39

Biochimica et Biophysica Acta, 559 (1979) 3 9 - 6 9 © Elsevicr/North-ttolland Biomedical Press

BBA 85192 THE APPLICATION OF CHEMICAL CROSSLINKING FOR STUDIES ON CELL MEMBRANES AND THE IDENTIFICATION OF SURFACE REPORTERS

TAF H. JI

Department oJBiochemistry, University of Wyoming, Laramie, WY 82071 (U.S.A.) (Received July 31st, 1978)

Contents I.

Introduction

11.

General approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Choice of reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Crosslinking conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Analysis of crosslinked products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

41 41 41 42

1II.

Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Homobffunctional reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Photosensitive heterobifunctional reagents . . . . . . . . . . . . . . . . . . . . . . . . . ( l ) Photosensitive groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Photolysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Conventional Functional Groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Cleavability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

43 43 44 48 48 49 49

IV.

Biological systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Identification of surface receptors: macromolecular affinity labels B. Erythrocyte membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (1) Spectrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (2) Transmembrane linkage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (3) Band 3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . (4) Other proteins and glycoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . (5) lfeteropolymers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Sarcoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Mitochondria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Membrane lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Lymphocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

V.

............................................

Possible artifacts of crosslinking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

40

...........

53 53 55 55 58 59 60 60 61 61 62 62 63

Abbreviations: EGTA, ethylene glycol bis(#-aminoethyl ether)-N,N'-tetraacetic acid; SDS, sodium dodecyl sulfate.

40 VI.

Concludingremarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

64

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

65

I. Introduction

It is now welt established that cell membranes are an assembly of various molecules interacting with one another within or at the inner or outer surface. In order to understand these interactions and the molecular organization of cell membranes, chemical crosstinking has been developed: this technique is unique in that chemical evidence of in situ interactions can be obtained. Crosstinking has been a major tool for predicting subunit structures of molecules and molecular associations in biological membranes. Protein structural studies utilizing chemical crosslinking technique began with the work of Zahn during the 1950's [1-3] and continued in the 1960's primarily with the work of Wold and his colleagues [4 12]. The first chemical crosslinking studies of cell membranes were performed on erythrocytes; after treatment with either difluorodinitrobenzene [13] or diethylmalonoimidate [t4], the red blood cells became resistant to hemolysis. Although this is now thought to be due to crosslinking of integral and peripheral membrane proteins [ 15-17], the early studies are not very revealing. In fact, the usefulness of chemical crosslinking in elucidating membrane structure had not been realized until sodium dodecyl sulfate-polyacrytamide gel electrophoresis was applied for the systematic analysis of crosslinked products [18,t9]; it underscores the fact that the introduction of new methodologies has been vital to the progress of chemical crosslinking studies. By gel electrophoresis in detergent buffers, crosslinked products which are refractory to separation during detergent solubilization can be identified. The introduction by several laboratories [ 15,17,20-24] of cleavable reagents containing a disulfide bond or a glycol bridge has made the general analysis of components of crosslinked products simple and easy. This is accomplished with two-dimensional gels where the crosslinked products are separated in the first dimension and cleaved in the gel. and the cleaved components are analyzed directly in the second dimension [15]. Successful formation of crosslinks depends on the availability of suitable reactive groups located within the effective range of the reagents; due to this limitation, not all adjacent or interacting components may be crosslinked. However, an increasing variety of available crosslinking reagents, especially nonspecific reagents such as azides, and careful attention to crosslinking conditions should enhance the efficiency of crosslink formation. For example. Liu et al. [25] have reported that difficulties in direct crosslinking of integral to peripheral membrane proteins in erythrocyte membranes [15-17,26] can be avoided by appropriate choice of pH and temperature. On the other hand, too many crosslinks can impose problems. It happens because all susceptible molecules present in membranes or cells are subjected to crosslinking when crosslinking reagents are introduced to the systems and the result may disclose a complex array o f subunit structures and molecular associations. However, such indiscriminate formation of crosslinks can result in a high multiplicity of crosslinked products (a difficult task for analysis) and crosslinked products of low yield may go undetected To eliminate this problem, more selective crosslinking with macromolecutar photoaffinity labels, utilizing photosensitive heterobifunctional reagents, has been introduced [27,28]. Specific crosslinking o f surface

41 receptors with ligands has been successful in a limited number of systems [27- 29]. A major objection to the usefulness of crosslinking techniques in membrane studies has been the possibility that random collision-dependent crosslinks could occur at a significant frequency [17]; the presumption being that molecules are crosslinked nonspecifically during random collisions in fluid membranes [30,31]. Rapid (millisecond) crosslinking with photosensitive reagents can overcome this problem in at least some systems, such as the erythrocyte membrane [17], where the lateral diffusion rates of proteins are known to be relatively low [32,33]. In this review, recent developments in chemical crosslinking studies, in particular, the use of photosensitive heterobifunctional reagents, are presented. It will also attempt to analyze the lessons learned from and problems raised by crosslinking studies on a number of selected membrane systems. For readers who are not familiar with chemical crosslinking techniques, a brief section is devoted to the general approach.

I1. General approach Technically, it is not difficult to cross/ink cell membranes because it can be achieved simply by mixing membranes with reagents. What is time-consuming and not easy is to find optimal conditions for crosslinking so that crosslinked products and cleaved components can be clearly demonstrated. Crosslinking conditions vary depending on reagents, and there is a variety of reagents.

IIA. Choice of reagents Considerations for choosing reagents include: (a) cleavability, (b) photosensitivity, (c) homo- versus heterobifunctionality, (d) hydrophobicity or hydrophilicity, (e) charge, (f) specificity, and (g) molecular dimensions of reagents. Cleavable reagents are necessary to identify protomers of a crosslinked product. Photosensitive reagents are convenient when reactions need to be controlled and rapid crosslinking or nonselective reaction is desirable. Heterobifunctional reagents offer a versatility in the reactivity and control of reactions. In particular, photosensitive heterobifunctional reagents are useful in the formation of macromolecular affinity labels. Hydrophobic or hydrophilic reagents may reveal the environment around a crosslinked point, but as yet, no systematic study has been carried out. A reagent can be neutral, or charged positively or negatively and this will influence its permeability and interaction with sites on the membranes which may also bear charges. Reagents with differing specificities are sometimes useful in examining a single component of a complex system. In erythrocyte membranes, for example, sulfhydryl reagents favor the crosslinking of an integral membrane protein (band 3) whereas a peripheral membrane protein (spectrin) is crosslinked preferentially by reagents specific for amino groups, such as imidates. However, these crosslinks are not mutually exclusive. Finally, the molecular dimensions of the reagent also have an effect on crosslinks. For example, an imidate shorter than 5 A usually yields few or no crosslinks, whereas extensive crosslinks can be achieved when the length is more than 11 A.

liB. Crosslinkh,g conditions Generally, crosslinking of cell membranes is dependent on time period, temperature, pH, reagent concentration, and buffer composition. Crosslinking has been carried out for

42 several minutes to an hour, regardless o f reagents. Exceptions are crosslinking with photosensitive reagents for milliseconds [ 17] and crosslinking o f glycoproteins for several hours [34]. Crosslinking by most reagents proceeds over the temperature range 0-37°C [26, 35], and at neutral to alkaline p H [17,25,28,36]. The optimal concentration Of reagents varies depending on the reactivity and hydrolysis rate. Some reagents are hydrolyzed readily [ 3 7 - 4 0 ] and therefore an excess o f reagent may be required [41]. However, a single crosslink is as good as many and a minimal crosslinking is more desirable, since reagent excess can cause monofunctional reactions and adverse effects. Crosstinking studies have generally been carried out in simple buffer systems, such as phosphatebuffered saline or isotonic phosphate, but it is possible that careful modification of the buffer composition may produce diverse results reflecting changes in the molecular interactions.

