230

Biochimica et Biophysica Acta, 470 ( 1 9 7 7 ) 2 3 0 - - 2 4 1 © E l s e v i e r / N o r t h - t t o l l a n d Biomedical Press

BBA 7 7 8 1 6

THE S T R U C T U R A L T R A N S I T I O N S OF E R Y T H R O C Y T E MEMBRANES INDUCED BY CYCLIC AMP

S.V. K O N E V a, I.D. V O L O T O V S K I I a, V.S. F I N I N a, A.V. K U L I K O V b, V.A. K I R I L L O V a a n d E.I. Z A I C H K I N c

a Institute o f Photobiology, Academy o f Sciences o f the BSSR, Minsk; b Institute o f Chemical Physics, Academy o f Sciences o f the USSR, Moscow and c Institute o f Biochemistry and Physiology o f Microorganisms, Academy o f Sciences o f the USSR, Puschchino-on-Oka (U.S.S.R.) (Received J a n u a r y 21st, 1 9 7 7 ) (Revised m a n u s c r i p t received May 5th, 1 9 7 7 )

Summary The generalized structural transitions of e r y t h r o c y t e membranes induced by cyclic AMP were registered by ESR, fluorescence, freeze-fracture and circular dichroism methods. Two transitions different in nature were revealed. One, which arises at 1 0 - ~ - - 1 0 -~° M cyclic AMP, is cooperative and may be considered as a consequence of interaction of cyclic AMP with a receptor. It was calculated th at a structural rearrangement in one e r y t h r o c y t e ghost is induced by three cyclic AMP molecules. As a result of it the membranes are " l o o s e n e d " . The o t h e r transition arises at 10-1°--10 -8 M cyclic AMP and depends on the activity o f the protein kinase system. This transition was shown to be noncooperative and due to p h o s p h o r y l a t i o n of m e m b r a n o u s proteins. During this rearrangement the membranes are " s t i f f e n e d " . Both transitions were d e m o n s t r a t e d to relate to the m e m b r a n e integrity.

Introduction The problem o f biological amplification and regulation o f biochemical processes is one o f the considerations of m o d e r n cell biology. Cyclic adenosine 3 ' , 5 ' - m o n o p h o s p h a t e is k n o w n to be a universal mediator for d if f er en t h o r m o n e s and o t h e r biologically active c o m p o u n d s [1--3]. The participation o f cyclic AMP in the regulation o f cell growth, m e m b r a n e transport, b y o s y n t h e t i c reactions has been proved [4]. However, a concrete mechanism of coupling between cyclic AMP and the Abbreviation: IPK, inhibitor of cyclic AMP-dependent protein kinase.

231 subsequent biochemical and physiological events in the membranes and cells remains to be elucidated. The hypothesis of induction of the cellular protein kinase system does not explain the pleiotropic and polyfunctional action of cyclic AMP [5]. Recently a great deal of data emphasizing an important role of generalized structural transitions of cellular membranes in the regulation of life processes have been accumulated [6,7]. The structural transitions of biological membranes are initiated by factors both exogenous {physiologically moderate temperatures, light etc.) and endogenous (metabolites, hormones including cyclic AMP) [6--10]. The present paper summarizes experimental results relating to the nature and character of the structural transitions induced by cyclic AMP in erythrocyte membranes. The data obtained show that cyclic AMP causes the structural transition of the membrane, involving mainly its protein phase. The existence of two transitions was demonstrated. Materials and Methods Erythrocyte membranes were prepared by the technique of Dodge et al. [11] from fresh donor blood. The membranes were resuspended in hypoosmolar buffers to prevent resealing of ghosts. As was shown by the control experiments, formation of resealed ghosts did not take place. This excluded involvement of the permeability effects. For registration of structural transitions ESR, protein fluorescence, circular dichroism and freeze-fracture methods were used. ESR spectra were recorded in the X-band of frequencies on the spectrometer EPR-2. As spin probes the nitroxide derivatives of stearic acid (probes I and II) and benz-3,-carboline {probe III) were used. The average values from 5--7 experiments are given (S.E.M. < 0.7%). The incorporation of probes into the membranes was achieved by mixing them with ethanol probe solution (10 -2 M). The unbound probe was separated from incorporated material by centrifugation at 15 000 X g, 20 min. The protein fluorescence measurements were carried o u t on a double-beam absolute spectrofluorimeter FICA-55 and circular dichroism measurements on spectropolarimeter " S p e c t r o p o l - l " equipped with a special attachment. In these experiments we registered tryptophan fluorescence intenstiy, which reflects a quantum yield of protein emission. Also the half-widths of differential spectra associated with shifts of the fluorescence spectra were measured. In circular dichroism experiments the molar ellipticity at 222 nm, reflecting mainly the a-helix content of membranous proteins, was determined. Freeze-fracturing or freeze-etching was done by standard techniques [12]. Membrane samples for freeze-fracturing were pelleted and frozen in their reaction medium without special cryoprotectors on bare copper grids which were put into propane at liquid nitrogen temperature. Freezing, fracturing, etching and shadowing were realized in JEE-4c apparatus (JEOL). Platinum-carbon replicas were investigated in a JEM-100B (JEOL) electron microscope. The protein concentration in the samples was measured by the method of Lowry et al. [13].

