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Biochimica et Biophysica Acta, 576 (1979) 61--70 © Elsevier/North-Holland Biomedical Press

BBA 38083

CROSS-LINKING OF THE (Ca 2÷ + Mg2÷)-ATPase PROTEIN

RONALD J. BASKIN and STEPHEN HANNA *

Department of Zoology, University of California, Davis, CA. 95616 (U.S.A.)

(Received January 19th, 1978) (Revised manuscript received August 8th, 1977)

Key words: (Ca ~+ + Mg2+)-A TPase; Cross-linking; (Sarcoplasmic reticulum membrane)

Summary

The addition of cupric-l,10,-phenanthroline, a cross-linking catalyst, to sarcoplasmic reticulum membranes caused protein sulfhydryl groups to form disulfide bridges. Following a short exposure to the catalyst (15 s, 22°C) most of the protein was in a dimeric form (Mr = 248 000). Longer exposure times resulted in the formation of trimers, tetramers and other oligomers too large to enter the get. At low temperatures {4°C) dimer formation predominates, even for exposure times as long as 5 min. Cross-linking in the presence of 7.5 mM Triton X-100 (a concentration that resulted in clearing of the membrane sus- pension and thus solubilization of the membrane components) showed the appearance of a considerable dimer fraction, however, most of the (Ca ~÷ + Mg~÷)-ATPase protein appeared as a monomer. Following 1 min of cross-linking at 22°C, freeze-etched membranes showed no alteration in ithe number or appearance of 80 £ intramembranous particles. Thus extensive cross-linking of the (Ca 2÷ + Mg~÷)-ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.

Introduction

Sarcoplasmic reticulum membranes contain a transport system capable of actively accumulating calcium and serving as a control mechanism for muscle relaxation. The central element in this control system is the (Ca 2÷ + Mg2÷)-ATPase protein located within the membrane. Freeze-fracture studies of fragmented sar- coplasmic reticulum of developing muscle [1] as well as studies of the 'purified' (Ca~÷+ Mg~÷)-ATPase protein [2] have established that the 80 A freeze- fracture particles represent the ATPase protein. It appears, however, that the

* A port ion of this w o r k was done dur/ng the tenure o f a Research Fe l lowship o f Muscular D y s t r o p h y Assoc ia t ions of Amer/ca . Present address: Department o f Phys io logy , University o f California Medical Center; Los Angeles, CA 90024, U.S.A.

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membrane density of these particles (approx. 5700/pm 2) is not sufficient to account for the quanti ty of ATPase protein known to be present in the mem- brane. One explanation for this discrepancy is to regard the 80 )k freeze- fracture particles as representing oligomers of the ATPase protein. Preliminary calculations [3] indicate that two, three, or four molecule oligomers per 80 £ particle all would fit the available data. The cross-linking studies presented here represent an a t tempt to determine the number of (Ca 2÷ + Mg2÷)-ATPase mole- cules per 80 ~ particle. The rationale of the present s tudy is to observe using sodium dodecyl sulfate polyacrylamide gel electrophoresis, the results of cross- linking the protein both when it is located in the membrane and following solubilization with Triton X-100. In other experiments cross-linking has been carried out at low temperature in order to minimize random interaction of protein molecules. (A preliminary report concerning this work has been published [4].)

Experimental procedure

Preparation of sarcoplasmic reticulum membranes The abdominal muscles from a lobster (Homorus americanus) were

homogenized with 4 vols. of cold {4°C) 10 mM N-tr is(hydroxymethyl)methyl- 2-aminoethane sulfonic acid buffer, pH 7.0 (N-tris(hydroxymethyl)methyl-2- amioethane sulfonic acid, Sigma Chemical Co., St. Louis, Mo.) for 40 s in a Waring blender. Myofibrils were removed by centrifugation at 3000 × g for 20 min. The supernatant was filtered through glass wool to remove lipids and centrifuged at 8000 × g for 20 min. The supernatant was centrifuged for 1 h at 2 8 0 0 0 × g . The pellet was suspended in 10 ml of 0 .6M KC1 in 10 mM N-tr is(hydroxymethyl)methyl-2-aminoethane sulfonic acid, pH 7.0 using a Potter-Elvehjem homogenizer. After a 30 min incubation at 4°C, the suspen- sion was centrifuged for 1 h at 28 000 × g. This pellet was resuspended in 5 ml of 10 mM N-tr is(hydroxymethyl)methyl-2-aminoethane sulfonic acid, pH 7.0. Final concentrat ion was between 7--10 mg/ml.