IIC. Analysis of crosslinked products Crosslinked products have been identified on one-dimensional gels [ 19,34,42-44] and protomomers can be identified after cleaving either on one-dimensional [19,45,461 or

S

b

Fig. 1. Gel profiles of erythrocyte membrane proteins, before and after crosslinkirlg and of ~he components cleaved from crosstinked complexes. (a) Membrane-proteins, nonerosslinked, were sotublized in SDS and electrophoresed on agarose-polyaerylamide gel. (b) Same as (a) except that membranes were crosslinked before solubilization. (c) Aduplieategel identical to get (b) was subjected to cleaving and two-dimensional electrophoresis ona polyaerylamide gel. For comparison of cleaved products, the photograph of the one-dimensional agarose-polyacrylamide gel pattern of crosslinked membrane proteins, in the direction of the first dimension, and the photograph of the one.dimensional polyacrylamide gel pattern of noncrosslinked membrane proteins, in the direction of the second dimension, are shown along with the two-dimensional electrophoresis of cleaved products on polyacrylamide gel. 3X, 4X, 5X and 6X are components cleaved from cro~linked homopolymers of bands 3, 4.2, 5, and 6, respectively.

43 two-dimensional gels [15,22,24,47] (Fig. 1). Usual sodium dodecyl sulfate-polyacrylamide gels have an exclusion limit at M r = 5 • I0 s. As a result, most crosslinking studies of membranes have shown an intense band of crosslinked products retained at the top of the gels. The exclusion limit can be extended significantly up toMr = 3 - 5 " 106 and the resolution of larger crosslinked products can be improved when agarose-acrylamide gels are used [17,191 . New bands appearing on gels after crosslinking are certainly candidates for crosslinked products. However, care must be exercised since they may represent molecules with altered electrophoretic mobility or non-crosslinked simple iaggregates. Frequently the electrophoretic mobility of polypeptides on gels increases when crosslinked because of intra-chain crosslinks which results in a more compact protein in detergent solution. Cleavage of crosslinks or reactions with monofunctional reagents can clarify this question. The homogeneity of bands of crosslinked products is critical and has to be examined, especially when multiple crosslinks are suspected. Any bands which diminish in intensity or disappear upon crosslinking are likely to have been crosslinked, but may be the result of extraction, proteolysis, or reduced staining facilitated by the crosslinking conditions. Careful attention to extracted components, proteolytic products, and total intensity of staining may resolve this problem. Because not all membrane components are readily visualized by a specific stain or method [48,49], unstained components should be considered for their involvement in crosslinked products and, therefore, examined by more than one staining method. Cleavage of crosslinks and the identification of protomers of crosslinked products are now an accepted general procedure, and a variety of cleavable reagents are available (see the following section). Once protomers are identified, the con> position or stoichimetry of a crosslinked product is often evaluated by (a) the molecular weights estimated on the basis of relative electrophoretic mobility, (b) the alignment of cleaved components on two-dimensional gels [15], and (c) the relative intensities of the bands. However, although these tests may be sufficient in a simple system, they are indirect and not foolproof. Therefore, additional evidence may be required in more complex systems with multiple crosslinks for unequivocal demonstration of the composition of each crosslinked product, perhaps by specific staining with lectins [50- 52] or antibodies [53,54].

III. Reagents Chemical crosslinks can be established either by introducing a bridge with bifunctional reagents or by catalyzing the formation of a new chemical bond between interacting components. In the latter case, chemicals like cupric penanthroline or enzymes such as transglutaminase have been used [12]. In the former case, crosslinking reagents can be classified into two groups, homo- and heterobifunctional reagents.

IliA. Homobifunctional reagents Bifunctional reagents carrying two identical reactive groups belong to this category. Among a variety of available homobifunctional reagents, only imidates and N-hydroxysuccinimide esters are discussed in this review, for they are frequently used in crosslinking studies on membranes. Bisimidates introduced by Hartman and Wold [6,7] and Dutton et al. [14] are easy to synthesize [55] and the chain length can be varied [56]. They are highly specific for amino groups [7,37,57,60]; the chemistry of imidates has

44

been reviewed recently by Peters and Richards [60]. Most bisimidates are permeable to membranes. They are readily soluble in water and hydrotyze rapidly with a half life of several minutes to approximately half an hour depending on the pH [37-40]. Up to 10% fold excess of reagent (a concentration range of 0 . 1 - 1 0 mM) Is required for complete reaction with available amino groups in erythrocy te membranes. To circumvent the degradation problem, incremental additions of reagents are desirable, lmidates react over a wide range of pH (pH 7 10) [t7,25,28,37,61], and temperature (0 40°C) [26,35,37]. An alkaline pH above 9.5 favors both the required deprotonation of the e-amino groups of proteins and the reaction of imidates with amines (Scheme I: amidination [37.40.57. 60]). It takes 1 0 - 2 0 rain to reach the half maximum amidination of concanavalin A al room temperature [28] and less than 10 rain for phosphofructokinase at 4°C [62]. The reaction rate decreases severalfold as the temperature drops from 39 to 25°C and then again from 25 to near 0°C [37]. Near or below 0°C amidination occurs at considerably slower rates and requires longer reaction times of from several hours to overnight [14,26, 35,63]. NH~

NH~

R-C-O-CH3 + NH2-P -+ R-C-NH-P + CHBOH 0 R-

-O-

0 + NH2-P

~ R-C-NH-P

+ HOI

o

o

Scheme I An amidine bond is stable to the acidic condtions normally used for peptide bond hydrolysis [6,7]. The hydrolysis rate in 6 M HC1 at 100°C is equivalent to a half life of 46 h [64]. The original positive charge of amines is preserved after amidination and the pK a shows an alkaline shift of approx. 2 pH units [37]. In general, cleavage of amidines with no side reactions has not been easy, primarily due to low yields and inconsistent results [28,60]. tmidates can be readily quenched with various amines including amino acids. Ammonia is a less efficient quencher because its reaction with an imidate is reversible [60]. In addition to imidates, N-hydroxysuccinimide esters are becoming popular. They are known to react primarily with amino groups to form an amide bond (Scheme I) but also with the imidazole group of histidine and the sulfhydryt group of cysteine [65,66]. They are unstable in aqueous media, with a hatflife on the order of 10 min at pH 8.6 and 4°C [66] and several hours at pH 7.0 and O°C [67]. Reactions are complete within 10 min at 4 0°C [66,67]. A number of other reagents have been used in similar studies and are covered in several general reviews [ 10,12,60].

IIIB. Photosensitive heterobifunctional reagents Bifunctional reagents carrying two dissimilar groups, one photosensitive and one convential group, such as an imidate, belong to this category. In the course of studying the structure of cell membranes by chemical crosstinking, it has become clear that the utility of conventional homobifunctional reagents is limited due to several potential inherent

TABLE I

PHOTOSbNSITIVE

specific

!l

N3

1161

(171

_

.____

E.1:. and

E.F. and Ji, T.H.

[29] and Vanin, Ji, T.H.