232 P r o t e i n kinase i n h i b i t o r ( I P K ) was isolated f r o m r a b b i t muscles a c c o r d i n g to t h e t e c h n i q u e o f Walsh et al. [ 1 4 ] . E x p e r i m e n t s w e r e carried o u t in 50 m M Tris - HC1 b u f f e r , p H 7,0. T h e pH value r e m a i n e d c o n s t a n t u p o n a d d i t i o n o f effectors.

Results

ESR spectroscopy of spin probes ESR spectra of probes I, II, III in erythrocyte membrane are presented in Fig. 1. Fig. 1 gives an idea of the basic parameters from which T, the motion parameter (probe I) [ 15], S, the order parameter (probe II) [ 16] and parameter P (probe III) were calculated. The parameter P is a ratio of amplitudes of the restric-

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III

into the erythrocyte membrane. Mea50 mM Tris • HCI buffer, PH 7.0; 50

233

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(°Io)

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1 0 -1 0 10 9 1 0 43 Concentration (M)

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Fig. 2. D e p e n d e n c e o f t h e m o t i o n p a r a m e t e r o f p r o b e I (r, o p e n a n d c l o s e d t r i a n g e l s ) a n d t h e o r d e r p a r a m e t e r o f p r o b e II (S, o p e n s q u a r e ) in t h e e r y t h r o c y t e m e m b r a n e o n t h e c y c l i c A M P c o n c e n t r a t i o n . 1 a n d 3, e r y t h r o c y t e g h o s t s o n l y ; 2, e r y t h r o c y t e g h o s t s w i t h 2 0 p g / m l o f I P K . Fig. 3. D e p e n d e n c e o f t h e m o t i o n p a r a m e t e r (T) o f p r o b e I in t h e e r y t h r o c Y t e m e m b r a n e o n c y c l i c A M P c o n c e n t r a t i o n . 1, e r y t h r o c y t e g h o s t s w i t h 1 m M MgCI 2 a n d 0 . 2 m M A T P ; 2, t h e s a m e as in 1 b u t w i t h 2 0 pg/ml IPK.

ted and more restricted components of the first derivative of the low-field of the ESR spectrum that correlates with membrane fluidity. The data obtained with membranes without Mg • ATP are presented in Fig. 2. At the cyclic AMP concentration of 2 • 10 -11--10-I° M the probe I mobility in the membrane is greatly enhanced. In the presence of IPK (curve 2), AT, after reaching its maximal value, is not changed any more with subsequent increase in the cyclic AMP concentration. Without IPK {curve 1) a decrease (10 -9 M) following some increase in the probe I mobility is observed. Addition of ATP and Mg 2÷ required for phosphorylation transforms the dependence r = f (cyclic AMP). In this case cyclic AMP decreases the probe I mobility. The whole process is described by a complex curve with two peaks at 2 • 10 -~° and 2 • 10 -8 M separated by the minimum at 2 • 10 -9 M (Fig. 3, curve 1). In the presence of IPK (curve 2) the probe I mobility first drops (minimum at 10 -9 M) and then rises (maximum at 8 • 10 -9 M). Quite different data were obtained with probe II. The order parameter (S) remains practically unchanged under all cyclic AMP concentrations used (Fig. 2, curve 3). The data with probe III associated with the hydrophobic zones of the membrane are shown in Table I. The parameter (P) of ESR spectra increases at 10 -7 M cyclic AMP. The essential influence upon structural response of the mem-

