, Redrawn from ref. 169.
224 TABLE IV VISCOTROPIC REGULATION
Selected examples for the correlation between functional acivity of membrane enzymes and motional parameters of the lipid phase are shown. Enzyme reaction (Na+-K+)-ATPase
Methods used
Refs.
Use of defined lipids, calorimetry
153,154.176. 221.222 93,216,217,223
(el" Fig. 4a), ESR spectroscopy
Cr,~-isoprenoid alcohol kinase Galactosyltransferase Cytochrome br, reductase Ca'-'+_ATPase
fi-Hydroxybutyrate dehydrogenase Glucose-6-phosphatase + U DPglucuronyl transferase Mitochondrial ATPase Cytochrome P-450 oxygenase system Lactose permease
Use of defined lipids. ESR spectroscopy Use of defined lipids Use of defined lilpids (of Fig. 4b) Use of defined lipids, ESR and laC-NMR spectroscopy Llse of defined lipids
81 169 100,103,159,198, 200,208,224 10,126,138
ESR-spectroscopy
225
Use of defined lipids, ESR spectroscopy, X-ray diffraction ESR spectroscopy, electron microscopy. fluorescence polarization ESR and fluorescence spectroscopy, calorimetry, X-ray diffraction
85,146,226,227 101,161,228 66.90.155-158
tional properties of reconstituted enzymes. Some representative studies are listed in Table IV. Discontinuous Arrhenius plots of membrane enzymes were only a few years ago almost exclusively interpreted in terms of the crystalline ~ liquid-crystalline phase transition of the lipid phase. A close correlation between the 'break' temperatures of Arrhenius plots and T
BBA 85185
REGULATION
OF MEMBRANE
ENZYMES
BY LIPIDS
H E I N R I C H S A N D E R M A N N , Jr. l n s t i t u t f i i r B i o l o g i e 1L B i o c h e m i e der P f l a n z e n , Universitiit Freiburg, D- 7800 F r e i b u r g i. Br. (G. F. R.)
(Received October 21st, 1977)
CONTENTS I.
Introduction
....................................................................
11.
Basic properties of m e m b r a n e proteins and phospholipids
210
210
.............................
A. M e m b r a n e proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. 2.
210 212
Thermal phase transition .................................................. Phase separations .........................................................
213
II1.
Arrhenius plots of some m e m b r a n e - b o u n d enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
213
IV.
Response of m e m b r a n e enzymes to lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
214
A. B. C. D.
214 215 216 218
V.
Obligatory lipid requirement versus lipid modulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solubilization of substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Solubilization of enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kinetic effects oflipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Effects of lipid structure
..........................................................
A. Lipid specificity, and possible experimental pitfalls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Viscotropic regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Interfacial regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI.
Summary and perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
VII. Appendix: Highly purified m e m b r a n e enzymes
......................................
219
..
219 223 225 228 229
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
231
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
231
210 1. INTRODUCTION
"Allein sehr viel sch6ner sieht man ihre Eigenschaften, wenn man die StOcke der Haut mit Wasser zusammenbringt.., so dringt es auf allen Seiten in Tropfen und F~iden heraus.., zu langen nervenartigen Gebilden." These observations were made by Virchow in 1854 [1], and probably represent the first demonstration of the (macroscopic) fluidity of membrane phospholipids ('Haut'). Membrane fluidity now appears to be of general importance for processes ranging from passive permeability and lateral diffusion to cell recognition, differentiation and malignant transformation. The fluid-mosaic membrane model regards biological membranes as two-dimensional solutions of oriented globular proteins in a fluid lipid bilayer phase [2]. It has also been recognized that the two-dimensional organisation of most biological membranes is heterogeneous. There appear to be areas of restricted mobility, caused by special protein-protein, lipid-protein or lipid-lipid interactions [2-9]. For a study of these complex interactions at the level of molecular parameters one may follow the traditional way of biochemistry, that is, isolation of the protein species involved, followed by their reconstitution with lipids. Reconstituted enzyme activities may then be taken as sensitive probes of lipid-protein interaction and protein conformation. The feasibility of this approach was probably first demonstrated in the case of the mitochondrial membrane enzyme, /3-hydroxybutyrate dehydrogenase. This enzyme activity was abolished upon removal of membrane lipids by detergent extraction, but it was restored by addition of lecithins containing at least one unsaturated fatty acid [10]. The lipid dependence of this and numerous other membrane enzymes has been reviewed [11-14], but previous studies were mostly performed at the cellular level or with particulate membrane fractions. The present review will concentrate on some general aspects which have emerged from recent biochemical and biophysical studies employing purified enzymes and chemically defined lipids. This review is incomplete in that a number of relevant studies, for example, on the use of phospholipases, are not covered. The following related topics have been covered in recent reviews; techniques for lipidprotein reconstitution [15-20], reconstitution of vectorial enzyme and transport activities [18-20], protein-lipid interactions [21-23] and properties of selected membrane enzymes [24]. II. BASIC PROPERTIES OF MEMBRANE PROTEINS AND PHOSPHOLIPIDS
1114. Membrane proteins Quite a number of tightly membrane-bound proteins have been highly purified in recent years, using detergents [22,25], chaotropic agents [26] or organic solvents [27] as solubilizing agents. These proteins are listed in the Appendix (Section VII).