Vanin,

1681

0

;H+2 cl-

-;-0-CHX

NH+* cl-

-&O-

S-S-(CH2)3-C-0-CH3

s-s-(CH~)~

S-S-(CH,)2

::

Pyridyl-2,2’dithiobenzyldiazoacetate (PDD)

N3

N3

N3

N3

0

Reference

[I71

acid

REAGENTS

Structure formulae ~_--~~~

HETEROBIFlJNCTIONAL

N-(4-azidophenylthio)phthalimide (APTP)

di-N-(2.nitro&azidophenyl)cystamine-S,S-dioxide (DNCO)

Sulfhydryl

Methyl4(4_azidophenyldithio)butyrimidatc (MADR)

Methyl-3-(4-azidophenyIdithio)propionimidate (hlADP)

(NHS-APDP)

N-Hydroxysuccinimide ester of 3-(4-azidophenyldithio)propionic

Reagent _~ ~~ .__~~ Amino specific

CLEAVABLE

N-Hydroxysuccinimideester of (4.azido-2-nitrophenyt)-3'-aminobutyric acid (NHS-ANAB)

Methyl 2-(4-azidophenyl)acetimidate (MAPA)

Methyl 4-azidobenzoimidate (MABI)

2,4-Dinitro-5-fluorophenylazide (DNFA)

4-Fluoro-3-nitrophenylazide (FNA)

Amino specific

Reagent

NO2

)3-C-O-N~

O

NH2+CICH2 -C,-O-CH3

NHa *C1-

N3-~NH-(CH2

N3_ Q

F

N3- ~ N O 2

NO2

N3- ~ F

NO2

Structure formulae

TABLE II NONCLEAVABLEPHOTOSENSITIVEHETEROBIFUNCHONAL REAGENTS

II

O

Ott [73,74]

[27,281

127,281

[721

[69-71]

Reference

4~

NO2

4-(Bromoaminoethyl)-3-nitrophenylazide N3 ~ -

NH2 +C1-

II O

O II

Br

NH-CH 2 -CH 2 Br

NO2

N20 H II CF3 -C-CC1

N3©O

O

4-Azidophenyl maleimide (APM)

N3/O

N3 ~ C - C H 2

2-Diazo-3,3,3-trifluoro propionyl chloride (DTPC)

O

o

~k~.~C--NH--CH2 - C - O - C H 2 -CH 3

N3

-O-N~

NcO 2

p-Azidophenacyl bromide (APB)

Sulfhydryl specific

N-5-Azido-2-nitrobenzoylamino. acetimidate (ABNA)

N-Hydroxysuccinimide ester of 5-azido-2-nitrobenzoic acid (NHS-ANBA)

[ 80]

[ 79 ]

i781

[76,77]

[75]

1751

48

problems: possible random collisional crosslinks, long reaction time. difficulty in controlling reactions, and nonselective crosslinking. In photochemical crosslinking with photosensitive heterobifunctional reagents, crossllnking can be easily, rapidly, and sequentially controlled. Brief crosslinking reactions by rapid photolysis can provide a picture image of interacting systems at a desired moment. Cell membranes are thought to be functionally and structurally dynamic, and the image of dynamic structures of interacting systems can be captured by photochemical crosslinking but not by conventional crosstinking, unless the dynamic structure is frozen by other means during the entire period of crosslinking. A systematic application of this approach, however, has not yet been attempted. Selective crosslinking is another advantage for photosensitive heterobifunctional reagents (see 'Identification of surface receptors'). Photosensitive heterobifunctional reagents can be classified on the basis of their photosensitive group, conventional functional group, and cleavability.

IIIB (1). Photosensitive groups Two classes of photosensitive groups are currently available: an azide derivative and a diazo derivative (Tables I and II). Nitrenes are generated from azides and have a half life on the order of 10 - 2 - 1 0 -4 s [81,82] and therefore the crosslinking reaction is expected to be terminated within that time period. Arylnitrenes have a high reactivity with a tow activation energy [60,71 ] and the reactivity is essentially temperature-independent; thus, temperature-dependent structural changes of interacting systems can be monitored. Their reaction is nonselective and does not require a specific reactive group and this broad reactivity is advantageous when crosslinking is the primary purpose but may not be desirable when the exact reaction site is in question. When ribonuclease A was treated with N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate [83] and photolyzed for approx. 2 ms, the nitrene reacted with most amino acid residues, including valine and proline. Glycine was the only amino acid resistant to the reaction [84]. Carbenes are the second type of photosensitive reagents and can be generated upon photolysis of diazo derivatives [85]. They are indiscriminately reactive with solvents. inert C--H bonds and hydroxyls. Westheimer and his colleagues are responsible for the development of carbene derivatives [68,79,86,87]. One cleavable reagent, pyfidyl-2.2'dithiobenzyl diazoacetate, reacts efficiently with nearby hydroxyl groups of threonine in creatine kinase [68]. IIIB (2). Photolysis A common method used to photolyze azides is irradiation with a shortwave ultraviolet lamp, for example, mineralight USV-1 I. The half time of photolysis with this lamp varies depending on the reagents and is on the order of 10 50 s (ref. 28; Vanin, E.F. and Ji, T.H.. unpublished). An alternative method, which has several advantages, is flash photolysis for an extremely short period, normally on the order of milliseconds. Regular flash photolysis units are expensive and require a high voltage (several thousand volts d.c.} power supply. Recently it has been found that inexpensive electronic flash units made for cameras discharge an intense flash in the ultraviolet and visible range of milliseconds duration [17]. The intensity of the xenon flash lamp units vanes somewhat but normally they are capable of photolyzing approx. 20-40% of 10-4-10 -s M arylazides in aqueous buffer (Ji, T.H., unpublished). The absorption maximum of arylazides is at 265-275 nm andthe molar extinction coefficient is in the range of 2 • 104 M -1 " cm -1 [28]. This absorption band is one of those responsible for the activation of arylazide by photolysis. Other absorption bands at longer wavelengths, 3 0 0 - 4 6 0 rim. appear when the phenyl group is

49 modified with nitro groups [16,72,75]. One attractive feature of this type of nitroarylazide is that it can be photolyzed with long wavelength ultraviolet or visible light so that radiation damage to the target can be minimized [16,70,71,75,83,88]. However, in these cases, irradiation times have been on the order of minutes [16,36,75,83] or even hours [70]. This long irradiation time may be undesirable, because a useful number of photoaffinity labels or crosslinks are not likely to be formed over a period of many minutes without inducing some change in protein surfaces, even without radiation effects. Irradiation for as long as 10 min with an argon laser (488 nm) at a power of I W resulted in thermal heating but an insignificant amount o f photolysis [84]. Although a systematic study has not been attempted to determine whether long irradiation at a long wavelength or a short pulse at a short wavelength is more effective in minimizing the denaturation of the biological sanlples, some data are available. Xenon flashes from electronic camera flash units did not cause any change in the absorption spectrum of concanavalin A (Ji, T.II., unpublished) or in the activities of several enzymes in tbe erythrocyte membrane [17]. When ribonuclease A was subjected to a flash from a 7L6 xenon flash tube at 2 kV which was expected to be considerably stronger than a flash from any electronic camera flash unit, the antigenic activity remained intact and the enzyme activity was 90% of the original. However, the circular dichroism above 235 nm showed a slight change [84]. In our hands, the flash photolysis produces a better defined crosslinking pattern on gels than the irradiation with a ultraviolet lamp does.

IIlB {3). Conventional functional groups Currently available photosensitive heterobifunctional reagents are specific t'or either amino or sulfhydryl groups. The amino group specific reagents are either an imidate or N-hydroxysuccinimide esters (Tables I and 1I) and were discussed in the previous section. A number of sulfhydryl specific arylazides are available (Table 1 and If). One such compound is di-N-(2-nitro-4-azidophenyl)cystamine-S,S-dioxide [16]. It is prepared by oxidizing the disulfide of the parent reagent to the dioxide to enhance its reactivity as a disulfide exchange reagent (Scheme II). N-(4-Azidophenylthio)phthalimide is another reagent [17]. In our hands, this reagent can be readily transferred to unionized sulfhydryl groups in erythrocyte membranes (Scheme If), and this transfer can be blocked by prior treatment of the membranes with N-ethylmaleimide (Kiehm, D.J., Vanin, E.F. and Ji, T.H., unpublished). Both reagents must be dissolved in an organic solvent prior to the introduction to membranes, and the final concentration of organic solvent is approx. 1-5%.