234

TABI,E

I

F I , ' F F C T OF" C Y C L I C A M P ( ) N EI(YTII ROCYTE MEMBRANE

PARAMETER

t' OI," S P I N

PI,~()BE 11I I N C O l i P O I i A T I , : I )

l':xi)erimcntal material

Without

Intact membrane

0.41 + 0.01

[).55 ~ 0.02

After treatment of intact membrane with cyanuric chloride

0.33 + 0.01

0.37 + 0.02

After treatment of intact membrane with iodacotamide

0.31

0.36 + 0.01

eyeli~ AMP

+ 0.02

INTO Tile

With 10 -7 M ('b,'lic A M P

brane was exerted by group-specific protein reagents. After treatment of the membrane with cyanuric chloride and iodacetamide reacting with sulphydryl, imino and amino groups the effect of cyclic AMP on the P value decreases. Treatment with glutaraldehyde stabilizing the membrane structure exhibits the same effect. Cyclic AMP does not affect the mobility of probes after mild ultrasonic disintegration of membranes. It should be mentioned that recording of ESR spectra of spin probes in the membranes was carried out in the presence of the phosphodiesterase inhibitor (10 -3 M) theophylline. Owing to that, the concentration of cyclic AMP in the system was kept at a constant level. In the absence of theophylline the effects were observed at a much higher cyclic AMP concentration (by about three orders of magnitude) than in the presence of the inhibitor. F l u o r e s c e n c e and circular d i c h r o i s m data

Erythrocyte membranes reveal the typical protein spectra of fluorescence with emission m a x i m u m at 330--331 rim. This indicates the hydrophobic environment of t r y p t o p h a n residues in the membranous proteins. Fig. 4 presents the dependence of the intensity and relative half-width of the differential spectrum of t r y p t o p h a n fluoresence of e r y t h r o c y t e membrane proteins and molar ellipticity versus the cyclic AMP concentration. As is shown in Fig. 4, addition of cyclic AMP results in appreciable changes of these parameters. Fluorescence quenching, an increase in the half-width of spectra and a decrease in molar ellipticity at 222 nm are observed. The control measurements indicated that changes in the fluorescence parameters observed do not arise from such trivial optical effects as screening of excitation light, reabsroption of fluorescence or from light scattering. Similarly, the methodical artefacts which arise from superposition of dichroism of cyclic AMP and light scattering do not cause a decrease in molar ellipticity. This conclusion follows from the fact that cyclic AMP at the concentration used does n o t possess any optical activity, and light scattering of membranes with cyclic AMP remains practically unchanged. It is characteristic that noncyclic analog of the nucleotide 5-AMP did not induce any noticeable changes in fluorescence and circular dichroism parameters. The membranes treated with mild doses of ultrasound do not show the effects described.

235

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Concentration (M) Fig. 4. Dependence of fluorescence intensity (•340) (2), halfwidth of differential spectra (A~) (3) and molar ellipticity (0222) (4) of etythrocyte ghosts on the cyclic AMP concentration. Curve 1 shows the noncyclic 5'-AMP efficiency.

It should be emphasized that the effective cyclic AMP concentration necessary for the transitions differs for spectroscopic and spin probe experiments by a factor of ten. This paradoxal discrepancy is due to the following: (a) theophylline distorts the real effect because of its screening and optical activity; (b) totally hemoglobin-free ghosts are used in optical experiments in contrast to the ESR-spectroscopic ones. Hemoglobin depletion is known to be followed by the distortion of the ghosts' integrity and by partial removal of the corresponding receptor site [17]. Indeed, the control measurements using spin probes demonstrate that in the theophylline absence the structural response of maximally washed ghosts is shifted to the concentration range characteristic for optical probing.