211
Cyt. b5 Red~,~se
) ©TN 1111111Jll 111111111 Fig. 1. Proposed structures of amphipathic membrane proteins, a. Cytochromebs (left) and cytochromebs reductase (right). The highly schematic arrangement of the polar and non-polar domains of these proteins is based on indirect data [22,28,29,169].Redrawn from ref. 185.b. Bacteriorhodopsin. The structure shown was derived from X-ray and electron microscopicstudies, and was redrawn from ref. 36. There are seven trans-membrane a-helices per polypeptide chain of bacteriorhodopsin (molecularweight 26 000; ref. 36).
Primary structure determinations have in several cases indicated that the purified proteins consist of polar and non-polar domains, and by this amphipathic structure resemble lipids or detergents. The first such examples have been cytochrome b s [22,28] and cytochrome b 5 reductase [29]. The presumed membrane localisation of these proteins is depicted schematically in Fig. la. Non-polar domains have also been detected in intestinal aminopeptidase and maltases [30], glycophorin [31] and cytochrome P-450 [32]. These proteins will not bind to lipid membranes when the non-polar domains are removed, e.g. by partial proteolysis. Some membrane proteins have an exceptionally high overall hydrophobicity, e.g., C55-isoprenoid alcohol kinase [33], rhodopsin [34] or bacteriorhodopsin [35]. The latter tightly membrane-bound protein is the only one for which a tertiary structure has been derived with a resolution of about 7 A [36]. Seven transmembrane a-helices were detected per molecule of bacteriorhodopsin, as depicted schematically in Fig. lb. An amphipathic structure may also arise from the presence of non-polar subunits in membrane enzymes with quaternary structure. This situation is known, for example, for the mitochondrial, chloroplast and bacterial ATPases [37-39], mitochondrial cytochrome oxidase [40] and microsomal fl-glucuronidase [41 ]. Still other proteins contain covalently bound fatty acid residues, e.g., the bacterial Braun lipoprotein [42] and penicillinase [43], and the proteolipids of myelin [44] and of sarcoplasmic Ca2+-ATPase [45]. In principle, of course, a non-polar domain could also be generated by some special folding of a polypeptide of 'normal' amino acid composition. Lipid-embedded proteins can undergo rotational and translational diffusion [4]. It is also known that amphipathic as well as peripheral proteins of biological membranes are asymmetrically distributed, and similar observations have been made for the lipids [3,9,46].
212
liB. Phospholipids Phospholipids, like detergents, are typical amphipathic compounds. When placed in excess water above a certain critical temperature [47], phospholipids will spontaneously form bilayer structures. This process is driven energetically by nonpolar interactions of the fatty acid chains. In contrast, the interactions of the phospholipid polar head groups have been characterized as being repulsive [21,48,49]. It is important to realize that both types of interaction are essential for maintenance of an intact bilayer structure. Recent years have seen a dramatic increase in knowledge about the physical chemistry of phospholipids, mainly due to the development of sensitive calorimetric, X-ray and neutron diffraction and a variety of spectroscopic methods. A number of reviews have appeared [4-6,19-24,50-53]. The effects of temperature on bilayer structure are of special importance, since the sensitivity of membrane enzymes to structural changes of the surrounding lipid phase has often been demonstrated by temperature studies. IIB-1. Thermal#hase transition. Artificial as well as certain biological bilayer membranes undergo reversible transitions from an ordered, quasi-crystalline to a disordered, liquid-crystalline state at characteristic critical temperatures, To. Changes in the packing arrangement (Fig. 2a) are accompanied by increases in the mobility of the lipid fatty acid chains, in bilayer permeability and in the number of 'kinks' (packing defects arising from trans --+ gauche isomerization of C-C bonds). The hydration of the polar groups and the capacity for the incorporation of foreign compounds are also increased above To. Tc values increase with the length and decrease with the degree of unsaturation or methyl-substitution of the fatty acid chains. These values also depend on the state of ionization and hydration of the phospholipid polar head groups. Tc values are modified by incorporation of foreign compounds, e.g. cholesterol, or by binding of divalent cations or proteins, or by changes in pH or ionic strength. It is therefore possible to induce the phospholipid order --+ disorder transition at constant temperature by changes in these environmental parameters. Even above T~, phospholipid bilayers contain a motional gradient. Mobility appears to be highest at the ends of the fatty acid chains, then decreases to a minimum near the glycerol backbone and increases again somewhat in the polar head group. 0
0
0
0
0 Oeeee
0
0@0000 0000 000 o o
000 • • 000 000 00 log oooo oo oooo •
oo'0
0
•
0
o
o o
•
•
Oo o
oo
o
o
°
o o
o
" 0
0
0
o 0
o
o
•
•
e O o e Oe •
Oo o
o o e
0
Fig. 2. Schematic illustration of phospholipid phase changes, a. Crystalline----, liquid-crystalline thermal phase transition of a lecithin bilayer, b. Lateral phase separation. In this top view of a bilayer of phospholipid (O), the second molecular species (O) could be another lipid or a protein.