IIIB {4). Cleavability A number of cleavable homo- and heterobifunctional reagents are available which contain a disulfide linkage (Refs. 16,17,20,23,36,67,68,89; Vanin, E.F. and Ji, T.H., unpublished) or a glycol bridge [22,24], as seen in Tables I and llI. All cleavable heterobifunctional reagents contain a disulfide bridge and none with a glycol bridge has been synthesized. A disulfide bridge can be cleaved by reduction with 10 mM mercaptoethanol or other reducing agents for about 10 rain. The use of these disulfide reagents precludes the application of reducing agents during the isolation of crosslinked products and, in addition, they are susceptible to disulfide exchange. Reagents containing a glycol bridge offer an alternative approach to cleavage and are stable under reducing conditions. This latter type of reagent is known to be quantitatively cleaved by soaking gels in 15 mM sodium periodate for 4 - 1 0 h [22,24], but has not been fully exploited. The fact that the aldehydes produced are intrinsically reactive may be a disadvantage [60].

Tartryldi(methyl-kminopropionimidate) (TDAP)

(DTBH)

(DTBV)

Dimethyl-5,5’-dithiobisvaierimidate

Dimethyl-7,7’dithiobisheptanimidate

(DTBB)

(DTBP)

DimethyI_rl,4’&thiobisbutyrimidate

Digethyl-3,3’-dithiobispropionimidate

Amino specific

rH2 ‘Cl-

-C-0-CH3

s;JH2 ‘cl-

-C-0-CH3

2

]2

12

(-bH-&-NH-(CH2)2-;-O-CHs]2 NH2+C1-

OH 0

[-S-(CH2)6-C-O-CH3]2

y+c1-

I-S-(CH2)4-C-o-cH3]

[ -S-(CH2)3

[ -S-(CH2)2

yHz +Cl-

CLEAVABLE HOMOBIFUNCTIONAL REAGENTS ~.--~----_--~-_._. _- ___-. Structure formulae Reagent

TABLE III

-..

17

22

17

14

12

Length (A)

1241

WI

WI

l891

[20,231

Reference

-

Periodate

Mercaptan

Mercaptan

Mercaptan

Mercaptan

Cleaving agent

._-.-.__

51

”

o=

07 0

o=i,

=o

Scheme II.

N3

o

O

+ HS-P -- N 3 - ~ S

NO2

NH-(CH2)2 -S-S-(CH2)2 -NH

NO2

II O

N3 sN

O II

NO2 N3 + HS-P -- N3 - ~ ) - N H - ( C H

rl

O

-S-P + H N ~

O II

2)2 S - S - P

+ HSO2 -(CH2 )2 - N H ~ N 3 / NO:

53 IV. Biological systems Chemical crosslinking can be performed either to a selected number of species of molecules or nonselectively to most species of molecules present in a membrane. A good example of selective crosslinking is the crosslinking of ligand proteins to their surface receptors in which ligand proteins are used as macromolecular affinity labels [27,28]. By nonselective crosslinking, most species of molecules of cell membranes are subjected to crosslinking, which can reveal the interactions of the molecules in the membranes.

IVA. Identification of surface receptors." macromolecular affinity labels Surface receptors have been identified primarily by affinity column chromatography. This technique has been found to be of limited use in studying membrane receptors due to its inherent technical difficulties. The major drawback is that the solubilization of membranes and receptors (irreversible disruption) is required prior to the isolation of receptors and that the behavior and dynamic properties of receptor-ligand complexes on cells cannot be studied by this technique. Chemical crosslinking of receptors and ligands on membranes is another approach. In this approach, ligand - receptors are complexed on membranes, crosslinked prior to solubilization of membranes, and studied. Conventional bifunctional reagents are inadequate for the purpose because they indiscriminately and extensively crosslink most membrane constituents and even then rarely crosslink ligands to receptors [27]. A better choice is photosensitive heterobifunctional reagents because at least one of the functional groups can be easily activated when desired. The general approach (Fig. 2) requires that the macromolecular ligands undergo reaction with only one of the functional groups of the reagent, yielding activated ligands. After purification, the activated ligands are incubated with intact cells or membranes. Ligand-receptor complexes formed on membranes are then crosslinked following the activation of the second functional group. This concept of macromolecular affinity labeling, introduced several years ago by Ji [27,28], was derived from earlier photoaffinity labels of small molecules by Knowles and colleagues [69-71] and specific membrane surface labels introduced by Maddy [90], Pardee and Watanabe [91] and Berg [92]. Photosensitive imidates consisting of an imi-

~-~ ,4- ~ reagent

I~

acti vation

ligand

UV =

I~

SDS

Fig. 2. The general approach of macromolecular affinity labelling. Ligand proteins are activated by attaching photosensitive heterobifunctional reagents, introduced to surface receptors of cells, crosslinked by ultraviolet irradiation, and solubilized in SDS solution for analysis. For details see text.

54 date at one end and an arylazide at the other were the first reagents successfully used for these studies [27,28]. The arylazide is chemically inert and is converted to the reactive arylnitrene only upon absorption of a photon. Due to its latency, this reactive group can be handled safely in the dark and the reaction begins only upon illumination. On the other hand, the irnidate group needs no activation and reacts as soon as it is introduced to the primary amines of proteins in solution. Taking the advantage of the differential reactivity of the two functional groups, the photosensitive heterobifunctional reagents are attached to ligand proteins (lectins, peptide hormones, antibodies, toxins, etc.) simply by mixmg them in solution. When the amidination is completed, the labelled molecules are purified from excess reagents and introduced to cells and membranes as macromolecular affinity labels. Up to this point, manipulations are carried out in the dark. Ligand complexes on the membrane surface are irradiated with light to activate azides. Any complexes successfully crosslinked may be isolated and the receptors can be identified under denaturing conditions, primarily on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Crosslinked complexes can be identified readily by the use of ligand proteins labelled with radioactive isotopes. However, it is difficult to identify receptors by this appraoch alone. Therefore, in the subsequent study, membrane proteins instead of ligand proteins are surface-labelled with radioisotope and crosslinked with nonradioactive tigand proteins. Surface receptors can then be identified after cleaving the ligand protein-receptor complexes. Alternatively, ligand proteins and receptors can be double-labelled Successful crosslinking of ligand protein-receptor complexes requires both a close proximity and reaction of the photosensitive groups of the ligand proteins with the receptors. Problems may arise when ligand proteins are labelled unequally, undergo conformationa~ change or denaturation upon labelling, or interact with receptors nonuniformly, and any one of these factors can partially or totally prevent completion of the crosslinks. Reagents attached near the binding sites have a better chance for crosslinking than those reagents located elsewhere and in particular, those at the hydrophobic center of proteins may not be useful at all. On the other hand, reaction at functionally hypersensitive groups near the binding site may critically alter biological activity. Because not all reagents which attach are expected to crosslink, an excess of reagents is desirable, but it must be kept in mind that one crosslink is sufficient. The optimal conditions for reaction with the imidates are pH 9.3, 30 60 min incubation at room temperature, a total reagent concentration of 1 5 mM with incremental additions every 1 0 - 3 0 min, and a lO0-fold molar excess of reagents over accessible amino groups in the proteins [17,28] (details can be found in Section IliA. Homobifunctional Reagent). To prevent denaturation of binding proteins, their substrates may be added. Attached reagents can be quantitated with radioactive reagents, the extinction coefficient of arytnitrenes [28], or by monitoring the decrease in reactive groups of proteins [17]. As in any chemical modification of proteins, the biological activity has to be carefully monitored. The existing evidence shows that the attachment of photosensitive imidate derivatives alone does not change the biological activities of proteins to a significant extent [28,29], but the subsequent photolysis may impose more serious effects. An example is phospholipase A2 which loses 85% of its activity [75]. It is conceivable thal most of the amino groups which react with the reagents are not functionally critical. This is not true for sulfhydryl groups, and reaction of the residues of gtyceraldehyde-3phosphate dehydrogenase [93,94] with p-azidophenacyl bromide inactivates the enzyme