Freeze-fracturing of erythrocyte ghosts Freeze-fracturing electron micrographs of erythrocyte membranes are presented in Fig. 5. The outer half of the fractured control membranes viewed from the outside of the cells (protoplasmic fracture face [ 18] ) demonstrates a random distribution of many intramembrane particles measuring 80--100 A in diameter. There are also some aggregates consisting o f several particles. According to the calculations there are about 3000 particles in pm 2 of the fracture face of the control samples (Fig. 5a). After the addition of Mg • ATP (2 • 10 -4 M) to the medium there are no changes in the quantity of intramembrane particles in the fracture face (Fig. 5c). Only the tendency towards association of the particles with a change in the form and distribution of particle-free zones is manifested. In contrast, addition of cyclic AMP (10 -s M) results in an essential change of freeze-fracturing data (Fig. 5b,d): the quantity of intramembrane particles per

236

Fig. 5. E f f e c t o f c y c l i c A M P o n scopy. The protoplasma fracture B, e r y t h r o c y t e g h o s t s w i t h 10 -8 a n d 0 . 2 m M A T P ; D, e r y t h r o c y t e

the e r y t h r o c y t e m e m b r a n e revealed by freeze-fracture electron microfaces are s h o w n . M a g n i f i c a t i o n X 52 0 0 0 . A, E r y t b r o c y t e g h o s t s o n l y ; c y c l i c A M P a n d 20 ktg/ml I P K ; C, e r y t h r o c y t e g h o s t s w i t h 1 m M MgCI 2 g h o s t s w i t h 1 m M MgCl 2, 0.2 m M A T P a n d 10 -8 M c y c l i c A M P .

p m 2 significantly decreases in t h e p r e s e n c e of Mg • A T P ( 1 7 6 0 + 70) or w i t h o u t it ( 1 8 0 0 + 70). S i m u l t a n e o u s l y t h e size o f particles increases. H o w e v e r , t h e i r dist r i b u t i o n d o e s n o t b e c o m e regular. As in c o n t r o l s a m p l e s t h e i n t r a m e m b r a n e particles are s c a t t e r e d o n t h e f r a c t u r e face, f o r m i n g aggregates c o m p r i s i n g 3--5 particles. T h e areas o n t h e f r a c t u r e face free o f particles increase c o r r e s p o n d ingly. Similar b e h a v i o u r is revealed f o r t w o e x p e r i m e n t a l v a r i a t i o n s (with and w i t h o u t Mg • ATP). A t t h e s a m e t i m e large h u m p - s h a p e d p r o t r u s i o n s o f a b o u t 1 0 0 0 A d i a m e t e r a p p e a r on t h e f r a c t u r e face a f t e r a d d i t i o n o f cyclic AMP a n d Mg - ATP.