213
IIB-2. Phase separations. When the physical state of lipid bilayers is measured with the aid of solubilized probe molecules, a lateral movement and aggregation of these molecules is usually induced below T~ (Fig. 2b). This type of lateral phase separation has, for example, been demonstrated in model membranes containing an electron-spin labeled steroid derivative [54]. A 'vertical' phase separation may also occur below T~, by preferential partitioning back into the water phase. This phenomenon has been demonstrated with another electron-spin labeled compound, TEMPO [55]. When binary phospholipid mixtures are studied, e.g., by calorimetry or electron-spin resonance spectroscopy, a co-crystallisation is usually observed belowT~. However, when the phospholipids are structurally so different that packing problems arise, lateral phase separation and crystallization of the individual phospholipid species will occur (solid phase immiscibility). The construction of phase diagrams for binary and more complex lipid systems is discussed in a number of recent reports [5,56-59]. From the biological point of view, phase separation phenomena occurring above T~ (fluid/fluid phase separations) are most interesting. Limited miscibility above T~is typically observed for mixtures of phospholipids and non-hydrating lipids, like triglycerides or cholesterol esters. A useful classification of hydrating and non-hydrating lipids is given by Small [60]. A lateral phase separation above T~ has also been described for the binary mixture of dipalmitoyl phosphatydylethanolamine and dielaidoyl phosphatidylcholine [57]. The formation of short-lived 'clusters' up to a temperature some 50 °C above T~ (-- 22 °C) has been reported for dioleoyl phosphatidylcholine [61]. Freeze-fracture electron microscopy [52,62] and electron-spin resonance spectroscopy [56,62] indicate that membranebound proteins can also undergo lateral phase separations below T~ of the lipid phase. However, some proteins (e.g. bleached rhodopsin, glycophorin, proteins of Staphylococcus membranes) appeared not to aggregate below Tc [52,56,62]. III. A R R H E N I U S
PLOTS OF SOME MEMBRANE-BOUND
ENZYMES
A correlation between the activity of membrane-bound enzymes and motional parameters of the surrounding lipid phase has frequently been sought by comparison of their temperature dependencies. The effects of temperature on the rate of enzyme reactions are extremely complex [63,64]. A number of auxiliary experiments have therefore been suggested in order to recognize some of the more trivial effects of temperature [63,64]. In most temperature studies of membrane functions this complexity has been ignored, and the logarithm of the experimental reaction rate has been plotted against the reciprocal of the absolute temperature. This treatment is based on the Arrhenius equation which may be written as dlogk = -
2.3 R
"d
where k is the reaction constant, E is the empirical activation energy and T is the
214 absolute temperature (K) [63]. Most soluble enzymes will yield a straight line, and in these cases the activation energy can be readily calculated from the slope of the curve obtained. Some soluble and many membrane-bound enzymes, however, will yield bi- or triphasic curves. Some examples for this behaviour will be discussed in Section VB (cf. Fig. 4). Usually these sudden changes in activation energy are not accompanied by a significant change in reaction rate. The change thus is in d V / d T and not in V. This behaviour is difficult to understand since it requires that the change in activation enthalpy be exactly compensated by a change in activation entropy [65,66]. It has been proposed, and has actually been demonstrated for the lactose permease system, that the discontinuities of biphasic Arrhenius plots may really extend over a broad temperature range and that a second discontinuity may be present at low temperature. The latter discontinuity may have remained undetected in may temperature studies [66]. The molecular mechanisms underlying bi- or triphasic Arrhenius plots have so far in no case been elucidated. It has, however, been shown that an explanation may become possible in terms of protein solubility in the lipid phase [6,67], so that enzyme/lipid phase diagrams should be constructed. An interesting attempt in this direction has been made for the lactose permease system ofEscherichia coli [66]. IV. RESPONSE OF MEMBRANE ENZYMES TO LIPIDS