55 [76]. Because of possible nonspecific binding following denaturation, putative crosslinked ligand protein-receptor complexes must be carefully examined, particularly when the yield of crosslinks is low or multiple crosslinks are apparent. Ji [27,28] has found that concanavalin A is crosslinked to an integral membrane protein (band 3) of erythrocyte membranes and the efficiency of this crosslinking, that is, the amount of concanavalin A crosslinked to the receptors versus total level of lectins bound to the receptors, is approximately 20%. Das et al. [29] have reported the size of the receptors for epidermal growth factor in 3T3 cell lines to be about Mr = 190 000, based on the electrophoretic mobility of a complex containing the x2SI-labeled hormone. It is not known, however, whether the receptor is a single polypeptide or a complex. A sure way to answer this question is to cleave and identify the receptor on a second twodimensional gel. Alternatively, since epidermal growth factor has only one amino group (at the N-terminus) and two disulfide bonds [95], the number of photosensitive heterobifunctional reagents attached to the hormone either via an amidine linkage or via disulfide exchange [60] can be taken to reflect the maximum number of receptors crosslinked to the hormone. One problem with this method, however, is that an arylnitrene, although putatively monofunctional, can react bifunctionally [60]. Several cleavable photosensitive heterobifunctional reagents are currently available and all have a disulfide bridge (Table lI). Although they are preferred to noncleavable ones, they cannot be used in a reducing condition and are susceptible to disulfide exchange [60,68]. Therefore, the transfer of photosensitive groups to different molecules by this process should be examined. Currently available photosensitive heterobifunctional reagents are either specific for amino groups or for sulfhydryl groups (Table II). The sulfhydryl reagents are more hydrophobic than the imidate derivatives and are suspected to react near the hydrophobic domain of proteins. For maximum crosslinking, it is desirable to have the photosensitive group at the surface of molecules. It has been reported that p-azidophenacyl bromide and 4-fluoro-3-nitrophenylazide were not useful in identifying receptors [27]. Besides the identification of surface receptors, photosensitive heterobifunctional reagents are useful for elucidating the fate and role of protein ligand-receptor complexes in endocrine and immune systems.

IVB. Erythrocyte membranes After solubilization in detergent, the proteins and glycoproteins of erythrocyte membranes can be resolved into approximately 15 discrete bands on sodium dodecyl sulfatepolyacrylamide gels (Fig. 3). Chemical crosslinking studies have demonstrated that most of the polypeptide species are subunits of larger proteins and there are extensive interactions between different species of proteins. Crosslinking of proteins to lipids, or lipids to lipids has been attempted.

IVB (1]. Spectrin Spectrin, a major protein which comprises 25% of the total erythrocyte membrane proteins [49], consists of two subunits, a heavy chain, Mr = 240 000, and a light chain, Mr = 220 000 [49,96]. It assumes a fibrous structure localized on the inner membrane surface [97]. Crosslinking studies have been attempted to answer two general questions; (a) how the spectrin fiber is formed by the two subunits, and (b) what is the nature of the interaction of spectrin with other membrane proteins, in particular with surface recep-

56

!

Fig. 3. Band designation of erythrocyte membrane proteins separated by polyacrylamide get electrophoresis.

tors. The latter question will be discussed in the following section. With respect to the first question, crosslinking studies have raised several more specific questions: (a) whether the heavy and light chains are crosslinked to each other to form heterodimers [ 15,25,42, 89,98,99] or crosslinked each to itself to form homodimers [19,100]; (b) why crosslinked dimers appear in discrete multiple bands [17,19,42,89,98]; and (c) why spectrin on membranes is crosslinked to form oligomers no larger than tetramers [17] whereas higher oligomers are seen when spectrin is crosslinked in solution [25]. On the question of dimer formation, most laboratories favor the formation of heterodimers [15,25,42,89,98,99]. This is based on the observations that approximately equimolar amounts of the two chains (a) are present on membranes [49,98], (b) disappear upon crosslinking [19,42,89,98], and (c) are produced when crosstinked dimers are cleaved [15,17,25,42,89]. The last observation is most critical and is based on the presumption that each band of crosslinked dimers is homogenous and has an identical composition, one heavy and one light chain - a point which has never been rigorously tested. On the other hand, Steck [19,100] has favored the contrary view that spectrin is crosslinked to form homodimers, on the basis of the observation that the heavychain is produced in a 2-fold molar excess over light chain when a get slice containing the dimer crosslinked with cupric phenanthroline was cleaved [19]. The converse experiment hasshown a similar result that heavy and light chains are not always crosslinked in an equimolar ratio (Kiehm, D.J. and Ji, T.H., unpublished observation). There is evidence of independent activity of each chain of spectrin [ 101 ]. For example, the heavy chain is extensively

57 crosslinked, preferentially over the light chain, with other proteins, bands 3, 4.2, and 5 [25]. Compounding this problem is the multiplicity of crosslinked dimer bands [17,19, 42,89,98] and the difficulty in isolating each of them because of their close proximity on gels. Despite a better separation on agarose-acrylamide gels [ 17], analysis of sequential gel slices of the dimer band area, in an attempt to purify the material in each band, has revealed cross-contamination (Kiehm, D.J. and Ji, T.H., unpublished results). Alternatively, crosslinked dimers can be cleaved on gels and analyzed directly on a two-dinlensional gel [15]. The heavy and light chains cleaved in this way usually streak and appear in tight associations, making a critical and unequivocal quantitative analysis difficult. Finally, the homogeneity of each dimer band has yet to be established. The multiplicity of crosslinked spectrin dimers is another point of interest. The number of dimer bands varies from one to three in the range of Mr = 450 000-500 00 when crosslinked either on ghosts [15,17,19,25] or in solution [19,89,98]. Their intensity is not always equal, and two dimer bands of unequal intensity, one several fold higher than the other, are seen when ghosts are crosslinked with several photosensitive crosslinkers [17]. The disparity in the intensity of the two bands can be reversed without changing the depletion rate of the monomeric heavy and light chains (Kiehm, D.J. and Ji, T.H., unpublished observation). The simplest explanation of these results and the observation that all dimer bands produce, after cleaving, both heavy and light chains [15,17,19,25,89] is that the crosslinked dimer bands differ in the mode of crosslinking. Dimers crosslinked at different points may differ in their hydrodynamic characteristics, especially in their Stokes radius, and might be separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis [15,16,19,20,60,102,103]. It is unlikely, although possible, that intra-subunit crosslinks caused the multiplicity of dimer bands [16], because the electrophoretic mobility of the monomer bands remained unchanged after treatn~ent with the reagents [17.19, 89, 98]. Whether or not the discrete dimer bands represent crosslinks at a limited number of distinctive points of contact is not known. No clear correlation of the various patterns of crosslinked dimer bands has been found with respect to the specificity of the reagents, their reactivity towards amino or sulfhydryl groups, or their solubility in water or organic solvents. Steck [19] has observed that glutaraldehyde produced one dimer band from spectrin in solution and two bands from ghosts. Therefore, the structure of spectrin appears to be one of the factors influencing the mode of crosslinking. There are reports that the distance between two functional groups in bisimidates affects the crosslinking of spectrin [26,89,104]. In addition to dimers, spectrin is crosslinked to higher homopolymers. Kiehm and Ji [17] have investigated spectrin crosslinking under conditions that permit extremely rapid crosslinking with minimal movement of spectrin [33]. When the crosslinked complexes were analyzed on agarose-acrylamide gels which had an exclusion limit of Mr = 3 - 5 • 106, discrete bands of trimers and tetramers appeared in addition to dimers. The rate of their formation is noteworthy. The accumulation of dimers preceded the appearance of tetramers, and the dimer bands diminished as the tetramer band increased. The trimer band is always minor and therefore difficult to quantitate. The next bands which appeared represented oligomers which were crosslinked to very high molecular weight complexes (Mr = 3 - 5 • 106), while the formation of other intermediate sized spectrin oligomers was not observed. Normally, intermediate oligomeric bands, e.g. pentamer, hexamer, etc., either do not appear at all or appear in extremely low yield, regardless of the specificity of the reagent towards amino or sulfhydryl groups (Kiehm, D.J. and Ji,