Cross-linking Cross-linking was performed in a solution of oxygenated 10 mM N-tris-

(hydroxymethyl)methyl-2-aminoethane sulfonic acid with 2 mg/ml lobster sarcoplasmic reticulum. Cupric-l ,10-phenanthroline was added from a stock solution of 5 mM cupric sulfate and 15 mM 1,10-phenanthroline (Sigma), to a final concentrat ion of 0.05 mM cupric sulfate and 0.15 mM phenanthroline. The reaction was terminated by the addition of N-ethylmaleimide to 16 mM and ethylene diamine tetracetic acid to 1 mM.

Cross-linking activity The assay for cross-linking activity was performed according to Kobashi [ 5].

1 mg of 5,5'-dithiobis-(2-nitrobenzoic acid) was added to 10 ml of oxygenated 10 mM N-tr is(hydroxymethyl)methyl-2-aminoethane sulfonic acid, and was subsequently reduced by the addition of 1 mg sodium borohydride. 1 ml of the solution was added to 1.5 ml oxygenated (95% O2, 5% CO2) 10 mM N-tris{hydroxymethyl)methyl-2-aminoethane sulfonic acid (pH 7 . 0 ) i n a glass

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cuvette, and the reaction was initiated by the addition of cupric sulfate (0.05 mM final concentration) and phenanthroline (0.15 mM final concentration). The reaction was monitored by measuring the change in absorbance at 412 nm.

For measurements of the effects of cross-linking on calcium uptake and ATPase activity, lobster sarcoplasmic reticulum was cross-linked as described, and the activity measured on a radiometer pH meter equipped with a dual electrode, as described by Deamer [6]. Cupric-l,10-phenanthroline activity was monitored under all conditions. Deoxycholate was not used as a detergent since it inhibited cupric-l,10-phenanthroline activity. Triton did not inhibit cupric-l,10-phenanthroline activity. In experiments run at 4°C the concentra- tion of cupric-l,10-phenanthroline was increased 4-fold such that its activity was the same as at 20°C.

Polyacrylamide gels Polyacrylamide gels contained 4% acrylamide, 0.11% methylene bis-

acrylamide, 0.1% sodium dodecyl sulfate, 0.05% tetramethyl ethylenediamine (v/v), 0.1% ammonium persulfate, and reservoir buffer. Reservoir buffer con- sisted of 0,04 M Tris, 0.02 M sodium acetate, and 0.002 M ethylene diamine tetraacetic acid, pH 7.4. Samples were solubilized by the addition of glycerol and sodium dodecyl sulfate to final concentrations of 5 and 1%, respectively. The solubflization mixture also contained reservoir buffer and pyronin Y. Samples of 0.02 ml, containing approx. 0.03 mg protein, were added to each gel slot. Gels were run on a 10 cm × 14 cm slab gel apparatus (1.5 turn thick) at 10 mA for 15 rain, then at 40 mA until completion. The standard proteins run were (a) trypsin, 23 300 daltons, (b) lactate dehydrogenase, 36 000 daltons, (c) pyruvate kinase, 57 000 daltons, (d) bovine serum albumin, 68 000 daltons, (e) phosphorylase a, 97 000 daltons, and (f) myosin, 220 000 daltons.

Mobility relative to bovine serum albumin was plotted as a function of log molecular weight. The six standard molecules were used to generate a function which was fitted by the method of least squares. The correlation coefficient was 0.996.