1VA. Obligatory lipid requirement versus lipid modulation The classical way to demonstrate the lipid dependence of an enzyme has been the observation of a loss of activity on removal of lipid, followed by the restoration of activity upon re-addition of lipid [10-13]. It now appears that these criteria are not always sufficient proof for an obligatory lipid requirement. For some enzymes, a lipid requirement is only observed for reactions involving non-polar substrates. The mitochondrial succinate dehydrogenase may serve as an example. The highly purified and lipid-free enzyme is active with water-soluble substrates [68]. A lipid dependence exists, however, when the purified enzyme is used for reconstitution of partial reactions of oxidative phosphorylation, where water-insoluble ubiquinone and cytochromes act as electron acceptors [68,69]. Further examples for the role oflipids in the 'activation' of non-polar substrates are discussed below (Section IVB). A few membrane-bound enzymes will respond to phase changes of the surrounding lipid phase, but will remain active with water-soluble substrates in lipidand detergent-free form. This has, for example, been reported for cytochrome b 5 reductase [29]. In addition this enzyme and several other amphipathic membrane enzymes will remain active when the hydrophobic domain of these proteins is removed by partial proteolysis (e.g. cytochrome P-450 reductase, aminopeptidase, sucrase, isomaltase, bacterial penicillinase). Still other enzyme reactions initially appeared to require the presence of lipid, but more detailed studies have then indicated that only partial reactions of the
215 overall reaction pathway were lipid dependent. Such kinetic effects oflipids will be discussed in Section IVD. A special situation is offered by the mitochondrial, chloroplast and bacterial ATPases. The lipid-free 5-subunit enzymes (FI) are water-soluble and catalytically active [38,39,70]. The complete 8-11 subunit ATPases (F1-Fo) are water-insoluble and have amphipathic characteristics. These enzymes require lipid for full activity [38,39,70-73]. They are also sensitive to certain specific inhibitors and able to catalyze energy-dependent reactions. The above examples show that in some cases lipids may act as modulators of enzyme reactions, in addition to their role as obligatory lipid 'cofactors' for many other amphipathic membrane enzymes. IVB. Solubilization of substrate As noted above for succinate dehydrogenase, a lipid 'cofactor' may be required for the solubilization of water-insoluble substrates. These occur, for example, in reactions of lipid and glycolipid biosynthesis, in redox reactions involving long-chain ubiquinones or plastoquinones, or in glycosyl transfer reactions involving polyisoprenyl carrier lipids. In many of these reactions, a lipid dependence has been noted [12-14,74], and often certain detergents were able to substitute for phospholipid. The solubilizing effect of phospholipids has already been recognized in early studies, and the preparation of mixed micelles of phospholipids and ubiquinone or cholesterol was described in 1961 [75]. It must be emphasized that the term 'solubilization' is used here not in the sense of 'making water-soluble' or 'not sedimenting in the ultracentrifuge'. In the present context, solubilization means the incorporation of a non-polar foreign substance into a lipid bilayer or detergent micelle by a process analogous to mixed-micelle formation [21,25]. This definition also extends to the incorporation of amphipathic protein substrates (e.g., cytochromes) or the amphipathic enzyme proteins themselves (see Section IVC). The solubilizing lipid must fulfil the same structural requirements that are known for micelle or bilayer formation. The lipid concentration must be above the critical micellar concentration. The lipid must contain a suitable polar group so that lipid hydration can occur [21,60,76]. The temperature must be above the critical micellar temperature (Krafft point; refs. 15,25). In the case of phospholipids, this temperature is identical to Tc [47,50]. Below To, solubilization will be reversed, and a lateral or 'vertical' phase separation is likely to occur(see Section liB). The solvent properties of phospholipid bilayers have recently been studied in some detail. The partitioning ofn-hexane into lecithin bilayers or micelles of sodium dodecylsulphate appeared to be energetically somewhat less favorable than partitioning into bulk hydrocarbon [77]. Cholesterol linoleate [78] and squalene [79] did not appear to partition to a significant degree into lecithin bilayers. These bulky conpounds lack a polar group, and their incorporation into the bilayer interior would probably expose hydrocarbon to water. Cholesterol is similar in molecular