237

Discussion The results of the experiments with spin probe I are alone enough to conclude that cyclic AMP induced the structural transitions of erythrocyte membrane. The character of structural changes registered by the probe I mobility is modified by the activity of the protein kinase system. When the protein kinase system is "switched off", r of probe I is decreased under the cyclic AMP action. This corresponds to "loosening" of the membrane zones with which probe I is associated. On the contrary, when membranous protein kinases are active cyclic AMP addition results in r increase, i.e. "stiffening" of the membrane. Such complex character of the dependence of structural membrane state on "switching o f f " of protein phosphorylation does not explain the observed effects on the basis of the idea of only one structural transition. At the same time the simultaneous consideration of curves in Figs. 2 and 3 permits us to describe them satisfactory by t w o structural transitions of different nature. One of these is not connected with protein phosphorylation and results in a decrease in the probe mobility (the S-shaped curve). Another transition does depend on phosphorylation and is accompanied by an increase of the probe mobility (the cupola shaped curve). Curve 2 (Fig. 2) corresponds to the first transition. The minimum and high-concentration maximum of curves 1--2 (Fig. 3) probably represent the superposition of both transitions with variable contribution of phosphorylation. The relatively small maximum of curve 1 (Fig. 2) is due to the weak protein phosphorylation by endogenous ATP. What components of erythrocyte membrane are involved into these structural transitions? It is known that nitroxide derivatives of stearic acid might interact both with proteins and with phospholipids [ 19]. The observed modification of ESR spectra may be thought to be due to the change of the structural state of both protein and lipid phases of the erythrocyte membrane. However this is not likely to be the case. For probe I only the aliphatic ends of stearic acid penetrate into the lipid bilayer, and nitroxide radicals together with polar end groups remain near the membrane surface where the probability of limitation of their motion by proteins is naturally higher. On the contrary, the nitroxide group of probe II is buried into the lipid bilayer of the membrane together with the nonpolar hydrocarbon chain of stearic acid. The data of Bieri and Wallach [19] are in favour of such a suggestion. These authors showed that probe 5NS (stearic acid with nitroxide group in the 5th position) interacts with the proteins of erythrocyte membrane much stronger than probe 16NS (16th position). Taking that into account, it is reasonable to suggest that the appreciable change in the probe I mobility is due to the modification of the structure of the membrane protein system. At the same time it is possible that parameter r of the label I also reflects a state of polar heads of phospholipids localized at the membrane surface. It is worth noting that upon ESR cross scanning of membrane the amplitude of changes decays in direction from surface to the center. Thus at 10 -7 M of cyclic AMP the change in mobility of probe I (iminoxyl radical in the stearate polar head) is about 9%, that of the parameter S {free radical at C-5 position) is about 1% [10] while the S parameter of probe II (radical at C-10) is not changed at all. This means that the main changes of fluidity are localized in hy-

238

drophilic surface of membrmle rather than in the h y d r o p h o b i c lipid bilayer. There are o th e r data indicative of the presumably protein nature of cyclic: AMP induced structural transition. The structural response of the m em brane is repressed by group-specific bifunctional reagents mainly to the protein NH2 groups. Such inhibition could be due to the stabilization o f the protein framework o f the membr a ne by some kind of molecular cross-links caused by cyanuric chloride and glutaraldehyde. In similar way SH group blockage by iodacetamide prevents the m em br ane modification induced by cyclic AMP (Table I). It is worthwile to n o t e that the major qua nt i t y o f NH: and especially SH groups belongs to the proteins rather than lipids. Also the fluorescence and circular dichroism data indicate the modification o f the protein phase of the membrane. It is known that their parameters d e p en d on the structural state of protein macromolecules in a m em brane [20]. For exmnple the short-wave shift o f the fluorescence spectrum suggests a polarity decreased in the m i c r o e n v i r o n m e n t of t r y p t o p h a n y l s which may be con"sidered as statistically distributed natural probes sensitive to protein conformation. Broadening of spectra and fluorescence quenching probably reflect the growth o f t r y p t o p h a n y l m i cr oe nvi r onm e nt heterogeneity. A drop in molar ellipticity observed as a response to the cyclic AMP action reflects a decrease in a-helix c o n t e n t of m e m b r a n o u s proteins and, as a consequence of that, a change in c o n f o r m a t i o n . Finally, a decrease in the quant i t y and distribution of aggregates o f in tr am e m br a ne particles on a fracture face of e r y t h r o c y t e membrane unequivocally indicates the structural changes in the protein f r a m e w o r k o f the m e m b r a n e [21]. The nature o f the intramembrane particles has been shown to be a protein one [22,23] and their aggregation is of physiological importance [24,25]. F u r t h e r m o r e , the large h u m p - f o r m e d protrusions on the fracture face o f the e r y t h r o c y t e m e m br a ne induced by cyclic AMP and Mg. ATP (i.e. u n d er the optimal conditions for a second phosphorylation-depend e n t transition) may be an indication of a large-scale structural modification of the membrane. It is very likely that the contractile elements of e r y t h r o c y t e m e m b r a n e are involved in the f o r m a t i o n of these protrusions. It is also known that mobility o f i nt r am e m br a ne particles is restrained in the membranes by spectrin [26] viewed as one of the main substrates of protein kinase in erythrocyte m e m b r a n e [271. Thus the data obtained allow us to make the conclusion that cyclic AMP induced at least two structural transitions in the protein phase of the erythrocyte membrane. One of t hem is at low concent rat i on (10-1'--10 '° M ) o f cyclic AMP and is n o t c o n n e c t e d with phosphorylation. It may be considered as a high-rate physical structural reaction. It is likely that a rapid change of the fluorescence parameters o f e r y t h r o c y t e membranes is due to such a reaction. The second transition is under the control o f the protein kinase system. It is greatly influenced by phos phor yl at i on of m e m b r a n o u s proteins and may be viewed as chemical structural reaction, i.e. a structural rearrangement associated with mechanochemical displacements of the m e m b r a n e molecular components after binding of inorganic phosphorus to contractile proteins. On the whole our interpretation of this structural transition is close to that o f Kury and McConnell [ 10] with sufficient difference, however, for us to emphasize the p r e d o m i n a n t role of proteins rather than lipids in generalization of