58 T.H., unpublished observation). This is consistent with the observation that spectrin on membranes is crosslinked up to tetramers via intrinsic disulfide formation [25]. Crosslinking of spectrin in solution contrasts that on membranes, in that rnultimers up to hexamers are formed in a gradualmanner [25], suggesting that the structure ofspectrin in solution is different from that on membrane enabling spectrin to be crosslinked to higher multimers. These results suggest a significant disparity either in the mode of the interactions or in the availability of reactive groups at the interacting points between tetramers, as compared with that between dimers or monomers. It is unknown whether or not the difference is due to extrinsic factors such as interaction with actin molecules. It has been reported that spectrin monomers exist in solution as tong cylindrical rods with a high axial ratio of approximately 48 [98] and that dinaers are twice as long [105]. Furthermore, it has been suggested that monomer interactions are likely to be end to end [42,105] with alternating heavy and light chains [42]. This model is not totally consistent with the existing evidence. According to this model, oligomers larger than tetramers are expected to be crosslinked as readily as dimers, trimers, and tetramers, a prediction contrary to the crosslinking results of spectrin on membranes [ 17,25]. It is more likely that (a) monomer-monomer interactions involve more than two modes and (b) dimer-dimer interactions involve more than one mode and/or there are more than one species of dimers. This argument leads to the supposition that dimers may not be the repeating building blocks of spectrin fibers, although they appear to be structurally stable as evidenced by their presence in solution [96,98,103,105]. The dimer and tetramer forms have been reported to. coexist in solution, but depending on the conditions of preparation, in particular temperature, one of them predominates; dimer at 37°C and tetramer at 4°C [106-108]. Contrary to previous reports [98,105], these dimers and tetramers are slightly or moderately assymmetric with axial ratios in the range of 2 - 1 0 [107]. Singer and colleagues [109,110] have proposed an interesting model for the mechanism of polymerization of spectrin fibers. The structural units of spectrin which are present under normal conditions, undergo polymerization when activated by an ATPdriven phosphorylation of the light chain. Whether the tetramers seen in crosslinking studies [17] correspond to the structural units in this model is yet to be seen. Other have suggested that the structural units of spectrin consist of heavy and light chains and actin [111]. Preferential crosslinking of heavy chain to actin has been reported [25]. This depends on the availability of reactive side-chains, amino or sulfhydryl groups. Obviously, some of the 135 lysine and 20 cysteine residues per monomer [96,1 t2] are located at the points of crosslinks but do not appear to be involved in intrinsic intersubunit covalent bonds, by disulfide or e-(/3-glutamyl) lysine or e-(7-aspartyl) lysine [ 105.113- t 15 ]. Most sulfhydryl groups in intact cells are normally maintained in the reduced state by glutathione (2.0-2.5 mM) or other unidentified cytoplasmic reducing agents [1t6], but are unprotected and susceptible to oxidation on ghosts [ 117,118].

IVB (2~. Transmembrane linkage Interactions of spectrin with other membrane proteins have drawn considerable interest, since Nicolson et al. [119] demonstrated the existence of transmembrane linkages between spectrin and receptor glycoproteins. Subsequently, Ji and Nicolson [28] have shown that both the heavy and light chains, along with band 4.1, are more readily crosslinked when ghosts are treated prior to crosslinking with Ricinus communis agglutinin, a lectin which binds specifically to band 3 [ 120]. Though the nature of the involvement of band 4.1 is not known, it is of interest to note that the band splits into a doublet

59 similar to the spectrin doublet on Laemmeli gels (refs. 121,122; Ji, T.H., unpublished observations). It was concluded from the result that spectrin recognized and responded to the binding of the lectin to the receptors. Branton et al. [123,124] have confirmed the presence of such linkages by the reassociation of spectrin extracts with liposomes containing band 3 extracts. However, whether spectrin interacts directly or indirectly with the receptor glycoprotein was not answered. Attempts to crosslink spectrin directly to receptor glycoproteins have not produced a satisfactory answer. Wang and Richards [15[ have suggested that band 3 is crosslinked to spectrin on the basis of the observation that they are generated along with other proteins when the very high molecular weight crosslinked products retained at the top of the gel were cleaved. Since the cross/inked products were retained at the top of the gel, Kiehm and Ji [17] reexamined this result with the improved agarose-acrylamide gel system. The molecular weight of the very high molecular weight complexes is estimated to be more than 3 - 5 • 106 and they contain the heavy and light chains of spectrin, band 3, 4.2, 5, and 6. It was not possibly to determine which of these proteins is crosslinked directly to spectrin. Recently Liu et al. [25] have reported that band 3 was crosslinked directly to each of the two spectrin chains when ghosts were subjected to oxidation at 37°C and pH 4.5-5.0. The pH appeared to be critical, because outside the pH range, crosslinking did not occur. Since the pH coincides with the isoelectric point of spectrin and actin [125[ and their precipitation point [106,123,124], the crosslinks are ascribed to the reduction of electrostatic repulsion and more intimate contact among the proteins. The biological implications of the effects of changes in pH and temperature on the function of the membrane are not fully understood at this moment. Steck [100] has emphasized a point that band 3 is more likely to contact nonspecifically with spectrin by being trapped within the interstices of the fibrous spectrin reticulum seen under high-power scanning electron microscopy (Steck, T.L., personal communication), as opposed to specific interactions of the proteins [ 109",124]. IVB {3}. Band 3 Band 3 is a glycoprotein [126,127] of an apparent molecular weight of 90 000 [49] and spans the membrane [128,129[ at least twice [130-134] and possibly as many as four times [135]. Since Steck [19] showed its crosslinking to dimer in membranes and in solution, crosslinked dimer, trimer, and tetramer forms have been reported [ 15,16,25,36, 44]. Naturally, a question has been raised as to whether dimer or tetranler represent the natural stable oligomeric structure in membranes [15,60]. Recent reexamination of band 3 cross/inking by Kiehm and Ji [17], under minimal random collison conditions, has demonstrated that dimer is the only crosslinked homopolymer, independent of temperature (0-25°C) and time of crosslinking ( 1 - 1 0 ms), with over 80% yield of dimer (Kiehm, D.J. and Ji, T.H., unpublished). Dimer is the predominant species when crosslinked in Triton X-100 solution under nondenaturing conditions [ 136,137]. Similar results have been produced with a variety of crosslinking reagents: catalysts for the disulfide fom~ation, bisimidates, and photosensitive crosslinkers. Most likely, the dimer form represents the natural stable oligomeric structure of band 3 in membranes and it is conceivable that trimers and tetramers are crosslinked under abnormal conditions. Whether these larger oligomers are formed by the perturbation in the membrane structure prior to crosslinking or are an artifact of the crosslinking reaction per se is not known. Six sulfhydu1 groups per dinner are known to be present in the polypeptide segment of band 3 exposed at the inner membrane surface [130,132,135] and two are involved in the intersubunit disulfide formation (Guidotti, G., personal communication). Therefore,

60 the remaining four SH groups are responsible for crosslinking to other molecules. It is interesting that more than two potypeptide species, the spectrin heavy and light chains and actin have been reported to be crosslinked to band 3 [25]. This is possible only (a) when each monomer of the band 3 is crosslinked to a different polypeptide species, or (b) when heterogeneity exists among the copies of band 3 dimers with respect to their interactions and crosslinks to other molecules. The former case ~s contrary to some existing evidence that each monomer in a dimer binds equally to band 6 and aldolase [100,126, 137]. Finally, whether the reported crosslinking of band 3 to spectrin and actin reflects specific interactions [109,124] or nonspecific trapping [100} has yet to be determined.