216 weight to squalene. It is, however, quite well soluble in various phospholipids, apparently, because its hydroxyl group can be packed into the bilayer/water interface [80]. One may conclude that a phospholipid bilayer cannot be treated as a microscopic 'bulk' hydrocarbon phase. For substances approaching or exceeding the size of lipid fatty acid chains the presence of hydrophilic polar groups and the packing geometry appear to be crucial for solubilization. The role of a phospholipid 'cofactor' as a solubilizing agent has been clearly demonstrated for sugar transfer reactions of lipopolysaccharide biosynthesis in Salmonella [81,82]. The galactosyl- and glucosyl-transferases involved have properties of peripheral [2] proteins. The lipopolysaccharide substrate, however, had only acceptor activity when presented as a complex with phospholipids like phosphatidylethanolamine or phosphatidic acid. The mobility of the fatty acid chains was shown to be essential [81]. It should be noted that membrane lipids can also activate by the solubilization of lipophilic products. This is, for example, known for fatty acid synthetase systems, where inhibition by the long-chain fatty acid-CoA esters is relieved after their solubilization into liposomes [83,84]. I VC. Solubilization of enzyme The use of short-chain lecithins has in some cases allowed the demonstration that enzyme activation occurred near the critical micellar concentration of the phospholipid. Such results have been obtained for mitochondrial ATPase [85] and fl-hydroxybutyrate dehydrogenase [86,87] Amphipathic proteins will quite generally bind mild detergents near their critical micellar concentrations. It has also been shown that this cooperative binding process is related to the exposed non-polar surface of amphipathic proteins [22,23,88]. A positive cooperativity in the binding of fluorescent phospholipids to Ca2+-ATPase has also been demonstrated [89]. Optimal activation by long-chain phospholipids is, however, usually obtained in the concentration range of 0.1-2 mM, which is far above their critical micellar concentrations (10 -1° M for dipalmitoyl phosphatidylcholine; ref. 21). The use of short-chain phosphatidylcholines or detergents will lead to micellar protein complexes which have often been suitable for the demonstration of nonvectorial enzyme activities. Reconstitution with normal long-chain phospholipids will yield vesicular preparations which, of course, are required for the study of vectorial enzyme and transport reactions [15-20]. Amphipathic proteins may either be partially embedded in the vesicular bilayer phase, or they may traverse the entire bilayer once or more than once (see Fig. 1). Vesicular lipoprotein complexes have been. characterized by a number of physical methods, as shown in Table I. The structural requirements for solubilization by phospholipids have been discussed in Section IVB, and they are schematically summarized in Fig. 3 for the case of enzyme proteins. The solubilization of proteins may be particularly favored in a phospholipid phase containing phase boundaries. It has been suggested that such systems will display strong density fluctations and high lateral compressibility
217 TABLE i EXAMPLES FOR RECONSTITUTED PHYSICALLY CHARACTERIZED
LIPOPROTEIN
SYSTEMS
WHICH
Protein
Methods of characterization
Refs.
Cytochrome br,
Gel filtration, ultracentrifugation, electron microscopy. ESR and 'H-NMR spectroscopy, fluorescence polarization, intrinsic fluorescence Gel filtration, electron microscopy, ultracentrifugation Surface pressure (monolayer) Gel filtration, electron miroscopy, calorimetry, ESR and '3C-NMR spectroscopy Gel filtration, ultracentrifugation, electron microscopy Ultracentrifugation, electron microscopy. ESR and '3C-NMR spectroscopy, fluorescence transfer Electron microscopy, ESR spectroscopy, X-ray diffraction Electron microscopy, ESR spectroscopy
99,169. 186-191
Cytochrome b.~ reductase Galactosyltransferase Glycophorin Band 3 proteins Cae+-ATPase Cytochrome oxidase Rhodopsin
HAVE
BEEN
169,191 192 104. 193-195 195-197 103,111.124, 198-200 97,98,102 139,201 62
[5,51,67,90]. When visualized as a process analogous to mixed-micelle formation, enzymatically active lipoprotein complexes may be formed by hydrophobic interactions of non-polar amino acid residues with mobile fatty acid chains, while the appropriately hydrated lipid head groups line up with the hydrated polar amino acid side chains to form a lipoprotein/water interface [91]. The degree of dependence on the lipid physical parameters may well differ for various proteins and may, for example, depend on the depth of penetration of the protein into the lipid phase. The motional state of the lipid phase will then determine the rotational and lateral diffusion rates of the solubilized protein, and probably its ability to undergo conformational changes. Unfortunately, exact data on the conformational behaviour of functional amphipathic membrane proteins are not yet available. FUNCTIONAL LIPOPROTEIN SYSTEM SOLUB ILIZATION MOBILITY ~ OF LIPID FATTY ACID CHAINS" I
HYDRATION [ " OF LIPID POLAR GROUPS
4 ,
PHASE SEPARATION NON - FUNCTIONAL SYSTEM Fig. 3. Schematic relationship between the physical state of a lipid phase and lipoprotein function. Redrawn from ref. 93.