239 structural effects. Also, some data from ref. 10 might be considered as indirect evidence of ability of cyclic AMP to induce the membrane modification which is not causally connected with phosphorylation. Indeed, an increase of spin probe parameter S under the influence of cyclic AMP in the presence of Mg 2÷ and ATP was observed along with a decrease of S upon substitution of GTP for ATP. In the latter case phosphorylation has been blocked both because exgenious ATP and Mg 2÷ were absent and due to the inhibitory effect of GTP on protein kinase activity [27]. In other words, the conditions were as with the presence of protein kinase inhibitor without ATP and Mg 2÷ (Fig. 2, curve 2). It is quite evident that observed transitions are of a generalized nature because luminescence and circular dichroism parameters reflect the structural state of the whole pool of protein molecules rather than some of their fractions. Similar information is obtained from the ESR measurements. The effect of physical generalization is particularly demonstrated in those transitions which are not connected with the protein kinase system when the mechanism of chemical amplification through enzyme phosphorylation is "switched off". According to the calculations, the structural state of the membrane is changed after binding of approx. 3 molecules to the cyclic AMP receptor sites. At the same time the lipid bilayer of the membrane may also be incorporated to some extent in the structural rearrangements as follows from the negligible changes in the parameters of lipid spin probes according to Kury and McConnell [10]. There are other data suggesting the generalized nature of the transitions: (a) physical structural transition is realized at a comparatively narrow range of low cyclic AMP concentrations, and (b) the plot of the fluorescence intensity versus the cyclic AMP concentration is distinctly S-shaped. This means that the structural rearrangement obeys the co-operative law. In support of that the data in the Hill coordinates may be presented. So, the Hill numbers calculated for cur-

Ig

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-5

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Fig. 6. The Hill plot for t h e

(7")of s p i n

as a f u n c t i o n

The data

motion parameter of the cyclic AMP concentration.

Probe 1 incorporated

into the erythrocyte used.

ghosts

of Fig. 2 (curve 2) w e r e

Fig. 7. The Hill Plot for the f l u o r e s c e n c e i n t e n s i t y (1340) of e r y t h r o c y t e AMP c o n c e n t r a t i o n . T h e d a t a of Fig. 4 (cruve 2) w e r e u s e d