Results

Two systems were considered for study. The first was the (Ca 2+ + Mg2+)- ATPase protein present in rabbit saxcoplasmic reticulum. Initial experiments revealed that gels of rabbit fragmented s.arcomplasmic reticulum in which mercaptoethanol was omitted showed evidence of high molecular weigh bands without added cupric phenthroline. An analogous finding was reported by Chyn and Martonosi [7]. Thus higher molecular weight bands were either present initially or were formed during the normal preparation procedure for gel electrophoresis. Since the bands were present initially, no effect of added curpic phenanthroline could be observed except after long reaction times when extensively cross-linking had obviously occurred.

The (Ca 2÷ + Mg~÷)-ATPase protein present in lobster sarcoplasmic reticulum did not evidence this problem. Gels of lobster fragmented sarcop!asmic reticulum, prepared without mercaptoethanol, did not show high molecular

64

c 4 2 • ~= ,,

F- . ~ . <~ e ~ ~

'.~ .~ _ ~ ~ '.,. _ ~ o ~ .I ~ I ~ E ~ ~ 0 ~ ~.- ~.~._.~.~ ....

I - ~

i ~ i L I

I ~ ~ ~ ~

~ i ~ ~ C r ~ - I i n ~ i n ~

Fig. 1. Nf fee t of d u r a t i o n ( ra in ) o f e ross - l inMng ( 2 2 ° C ) w i t h e u ~ i e - l . 1 0 - ~ h e n a n t h r o l i n e o n eMeium s e n s i t N e A ~ P a s e a e t N i t y ( ~ ) a n d Ca 2 . u~ t aRe ac t iv i t y (~). The ~agio C a 2 + / A ~ P is Mso ~ lo* ted (~).

weight bands in the absence of cupric-l,10-phenanthroline. For this reason, all of the remaining work w~as done with the lobster enzyme.

Lobster fragmented sarcoplasmic reticulum has another critical attribute. 75--85% of the membrane protein is the 106 000 dalton (Ca ~+ + Mg2+)-ATPase protein (see Fig. 2a and e). On this basis and the observation that the other very faint b&nds were not affected by cross-linking we consider the products to be homopolymers.

Effect of cross-linking on A TPase activity and calcium transport Cross-linking with the catalyst cupric phenanthroline substantially decreases,

Fig. 2. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of lobster sarcoplasmic reticulum vesicles at 22°C and at 4°C for various durations of exposure to the cross-Linking reagent cup~ic-l,10- phenanthzoline (0,15 mM cup~ic-l,10-phenanthzoline at 22°C and 0.60 mM at 4°C). a, 22°C, no cross- linking~ b, 22°C, 15 sl c, 22°C, 1 mini d, 22°C, 5 mini e, 4°C, no czoss:linkingl f, 4°C, 15 s~ g, 40C, I mini h, 4°C, 5 mini i, bovine se~um albumin.

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but does not abolish calcium sensitive ATPase activity and calcium transport in lobster fragmented sarcoplasmic reticulum (Fig. 1}. (Calcium insensitive ATPase activity is negligible in lobster fragmented sarcopl~ .m!c reticulum.) The decrease in both ATPase activity and in calcium transport bcCurs rapidly; within the first 15 s after addition of the catalyst. Following this initial rapid inhibition, a low level (10% of initial value) of calcium transport and ATPase activity was maintained.

Time course of cross-linking Cross-linking of the lobster fragmented sarcoplasmic reticulum protein

occurred quite rapidly at 22°C. It was therefore necessary to stop the reaction initially after only 15 s of reaction. Cross-linking was also allowed to occur for 1 min and for 5 min (Fig. 2). Following 15 6 of cross-linking most of the protein was in dimer form with trimer and tetramer bands clearly visible. After 1 min, the protein existed largely as tetramer and higher molecular weight oligomers. By 5 min the protein was entirely in the form of oligomers too large to enter the gel. This behavior was highly consistent and several trials gave exactly the same results.

The mobilities of the monomer and the cross-linked oligomers have been determined in each of seven runs. The average values are listed in Table I, The molecular weight estimates, also listed in Table I are based on these average mobilities.