218 Sometimes (e.g. ref. 92) polypeptides are depicted as being completely buried in the hydrophobic interior of the bilayer so that lipid and protein polar groups cannot line up. This arrangement appears to be very unlikely (see Section IVB). There is strong evidence that solubilization by itself is not sufficient for the activation of lipid-dependent enzymes. For example, the detergent Triton X-100 is a good solubilizing agent, but it failed to activate the C55-isoprenoid alcohol kinase. Addition of small amounts of phospholipid to the detergent micelles, however, resulted in full activation [93,94]. Similar observations have been made for other enzymes. For example, cytochrome oxidase is activated only by certain detergents [95], and only a single detergent is known as an activator of Ca2+-ATPase [96]. Such observations indicate that in addition to the physical requirements for solubilization (Fig. 3), there also exist certain structural requirements for proper lipid-protein interaction (see Section VA). In this context, it is of great interest that spectroscopic and lipid titration studies have shown that solubilized proteins will surround themselves with a distinct layer of 'boundary' lipid [5,22,97-102]. These studies have been extended to cytochrome oxidase [97,98,102], cytochrome b~ [99], CaZ+-ATPase [100] and the cytochrome P-450 oxygenase system [101]. The boundary lipid layers (also called 'annulus'; ref. 100) appeared to be immobilized relative to the bulk lipid phase when studied by electron spin resonance spectroscopy [97-102]. An exchange between boundary and bulk lipid, which was slow on the electron spin resonance time scale, has been demonstrated for cytochrome oxidase [102] and Ca2+-ATPase [103]. A boundary lipid fraction has also been demonstrated for glycophorin [104] and rhodopsin [105] by 13C- and 1H-nuclear magnetic resonance spectroscopy, respectively. It should be noted that preferential solvation is a rather general phenomenon which is often encountered when the role of the solvent in kinetics is studied [106]. The vast excess of lipid or detergent usually employed in solubilization or reconstitution studies will in general lead to a disaggregation of amphipathic proteins. Many such proteins, however, will maintain strong protein-protein interactions. This is obviously the case for membrane enzymes with quaternary structure and for proteins forming lattice structures, like bacteriorhodopsin [36] or the acetylcholine receptor [107]. Oligomeric protein structures which persist in a fluid lipid or detergent environment have been demonstrated for other proteins, like (Na+-K+)-ATPase [108,109], CaZ+-ATPase [110,111], cytochrome oxidase [95], band 3 proteins of erythrocytes [112] or glycophorin [113]. An oligomeric structure may be essential for the function of these proteins. It should be added that the physical state of lipid-embedded proteins may be modified by the binding of peripheral proteins. The important functional consequences of such interactions have been reviewed [3,7,8]
IVD. Kinetic effects oflipids Most membrane-bound enzyme and transport systems utilize water-soluble substrates and take place near the polyelectrolyte membrane/water interface. Due,
219 for example, to local concentration and charge effects, these reactions will be influenced by microenvironmental parameters. Such effects and their kinetic treatment have been reviewed for purely micellar systems [114] and for model membrane and lipid systems [115-117]. The effect of lipids on the apparent cooperativity of membrane enzymes has also been reviewed recently [118]. Kinetic effects of lipids have been thoroughly studied for the peripheral enzyme, pyruvate oxidase of Escherichia coli [ 119-121 ]. This water-soluble enzyme behaved rather differently from amphipathic membrane proteins. Lipid was bound to high-affinity sites in monomeric form in a way reminiscent of serum albumin [25,122]. The kinetic effects induced by lipids may therefore have arisen by some allosteric effect rather than the mechanism ofmixed-micelle formation discussed in Section IVC. In a few cases, the overall reaction pathway of lipid-dependent amphipathic enzymes has been kineticaUy resolved, and the lipid 'cofactor' has been found to be involved in only partial reactions (Table II). The underlying molecular mechanisms have not been elucidated. The phenomenon by itself, however, may indicate that lipids can exert a subtle control of enzyme reactions. V. EFFECTS OF LIPID STRUCTURE
VA. Lipid specificity, and possible experimental pitfalls There appears to be no example of a lipid-dependent enzyme possessing a strict specificity for the chemical structure of the fatty acid chains of its lipid 'cofactor'. In contrast, much emphasis has been placed on the specific recognition of chemically defined polar head groups [11-14], and these results have also been incorporated into the fluid-mosaic membrane model [2]. However, a number of reported lipid specificities have been refuted or modified in recent years (Table III). It appears appropriate therefore, to discuss possible experimental pitfalls of lipid specificity studies in some detail. TABLE II KINETIC EFFECTS OF LIPID ACTIVATORS Enzyme reaction
Proposed lipid-dependent step
Refs.
/3-Hydroxybutyrate dehydrogenase Cytochrome P-450 oxygenase system Cytochrome oxidase
Binding of NADH (and NAD)
202
Electron transfer between cytochrome P-450 ajnd reductase Electron transfer between cytochromes a and a 3 K+-Phosphatase regulated differently from ATPase Dephosphorylation of enzyme (Contrary reports:
203
(Na+-K +)-ATPase Ca2+-ATPase
204 205,206 207-209 129.210)
220 The aqueous dispersion of certain phospholipids, e.g., phosphatidylethanolamine, may be difficult and, in any case, it must be performed above T~..Reconstitution often requires certain optimal lipid concentrations which can vary considerably for different lipids. Specificity studies should therefore be done at a range of lipid concentrations. Some lipids, e.g., dioleoyl phosphatidylcholine, decompose easily, and decomposition products, like lysolipids or free fatty acids, are highly surface active. Peroxidation may occur with lipids containing highly unsaturated fatty acids. The enzyme protein itself may be unstable under some conditions. Protein-protein interactions of enzymes with quaternary structure may be affected during isolation or reconstitution. The enzyme preparation may contain residual lipid or detergent or foreign proteins. For example, the proteolipid contents of Ca2+-ATPase is usually ignored in reconstitution studies although this polypeptide may act as a Ca2+ATPase is usually ignored in reconstitution studies although this polypeptide may act as a Ca z+ ionophore [96,123]. Lipid specificity profiles will often greatly differ between assays performed in