IVB (4). Other proteins and glycoproteins No general agreement can be found among laboratories on crosslinking of other proteins and glycoproteins. Differences in reagents used and conditions for ghost preparation and crosslinking are most likely to contribute to the discrepancies. Crosslinked tetramers of band 4.2 and 6 (glyceraldehyde-3-phosphate dehydrogenase) [17,19] are consistent with their presence in a nondenaturing solution [137]. Crosstinked band 4.2 dimer has been reported [15,25]. The approximate equimolar ratio of the band 4.1 doublet as resolved on Laemmli gels (ref. 122; Ji, T.H., unpublished) and their concomittant decrease on crosslinking [26] suggest, but do not prove, that they are subunits. There are indications of the presence of crosslinked homopolymers of bands 4.5 [ 15,25], 4.9 [25], 5 [15,17,25], and 7 [15]. The major glycoprotein, glycophorin [138] is quite resistant to crosslinking and has been crosslinked under exhaustive condtions. This usually happens after most of the proteins are crosslinked and shifted from their original positionson the gel to those of very high molecular weight complexes at top o f the gel 134,45,104]. Niehaus and Wold [9] have reported that crosslinked glycoproteins remain with an insoluble membrane fraction.

IVB (5). Heteropolymers The evidence of association between various functional units, e.g. band 3 with glyceraldehyde-3-phosphate dehydrogenase [136,137], the glucose transport system [44,139, 140], and aldolase [141,142] increases the interest in the mode of their interactions. In view of the evidence that most membrane proteins, including glycoproteins [9,34], are involved ultimately in forming large crosslinked complexes insoluble in detergent [9.15, 110,34,36,45], a variety of crosslinked heteropolymers are expected but have not been readily detected probably because of multiplicity and low yield. Nonetheless, crosslinks of band 3 to 4.1,4.2, 4.4, 5, and 6, in addition to spectrin [16], and spectrin to 4. t. 4.9 and 5 175] have been reported, and this result may reflect extensive interactions~either direct or indirect, of most proteins with one another. Further isolation and careful analysis of each complex may reveal the quantitative relationship and biological significance. In conclusion, chemical crosslinking studies have demonstrated that most membrane proteins, if not all, are composed o f subunits. Filamentous spectrin and actin appear to have repeating structural units. Further, most membrane proteins apparently interact with each other to form an enormous network. It is thought that this network is responsible for the resistance to hemolysis and solubilization in detergent solution [13]. Hemoglobin interact with [143-151] and is erosslinked to erythrocyte membranes [43]. When washed intact erythrocytes were incubated with dimethyldithiobispropionimidate for 30 rain at room temperature, most of the membrane proteins except for band 5 were shown to be crosslinked to hemoglobin and were inferred to be exposed to

61 the inner membrane surface. At a pH greater than 6 and a salt concentration of 20 raM, binding of hemoglobin to the membrane is reversible and weak [144,148 150]. There appear to be two classes of binding sites, specific high affinity sites and nonspecific low affinity one [149,150]. This binding study utilized the fluorescence quenching of a fluorescent fatty acid probe incorporated in the ghost membranes and revealed that the high affinity sites were reversibly competed by glyceraldehyde-3-phosphate dehydrogenase but not by aldolase, although both were known to be peripheral membrane proteins and to associate with band 3 [136,137,140,142,145,151,152]. Because the number of binding sites of glyceraldehyde-3-phosphate dehydrogenase and hemoglobin are equal, 1.2 - 106 sites per cell, and the enzyme is anchored to the membrane via band 3, it has been suggested that the enzyme is the hemoglobin binding site [149,150]. Macromolecular affinity labeling [27,28] of binding sites with hemoglobin modified with photosensitive heterobifunctional reagents may resolve this question. 1VC. Sarcoplasmic reticulum Ca2+-ATPase, a major protein component (70 80%) of sarcoplasmic reticulum membranes [153], has a molecular weight of approx. 100 000 and plays a key role in the accumulation and regulation of muscle relaxation. In the light of the observed oligomeric structure of Ca2+-ATPase (a) in nondenaturing solution [154], (b) on SDS-polyacrylamide gel electrophoresis without crosslinking [ 153], (c) by electron microscopy [155], and (d) by fluorescence resonance energy transfer [ 156], a question has been raised as to how many subunits form a molecule of the enzyme. Several laboratories agree that the enzyme is ultimately crosslinked to very high molecular weight complexes which are retained at the top of the gel [157-160], but have produced somewhat divergent results with respect to the size of intermediate crosslinked products, dimers to hexamers. Dimers and tetramers are formed when the membranes are aged, probably by the oxidation of sulfhydryl to disulfide [160]. Besides Ca2+-ATPase, there are two minor polypeptides in the range of Mr = 55 000-45 000 which can be radioiodinated on membranes by lactoperoxidase [160, 161] and readily extracted by EGTA at pH 8.0 [162]. They are resistant to crosslinking via S-S bonds, probably due to their low sulfhydryl content [163. 164], but are crosslinked by glutaraldehyde [160]. Their structural relationship with Ca2+-ATPase, which appears to be an integral protein, is not clearly understood. Crosslinking affects the enzyme activity regardless of which reagents are used [39,160, 165], and it has be~n suggested that the hydrolytic product of dimethyl suberimidate, dimethylsuberate, is responsible for the interference and not the crosslinking reaction per se [39]. IVD. Mitochondria Among the proteins of the mitochondrial inner membrane, two are best known: cytochrome c, a soluble peripheral protein and cytochrome oxidase, an integral membrane component consisting of seven distinct subunits [166-168]. In an attempt to determine which subunit of the oxidase binds to cytochrome c, 5,5'-dithiobis(2-nitrobenzoate) was attached to cytochrome c, making it a macromolecular affinity label. This reagent, when bound to a cysteine residue to give cysteinyl-bound thionitrobenzoate, becomes a good leaving group and promotes S-S formation provided free thiols are available for exchange [169]. In reconstituted complexes of cytochrome c and the oxidase, the labeled cyto-

62 chrome c is shown to be crosslinked to subunit IIt of the oxidase. Similar results are seen when the proteins are crosslinked by oxidation in the presence of cupric phenanthroline [ 170]. This result is consistent with the evidence by fluorescence resonance energy transfer studies that subunit III and cyt0chrome oxidase are in a close proximity [171]. Crosslinking of intact mitochondria and isolated inner membranes with dimethylsuberimidate has produced new higher molecular weight complexes with concomitant reduction in the intensity of a number of bands normally appearing [172-174]. Components of the apparently crosslinked products have not been identified.

IVE. Membrane lipids Among various membrane lipids, aminophospholipids are the primary target of crosslinking because of their reactive free amino groups. In erythrocyte membranes, both phosphatidylethanolamine and phophatidylserine are known to be primarily in the cytoplasmic half of the bilayer [41,175-181]. In earlier crosslinking studies, these phospholipids were refractory to crosslinking in ghosts and thought to be tightly bound to membrane proteins in the cytoplasmic half of the membrane [176,182-185]. Later it has been found, however, that in the previous studies the phospholipids reacted monofunctionaUy due to reagent excess and could be extensively crosslinked when less reagents were used [ 186]. When crosslinked with difluorodinitrobenzene, up to 33% phosphatidylserine and 4% phosphatidylethanolamine became resistant to extraction in chloroform/ methanol and they were considered to be crosslinked to proteins [186]. The considerable disparity in crosslinking of the two classes of phosphotipids is interesting but a meaningful interpretation is not easy at the present time, because (a) the converse experiment, the isolation of proteins crosslinked with the phospholipids has not been done, and (b) crosslinking has been accomplished under conditions of rapid lateral diffusion oflipids, diffusion coefficient being 10-7-10 -8 cm 2 -s -1 [187-190]. In addition to crosslinks to proteins, the phospholipids are thought to be crosslinked to themselves but there is a controversy as to whether each class of aminophospholipids is crosslinked exclusively to itself [184] or to itself as well as to each other [186,191]. A fraction of glycolipids, identified by periodate-Schiff staining and galactose oxidase dependent tritiation, has been crosslinked to a protein component but the identity of the glycolipids and proteins as well as the nature of the crosslinks have not been described [104]. Recently, lipid-lipid and lipid-protein crosslinking has been approached by Khorana and colleagues [192-194] with a new class of fatty acids and phospholipids containing a photosensitive group at different positions. The fact that these substituted fatty acids are taken up by an auxotroph of Escherichia coli and accepted into the biosynthetic pathway forming phospholipids and inserted into the membrane, promises to provide new insights on lipid-protein interactions in membranes.