Cross-linking at low temperature At 4°C cupric-l,10-phenalthroline activity was increased 4-fold to give the

same catalytic activity as at the higher temperature. Random interaction of membrane proteins should have been substantially decreased, however, due to the lipid phase change which occurs at about 18°C. The resulting pattern of cross-linking was significantly different than at 22°C. Cross-linking fo r 15 s 1 min and 5 min was carried out on the samples shown in Fig. 2e--h. Following 15 s of cross-linking at 4°C most of the protein was present as a dimer. After 1 min of cross-linking, most of the protein was still present as a dimer, but after 5 min, the presence of larger oligomers could be seen.

Cross-linking in the presence of Triton X-1 O0 Detergent solubilization of the fragmented sarcoplasmic reticulum vesicles

T A B L E I

E S T I M A T E D M O L E C U L A R W E I G H T O F O L I G O M E R S R E S U L T I N G F R O M C R O S S - L I N K I N G W I T H C U P R I C P H E N A N T H R O L I N E

Mobi l i ty * Moleculax w e i g h t

Monomer 0.766 106 000 Dimer 0.369 248 000 Trimer 0.233 333 000 Tetramer 0.159 394 000

* A v e r a g e o f s e v e n ~ n s .

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Fig. 3. S o d i u m d o d e c y l su l f a t e p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s of l o b s t e r s a r c o p l a s m i c r e t i c u l u m vesic les ( 2 2 ° C ) w i t h va r i ous c o n c e n t r a t i o n s o f T r i t o n X - 1 0 0 f o r v a r i o u s d u r a t i o n s o f e x p o s u r e to the c ro s s - l i nk ing r e a g e n t c u p r i c - l , 1 0 - p h e n a n t h r o l i n e . a, 7 .5 raM, n o c ross - l ink ing ; b, 7 .5 raM, 15 s; c, 7 .5 r aM, 1 r a i n ; d, 7 .5 raM, 5 r a in ; e, b o v i n e s e ru ra a l b u m i n ; f, 3 . 7 5 raM, n o crOss- l inking; g, 3 . 7 5 raM, 15 s; h, 3 . 7 5 m M , 1 m i n ; i, 3 . 7 5 raM, 5 m i n ; j, 15 raM, 1 5 s; k, 15 r aM, 1 ra in ; l, 15 m M , 5 min .

has the effect of dispersing the (Ca 2+ + Mg2÷)-ATPase protein throughout the detergent micells. Cross-linking may then be impeded by the screening effect of the detergent. Assuming that any oligomeric structure of the ATPase protein is not disrupted by the detergent (however, see Discussion), cross-linking among subunits of the oligomer would be favored over cross-linking resulting from random association of the protein due to its ability to diffuse within the membrane bilayer. In order to minimize disruption of the native (oligomeric) configuration of the (Ca~÷+ Mg~÷)-ATPase protein, a non-ionic detergent, Triton X-100, was chosen.

Although small amounts of Triton X-100 (>0.3 mm) completely inhibit calcium transport [8], calcium sensitive ATPase activity is present in solutions containing high concentrations (>7.5 mM) of Triton X-100. At the fragmented sarcoplasmic reticulum concentrations used in our experiments, a 7.5 mM Triton concentrat ion resulted in a visually clear solution. A 15 mM Triton X-100 solution showed, however, a 50% reduction in ATPase activity.

Using an aggregation number of 140 [9] 7.5 mM Triton contains 3.2 • 1019 micells/l. Sarcoplasmic reticulum at a concentration of 1.98 mg protein/ml solution contains 1 .2 .1019 molecules/1. Therefore, our solution contained almost 3 times as many Triton X-100 micells as ATPase protein molecules. Thus it is reasonable to assume that each protein monomer or oligomer was dispersed in a separate detergent micell.