the absence or in the additional presence of detergent. Detergent may, for example, relieve lipid dispersion problems or latency of a portion of the enzyme. MichaelisMenten kinetics may not be applicable to particulate enzyme preparations, and microenvironmental effects may have to be taken into account (see Section IVD). An apparent polar group specificity or fatty acid effect may depend on the particular method used for reconstitution. This has, for example, been observed for Ca2+-ATPase [124], (Na+-K+)-ATPase [125] and fl-hydroxybutyrate dehydrogenase [126]. An extreme case has been reported for the intestinal sucrase/isomaltase. Only the amphipathic form of this sugar translocating protein would bind to liposomes, without catalyzing sugar transfer [127], but the nonamphipathic form of the enzyme was claimed to translocate sugars in black lipid film experiments [ 127,128]. Different specificity patterns are usually obtained in non-vectorial and vectorial assay systems. The requirement for unidirectional incorporation and the variable leakiness of lipid bilayers will complicate vectorial assay systems. These effects are, for example, well documented for the Ca2+-translocating ATPase of sarcoplasmic reticulum [129,130]. A largely unidirectional incorporation of mitochondrial enzymes has recently been achieved by use of mixed liposomes containing acidic lipids [ 131 ]. A specific requirement for phosphatidylglycerol has been reported for enzyme II of the phosphoenolpyruvate transport system ofE. coli [ 132]. However, only experiments performed at single lipid concentrations were reported, and sodium dodecylsulphate could partially (about 20%) substitute for phosphatidylglycerol [ 132]. The experiments in which a specific requirement for phosphatidylglycerol was derived from the inhibitory effect of a phospholipase D preparation [133] have been refuted [ 134]. Previous reviews [11,23] have indicated a specific requirement of the galactosyl-transferase of lipopolysaccharide biosynthesis for phosphatidylethanolamine.
Phosphatidylserine, for review see Cholesterol Various proposals, for review see Phosphatidylethanolamine Cardiolipin, phosphatidylglycerol
(Na+-K+)-ATPase
Rhodopsin Cs.~-isoprenoid alcohol kinase
Cytochrome oxidase
Proposed specificity
Catalytic protein
40,95 34,211 215
20,125,143 219,220
Refs.
REFUTED OR MODIFIED LIPID SPECIFICITIES OF MEMBRANE ENZYMES
TABLE Ill
Various lipids or detergents (+ endogeneous cardiolipin) Various lipids, certain detergents Various lipids or detergents
Various negatively charged lipids or mixtures
Recent result
212-214 93,216-218
125,153,154, 176 95,204
Refs.
t-,J
222 However, the original report on the lipid specificity of this enzyme reaction clearly indicates that phosphatidic acid, phosphatidylglycerol, cardiolipin and bovine brain cerebroside were also active [81]. Lecithins appeared to be inactive [81]. A more recent study [82] indicates that the related glucosyl-transferase enzyme is activated by phosphatidylcholine. Previous reviews [13,14] have also emphasized the report [135] that malate dehydrogenase, a peripheral enzyme from Mycobacterium avium, was specifically activated by a single molecule ofcardiolipin. However, the original report [135] also stated that full activation was possible with lecithin or a neutral detergent, when another assay procedure was used. In recent work with highly purified enzyme from Mycobacteriumphlei, the specificity for cardiolipin could not be reproduced [136]. A strict polar group specificity for activation by lecithin has repeatedly been confirmed for the mitochondrial fl-hydroxybutyrate dehydrogenase. However, even this enzyme was not specific for the bilayer structure, ester linkages or detailed head group structure of phosphatidylcholine [86,87,137]. The chemically similar detergent, stearoyl phosphorylcholine [86], and the phosphatidylcholine analogue, N,N,N-trimethylphosphatidylbutanolamine [137], could substitute for phosphatidylcholine. In addition, it has been observed for this enzyme [87,138], for a number of reactions of oxidative phosphorylation [ 17,18,69,131,95,139-142] and for Ca 2+ translocation [130] that lipid mixtures will activate more effectively than any single lipid species tested. This may be of interest with regard to the lipid heterogeneity of biological membranes (see below). When presented in mixed micelles with inactive lipids, only 2.5 to 4 molecules of phosphatidylcholine per polypeptide chain were required for activation [87]. Possibly, fl-hydroxybutyrate dehydrogenase is activated by a combination of non-specific solubilization (see Section IVC) and specific binding to high-affinity sites (cf. pyruvate oxidase, Section IVD). Several enzymes have been found to retain phospholipids even after treatment with excess detergent. In the cases of (Na+-K+)-ATPase [143] and Ca 2+ATPase [144] the retained phospholipid was very similar in composition to the bulk phospholipid. It is remarkable that detergent-treated (Na+K+)-ATPase [145] and mitochondrial ATPase [146] still gave X-ray reflections typical for lipid bilayer structure. Other proteins will retain specific phospholipids: sphingomyelin in the case of 5'-nucleotidase (ref. 147, but see also ref. 148 for a contrary report), cardiolipin in the cases of cytochrome oxidase [40,95], NADH-ubiquinone reductase [142] and acetylcholinesterase [149], and phosphatidylserine [150] or diphosphoinositide [151] in the case of glycophorin. These preferential lipid-protein associations may be related to the 'boundary' lipid concept (see Section IVC). Certain lipids may be bound preferentially in order to minimize a mismatch in the packing of boundary lipid around the irregular surface of the protein. The heterogeneous lipid composition of biological membranes may be necessary to satisfy the different packing requirements of the embedded proteins, and this may also explain the efficient reconstitution by mixed lipid phases (see above). At
223 present, however, there is insufficient data to substantiate these explanations. A recent example may illustrate the present uncertainty about the packing requirements of membrane proteins. It has been suggested that the activity of the sarcoplasmic CaZ+-ATPase is determined by an 'annulus' of about 30 phospholipid molecules, and that the enzyme activity responds sensitively to structural changes of these phospholipid molecules [100,103,130]. Recent results indicate, however, that most of the phospholipid 'annulus' can be replaced by the neutral detergent, dodecyloctaoxyethyleneglycol-monoether[96]. It should finally be noted that a preferential association with certain phospholipids is also shown by simple compounds. For example, cholesterol will preferentially associate with sphingomyelin in mixtures ofsphingomyelin and other phospholipids [ 152].