IVF. Lymphocytes HLA and Ia antigens are two important antigens found on human B lymphocytes [195,196]. When HLA is extracted in a nonionic detergent solution and crosstinked by dithiobis(succinimidylpropionate), a crosslinked complex (M r = 54 0t30) is produced. When cleaved, a heavy chain (Mr = 44 000) and a light chain (Mr = 12 000) are released in an equimolar ratio, demonstrating a heterodimer structure [t97]. When Ia antigen is extracted in a nonionic detergent solution and crosslinked, a complex of Mr = 64 000 is

63 produced, which on subsequent cleavage yields two subunits of unequal size (Mr = 34 000 and 29 000) [198]. Crosslinking studies on ribosomes and the ( C a > + Mf+)-ATPase of the inner membrane ofE. coli have been reviewed elsewhere [60].

V. Possible artifacts of crosslinking The introduction of crosslinkers to a biological system raises concern for possible perturbations in the system and subsequent crosslinking of artifacts. Often reagents are used at the millimolar concentration range or higher. For example, a 100-fold excess of imidates is required in order to complete reaction with all the available amino groups in erythrocyte membranes [4]. Sometimes, small quantities of organic solvents are required to dissolve reagents which are insoluble in water. A more serious concern is possible structural alterations induced by multivalent reagents similar to those by multivalent biological molecules: agglutination of cells and aggregation of surface antigens. In a fluid membrane [31], some molecules undergo rapid lateral diffusion [30,188, 189,199-202] and are expected to collide with one another [203,204]. A protein molecule of 2 nm radius and a diffusion coefficient of 1 • 10 -9 cm 2 • s-I is expected to collide once every millisecond [ 17] and 1 0 s - I 0 ~ times during crosslinking for several minutes to hours. The collision frequency of lipids is approximately four orders of magnitude faster [188]. During these collision, molecules are known to be linked together by multivalent ligands, antibodies, and lectins [205,206], a phenomenon termed biological crosslinking. Since multivalent biological ligands and chemical crosslinkers do crosslink molecules similarly, it has been questioned whether chemical reagents crosslink molecules during their collisions. It would be a serious problem to distinguish crosslinked products of natural stable complexes from those of collisional complexes. Since it is difficult to determine whether or not crosslinking occurs during random collisions with conventional crosslinking techniques, Kiehm and Ji [17] have approached this problem under conditions which minimize tile level of random collisions. This is achieved with millisecond crosslinking by utilizing recently introduced photosensitive heterobifunctional reagents and xenon flash photolysis. In this study, ethyl-4-(4-azidophenyldithio)butyrimidate was attached in the dark to amino groups in erythrocyte membranes via imidate-amine reactions and then crosslinking was accomplished by the activation of the photosensitive moiety with a xenon flash. The flash discharges within 1 ms and crosslinking is expected to occur within a similar time period [ 17,81,82,205]. Crosslinked products which appeared included band 3 dimers, band 5 dimers, band 4.2 tetramers, and band 6 tetramers. Spectrin appeared in dimer, trimer, and tetramer forms. These crosslinked products have been considered to be natural stable complexes on the basis of the following reasons: (a) a band 3 molecule is estimated, from the known diffusion coefficient ( 2 - 6 - 10 -12 cm 2 • s -1) [32,33], to collide less than once during the period ofcrosslinking at 25°C [17] and a substantial amount of band 3 dimers, sometimes over 80%, is found to be crosslinked; (b) the mean time between collisions, 1 ms [17], includes the periods for travelling as well as remaining in contact and the collision period is expected to be briefer than the travelling period because molecules are expected to aggregate spontaneously if the collision period is longer; (c) by lowering the temperature from 25°C to that of an ice bath, the diffusion coefficient and the collision rate are reduced by two orders of magnitude /200], but the crosslinking results remained essentially unchanged; (d) most of the crosslinked complexes, band 3 dimer and band 6 tetramer [136,137], and spectrin dimer and

64 tetramer [106-108] can be isolated in solution without prior crosslinking; (e) when erythrocyte membranes are crosslinked with glutaraldehyde, intramembranous particles do not aggregate [208] and band 3 is known to be a component of the particles by virtue of being identified on the particles as a lectin receptor [120]. These results indicate that random collisional crosslinks did not take place under the conditions of crosslinking, but they do not answer unequivocally whether it was due to the lack of random collision or due to the failure of crosslinking despite random collisions. A variety of factors could have been responsible for the latter case: the interference by boundary tipids o f a protein molecule [209,210], steric hindrance, or the absence of reactivity. It is more likely, however, that random collisions did not take place to a significant extent, due to the extremely short period of crosslinking, the relatively slow diffusion coefficient of proteins found in erythrocyte membranes, compared with that in other animal cell membranes [32,33,189,200], and other reasons. Intramembranous particles are known to be negatively charged, primarily with sialic acids, to be repulsive to one another and to aggregate when the sialic acids are removed [211 ]. Random collisions in other types of animal cell membranes may impose a more serious problem where diffusion rates are considerably faster (by one order of magnitude [ 189, 200], and the extent of collisional crosslinks has yet to be examined. Besides collision between integral proteins in fluid membranes, there can be other types of collisions and collisional crosslinks in intact cells, e.g., collisions of soluble cytoplasmic proteins to membrane-bound proteins. In intact erythrocytes, hemoglobins are crosslinked to spectrin, band 3, 4.1, 4.2, 6 and 7. and the results are used as for internal surface labelling [43]. The crosslinking was carried out at 37°C for 30 rain and it is not known whether fast photochemical crosstinking will show such collisional c rosslinks. Can chemical crosslinkers crosslink two cells in suspension to produce a complex of molecules originating from different cells? This question is raised in view of cell agglutinations by multivalent biological ligands, lectins and antibodies. Ji and Ji [46] have found no crosstinking between erythrocyte ghosts by bisimidate, at a concentration of 2 - 3 10 s ghosts per ml and also have demonstrated that crosslinking between the two opposed membranes of a ghost did not take place.

VI. Concluding remarks It is becoming increasingly evident that chemical crosslinking offers a unique approach to and useful for studying surface receptors and membrane structure. At the same time, it is clear that the current method is not adequate for unequivocal determination of the composition of a crosslinked complex and the stoichiometry of the protomer-comptex relationship. For complex systems, selective crosslinking is desirable, and macromolecular affinity labels prepared with photosensitive heterobifunctionat reagents are a choice. Mternatively, other selective crosslinking reagents, for example, membrane-permeable or -impermeable crosslinking reagents, will be useful. In addition t o selective crosslinking, certain components of crosslinked complexes can be selectively identified by isolation or staining by immunological or other biospecific procedures. Crosslinked points of complexes provide useful information, but the application of protein chemistry for the analysis has not been extensive. Procedures need to be established to relate the pattern of crosslinks of homopolymers to the mode of subunit interactions. Possible random collisional crosslinks can be avoided m some membrane systems by

65 rapid crosslinking with photosensitive reagents, and this rapid crosslinking (millisecond) is potentially useful if dynamic aspects o f interacting molecules and subunits are in question. Acknowledgements The untiring assistance o f D.J. Kiehm is gratefully acknowledged, and I thank Drs. C. Hubbard, C. Edson, and E.F. Vanin for their c o m m e n t s on the manuscript. The author is a scholar in cancer research o f the American Cancer Society. Support was provided by grants from the National Science F o u n d a t i o n and the National Institutes o f Health. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

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