Sodium dodecyl sulfate gels (Fig. 3) of cross-linking carried out in the presence of 7.5 mM Triton X-100 showed, after 15 s, the formation of a sub- stantial quant i ty of dimer and a small amount of trimer. More than half of the protein was in monomer form, however. The situation was essentially the same after 1 min of cross-linking, but after 5 min, all three bands were considerably

67

diminished indicating the formation of oligomers too large to enter the gel. Only a small amount of tetramer was formed in the presence of Triton X-100 regardless of the reaction time.

In order to further examine the effect of Triton X-100 on the cross-linking reaction, sodium dodecyl sulfate polyacrylamide gel electrophoresis was also carried out in the presence of 3.25 mM and 15.0 mM Triton X-100 (Fig. 3). At the lower concentration of Triton X-100, the gels resembled the control runs. After 15 s, 1 min and 5 min of cross-linking, the band patterns were similar to that was observed in the absence of Triton X-100 with the exception that more monomer was present. This was evident in the gels run with 15 mM Triton X-100 {Fig. 3). At this concentration most of the protein appeared as a monomer regardless of the duration of cross-linking, and ATPase activity was less than 50% of its initial value.

Following preparation by freeze-etching, fragmented sarcoplasmic reticulum vesicles that had been cross-linked for 1 min were examined using an electron microscope. In spite of extensive cross-linking, vesicle appearance including the size and distribution of 80 A particles was unchanged from uncross-linked

Fig. 4. E l e c t r o n m i c r o g r a p h o f l o b s t e r s a r c o p l s s m i c r e t i c u l u m p r e p a r e d by f reeze-e tching [1] fo l lowing a 1 m i n e x p o s u r e t o cupr ic -1 ,10-phenan~hro l lne (22°C) . N u m e r o u s vesicles con t a in ing d i s t i nc t 80 J~ par- t ic les are o b s e r v e d (arrows) . "/5 000× .

68

0 iSsec lrnin

' / 5rain

Fig. 5. Scan of s o d i u m dode c y l sulfate p o l y a c r y l a m i d e gel e l ec t rophores i s p r e p a r e d at 22°C (N), at 4°C (C) and at 22~C in the Presence of 7.5 m M T r i t o n X-100 (T) . Dura t ions of exposure to cupr i c - l , 10 - p h e n a n t h r o l i n e were , 0, (no exposu re ) , 15 s, 1 m in and 5 min , The a r row in each scan indica tes the peak due to the 106 000 da l ton (Ca 2+ + Mg2+)-ATPase pro te in . Th e top of each gel is ind ica ted by the p e a k on the lef t side of the scan.

controls (Fig. 4). Thus extensive cross-linking of the (Ca2+ + Mg2+)-ATPase protein molecule is not revealed in the appearance of the 80 h freeze-etch particles.

Gels of samples cross-linked at 22°C, 4°C, and in the presence of 7.5 mM Triton were scanned (Fig. 5). The top portion of the gel from the monomer (arrow) is shown for various durations of cross-linking. The large quantity of dimer present in low-temperature runs is clearly apparent. The rapid formation of oligomers in samples cross-linked at 22°C is also apparent. After only 1 min most of the protein is cross-linked into oligomers too heavy to enter the gel. The Triton X-100 results in contrast show a relatively small change with increased duration of cross-linking.

Discussion

This investigation has established the following: 1. Cross-linking of lobster sarcoplasmic reticulum by the catalyst cupric

phenanthroline proceeds by the formation of dimers, tetramers, and then larger oligomers. Trimers are only marginally present.

2. When the rate of cross-linking and the mobility of the protein in the lipid matrix is decreased by lowering the temperature of the system to 4°C, the major oligomer initially formed is a dimer. Prolonged incubation results in the formation of larger oligomers, however, only a small quantity of trimer forma- tion was observed.

3. Solubilization with the detergent Triton X-100 gives different results depending upon concentration. At low concentrations (approx. 3.5 mM) the

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sodium dodecyl sulfate polyacrylamide gel electrophoresis results resemble closely the results in the absence of Triton Xl00 . At a level of Triton X-100 sufficient to cause clearing of the solution (7.5 mM), the major oligomer formed is a dimer with a substantial fraction appearing as a monomer. At high Triton X-100 concentrations (15.0 mM) oligomers do not appear and all of the protein appears as a monomer.