VB. Viscotropic regulation Functional activities of biological or reconstituted membranes often show bior triphasic Arrhenius plots (see Section III). The Arrhenius plots for activation of (Na+-K+)-ATPase with various defined species of phosphatidylglycerol are shown as an example in Fig. 4a. The temperature values of the discontinuities differed for the various lipid species, and correlated with their T,. values [153]. The term 'viscotropic' has been introduced by Kimelberg and Papahadjopoulos to designate the "influence of fatty acyl chain fluidity on enzyme functions" [153,154]. The term 'fluidity' is rather vague and only loosely defined. Associated physical parameters include spectral order parameters, relaxation times, partition coefficients of probe molecules, lateral or rotational diffusion coefficients, T,. values, and so on. These parameters differ greatly in their physical meanings, yet they have been used to establish a general correlation between lipid motional parameters and the funcz.
3 ¥
2 ¥
T
;>-
~I,,
~-C>: ~-4' _J00 - , ,
(.9 O ....I
i
3.1
1 xlO' 3,6 "T
3,3
I_x10 ~ +
i
3.6
T
Fig. 4. Viscotropic regulation, a. Arrhenius plots for the activation of(Na+-K+)-ATPase by various species of phosphatidylglycerol. 1, dioleoyl phosphatidylglycerol; 2, dimyristoyl phosphatidylglycerol; 3, dipalmitoyl phosphatidylglycerol; 4, distearoyl phosphatidylglycerol. The calorimetric Tc values of three of these lipids were within the temperature range studied, and are indicated in the upper part of the graph, Redrawn from ref. 153. b. Arrhenius plots for the cytochrome b 5 reductase reaction in liposomes of dimyristoyl phosphatidylcholine. The molar ratio of cytochrome b 5 to cytochrome b 5 reductase was approx. 2 in curve 1, and 45 in curve 2. The Tc value of dimyristoyl phosphatidylcholine (23.7 °C) is indicated in the upper part of the graph, Redrawn from ref. 169.
224 TABLE IV VISCOTROPIC REGULATION
Selected examples for the correlation between functional acivity of membrane enzymes and motional parameters of the lipid phase are shown. Enzyme reaction (Na+-K+)-ATPase
Methods used
Refs.
Use of defined lipids, calorimetry
153,154.176. 221.222 93,216,217,223
(el" Fig. 4a), ESR spectroscopy
Cr,~-isoprenoid alcohol kinase Galactosyltransferase Cytochrome br, reductase Ca'-'+_ATPase
fi-Hydroxybutyrate dehydrogenase Glucose-6-phosphatase + U DPglucuronyl transferase Mitochondrial ATPase Cytochrome P-450 oxygenase system Lactose permease
Use of defined lipids. ESR spectroscopy Use of defined lipids Use of defined lilpids (of Fig. 4b) Use of defined lipids, ESR and laC-NMR spectroscopy Llse of defined lipids
81 169 100,103,159,198, 200,208,224 10,126,138
ESR-spectroscopy
225
Use of defined lipids, ESR spectroscopy, X-ray diffraction ESR spectroscopy, electron microscopy. fluorescence polarization ESR and fluorescence spectroscopy, calorimetry, X-ray diffraction
85,146,226,227 101,161,228 66.90.155-158
tional properties of reconstituted enzymes. Some representative studies are listed in Table IV. Discontinuous Arrhenius plots of membrane enzymes were only a few years ago almost exclusively interpreted in terms of the crystalline ~ liquid-crystalline phase transition of the lipid phase. A close correlation between the 'break' temperatures of Arrhenius plots and T