g h o s t s as a f u n c t i o n

of the

cyclic

240 yes 2 (Fig. 2) and 2 (Fig. 4) were found to be correspondingly equal to 2.1 and 2.3 (Figs. 6 and 7). The second transition (the chemical structural reaction) is nonco-operative, as judged by the smooth changes in probe I nlobility. The condition necessary for both structural reactions is integrity of membrane. The importance of the integrity, i.e. intactness of the native network of molecular interactions, is evident from the experiments with ultrasound-treated membranes. The mild ultrasonic fragmentation of erythrocyte ghosts totally removed the cyclic AMP induced rearrangements, as is seen from the results of ESR, fluorescence and circular dichroism measurements. For example, the rotational mobility of probe I remains unchanged in the sufficiently large membrane fragments clearly visible in the phase-contrast microscope. This emphasize the role of long-range forces in the realization of structural transitions. Finally, the question of the nature of the receptors from which the observed structural transitions are initated is of great importance. It is very likely that these receptors are different in nature. Besides the regulatory subunits of protein kinase being the well-known cyclic AMP-binding site [3] other receptors of protein nature possessing high affinity to cyclic AMP without any protein kinase activity have been demonstrated [28,29]. Summing up, we consider a possible biological role of the cyclic AMP induced structural transitions. One may suggest that both structural transitions are related to pleiotropic regulatory action of cyclic AMP. The location and mobility of membrane components which are under the control of the phosphorylation level of contractile proteins are of some importance for final regulation processes. The changes in passive permeability, transport of substances, membrane potential and activity of a number of enzymes may be a consequence of such transition. Acknowledgements We are especially grateful to Dr. R.I. Zhdanov for kindly presented samples of nitroxide derivatives of stearic acid. We are also thankful to Dr. S.L. Aksentsev and Miss T.S. Kortneva for their valuable assistance in the preparation of the English version of the paper. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

R o b i n s o n , G., B u t c h e r , R. a n d S u t h e r l a n d , E. ( 1 9 7 1 ) Cyclic AMP, A c a d e m i c Press, N e w Y o r k S m i t h , H.M. ( 1 9 6 8 ) A n n . N.Y. A e a d . Sei. 1 5 0 , 1 9 1 - - 4 6 7 B o w m a n , W.C. a n d N o t t , M.W. ( 1 9 6 9 ) P h a r m a c o l . Rev. 21, 2 7 - - 7 2 P o s t e r n a k , T. ( 1 9 7 4 ) A n n . Rev. P h a r m a e o l . 14, 2 3 - - 3 4 K u o , J. a n d G r e e n g a r d , P. ( 1 9 6 9 ) Proc. Natl. A c a d . Sci. U.S. 64, 1 3 4 9 - - 1 3 5 5 K o n e v , S.V., A k s e n t s e v , S.L. a n d C h e r n i t s k i i , E.A. ( 1 9 7 0 ) T h e c o o p e r a t i v e t r a n s i t i o n s of p r o t e i n s in a cell ( R u s s i a n ) N a u k a i T e k h n i k a , Minsk K o n e v , S.V., C h e r n i t s k i i , E . A . , A k s e n t s e v , S.L., M a z h u l , V.M., V o l o t o v s k i j , I.D. and N i s e n b a u m , G.D. ( 1 9 7 5 ) Mol. Cell. B i o c h e m . 7, 5 - - 1 7 M e y e r s , M. a n d Swisloeki, N. ( 1 9 7 4 ) A r c h . B i o c h e m . Biophys. 164, 5 4 4 - - 5 5 0 K o n e v , S.V., V o l o t o v s k i j , I.D. a n d Finin, V.S. ( 1 9 7 5 ) D o k l a d y AN SSSR ( R u s s i a n ) 2 2 3 , 1 4 7 3 - - 1 4 7 6 K u r y , P.C. a n d M c C o n n e l l , H.M. ( 1 9 7 5 ) B i o c h e m i s t r y 14, 2 7 9 8 - - 2 8 0 3 D o d g e , B.T., M i t c h c L C. a n d H a n a h a n , D.J. ( 1 9 6 3 ) A r c h . B i o e h e m . Biophys. 100, 1 1 9 - - 1 3 0 M o o r , H. ( 1 9 7 1 ) P h i l o s . T r a n s . R. Soc. L o n d . , Ser. Biol. Sci. 261, 1 2 1 - - 1 3 1 L o w r y , O . t t . , R o s e n b r o u g h , N.J., F a r r , A.J. a n d Randall, R.J. ( 1 9 5 1 ) J. Biol. C h e m . 193, 2 6 5 - - 2 7 5 Walsh, D . A . , A s h b y , C.P., G o n z a l e s , C., Calkins, D., Fischer, E. a n d Krebs, E.G. ( 1 9 7 1 ) J. Biol. C h e m . 246, 1 9 7 7 - - 1 9 8 5

241 15 16 17 18

19 20 21 22 23 24 25 26 27 28 29

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