4. Following 5 min exposure to the cross-linking catalyst most of the protein appeared on sodium dodecyl sulfate polyacrylamide gel electrophoresis in the form of large oligomers. The appearance of vesicles prepared by the freeze- fracture technique showed, however, no alterations. Both the number and distribution of 80 A freeze-fracture particles were similar to those observed in noncross-linked ~resicles. Thus extensive cross-linking of the (Ca2÷ + Mg2÷) - ATPase protein can occur without disruption of the normal position of the intramembrane portion of the molecule.

The non-serial appearance of oligomers reported earlier [10] is difficult to understand since one would expect some appearance of dimers and trimers even in a system normally existing in a tetrameric state. At short reaction times not all of the components of the system would cross-link in an ideal fashion. At longer reaction times oligomers would generally combine to form very large aggregates.

Since it is known [11] that the monomeric form of the enzyme shows normal levels of ATPase activity and phosphoenzyme formation the functional role of oligomer formation is open to question. It may be that calcium trans- port requires the presence of the correct oligomer; however since cross-linking with cupric-l ,10-phenanthroline sharply decreases the calcium transport activ- ity of the protein, it is not possible to decide this question on the basis of the present evidence.

Above the 18°C lipid phase transition temperature, cross-linking is so rapid that large amounts of both dimers and tetramers are seen initially. The nearly simultaneous appearance of both oligomers could result from the kinetics of cross-linking a quaternary structure or from the presence of both oligomers.

Below the lipid phase transition temperature, at 4°C, the dimeric from predominates. This could represent a shift in oligomeric structure from a predominantly tetrameric form to a dimeric form or it could represent simply altered accessibility of cross-linking sites.

The presence of only the monomeric form of the protein in solutions con- raining a large amount of Triton X-100 indicates either that the native organiza- tion of the membrane is based upon a monomeric protein or that Triton X-100 in high concentrations splits the native oligomeric state. Measurements of ATPase activity do not allow a resolution of this question since a substantial degree of activity (though much less than in the absence of detergent) is still present.

The absence of evidence of intramembrane disruption following 1 min of cross-linking is of interest. It would appear to indicate that cross-linking occurs in a region of the molecule extensing from the membrane surface and large enough to allow cross-linking without altering the base position of the mole- cule. Evidence based on trypsinization of the protein indicates that approxi- mately half of the molecule may extend past the membrane surface and

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assuming this portion of the protein molecule is cross-linked by cupric-l,10- phenanthroline, would explain the results observed in the present study.

References

1 Baskin, R.J. (1974) J. Ul t ras t ruct . Res. 4, 348 - -371 2 Baskin, R.J , (1977) in Membrane Proteins and their In te rac t ions with Lipids (Capaldi, R., ed.), pp.

151- -188 , Marcel Dekker , Inc., New York 3 Baskin, R.J. (1977) in Methodologica l Surveys in Biochemis t ry (Reid, E., ed.), Vol. 6, pp. 53--64,

H o r w o o d , Chiches ter 4 Baskin, R.J . and Hanna, S.D. (1977) Biophysical J. 17, 29a 5 Kobashi , K. (1968) Biochim. Biophys. Ac t a 158, 239- -456 6 Deamer, D.W. (1973) J. Biol. Chem. 248, 5 4 7 7 - - 5 4 8 5 7 Chyn , T. and Martosoni , A. (19~7) Biochim. Biophys. Ac ta 468, 114 - -126 8 Baskin, R.J. (1972) Bioenerget ics 3, 249 - -269 9 Helenius, A. and Siraons, K. (1975) Biochim. Biophys. Ac ta 415, 29- -79

10 Murphy , A.J. (1976) Biochem. Biophys. Res. C o m m u n . 70, 160- -166

11 Dean, W.L. and Tanford , C. (1978) Biochemis t ry 17, 1 6 8 3 - - 1 6 9 0