ARCHIVES
OF BIOCHEMISTBY
AND
Calcium-Binding
BIOPIiYSIC&
179,
302-309
Sites in Rat Myocardial CONSTANTINOS
University
of Minnesota
(1977)
JOHN
Sarcolemma
LIMAS
Medical School, Department of Medicine, Cardiovascular Memorial Building, Minneapolis, Minnesota 55455
Division,
Box 83, Mayo
Received October 3, 1976 Myocardial sarcolemmal preparations have the ability to bind Caz+; this binding shows saturation kinetics, has a pH optimum of 7.4 to 7.8, and is stimulated by ATP. Scatchard analysis reveals both high- and low-affinity binding sites. Both classes are inhibited by ruthenium red, whereas only the high-afiinity sites are affected by lanthanum and hydroxylamine. Electrophoresis in the presence of sodium dodecyl sulfate suggests that the high-affinity sites are associated .with a protein peak of molecular weight of about 100,000. Enzyme treatment of the membranes suggested that proteins accounted for most of the %a binding with phospholipids and sialic acids residues playing a secondary role. Interaction with different function group reagents indicated that the carboxyl residues were necessary for the calcium binding, whereas thiol, amino, and sulIhydryl~groups were unimportant.
calcium for binding sites) specifically inhibits excitation-contraction coupling in the heart, while it is much less effective in skeletal muscle (4). All of these observations suggest superficial binding sites as a major source of activator calcium in the heart. In addition, there is accumulating evidence that the major defect in spontaneous or induced heart failure may involve deficient excitation-contraction coupling (5). The present study was undertaken, therefore, to characterize the chemical groups responsible for Ca2+ binding by myocardial sarcolemma.
The interaction of contractile proteins is regulated in the myocardium, as in all types of muscle, by the amounts of calcium ion entering the cytoplasm from extracelMar and intracellular storage sites in response to membrane depolarization. The relative contributions of intra- and extracellular sites are a matter of debate. It seems increasingly evident, however, that a difference exists between skeletal and cardiac muscle; whereas sarcoplasmic reticulum and to some extent mitochondria, appear to play the predominant role as source of activator calcium in the former type of muscle, sarcolemma is more important in the heart (1). Structural and functional observations support this conclusion. The sarcotubular lateral sacs are less developed in the myocardium and may constitute as little as 2% of the whole cell volume. By contrast, the volume of the T system is proportionately increased (2). The force of contraction is rapidly diminished when cardiac muscle is perfused with calcium-free fluid with a half-life of less than 2 min, suggesting that the activator calcium is in rapid equilibrium with the extracellular space (3). Finally, lanthanum (which does not enter the intracellular space and competes effectively with
MATERIALS
AND METHODS
Experiments were conducted on 4- to 6-month-old male Wistar rats, weighing 250 to 350 g. Isolation of sarcolemma. The hearts were rapidly excised, washed in cold 10 mM NaHCO,, minced, and blotted dry. Homogenization was performed in 10 vol of 0.34 M sucrose using a Polytron PT-20 (rheostat setting, 5; duration, 30 9. The homogenate was filtered and centrifuged at 2000g for 10 min. The resultant sediment was suspended in 10 vol of 10 rnr.4 Tris-HCl, pH 6.0 and centrifuged at 2OOOg for 10 min. This step was repeated three times. The final pellet was suspended in 5 vol of 10 mu Tris-HCl, pH 8.0 and 4.0 M LiBr was added to bring the final concentration to 0.4 M. The solution was stirred gently for 4 to 5 h and was then diluted fourfold with 302
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0993-9861
CALCITJM-BINDING
SITES IN RAT MYOCARDIAL
buffer. It was then centrifuged at 2OOOgfor 10 min and washed with 10 vol of buffer, and the centrifugation was repeated. The sediment was washed with 8 vol of buffer and centrifuged at 2000g for 10 min; this step was repeated once and the final pellet was suspende’ddin distilled water at a protein concentration of 2 to 5 mg/ml. Phase contrast microscopy at this level showed predominantly empty sarcolemma1 sacs which had vesicular appearance under the electron microscope. Mitochondria and sarcoplasmic reticulum were also prepared and enzyme activities of Na+, K+-ATPase, adenylate cyclase, cytochrome oxidase, glycose 6-phosphatase, and acid phosphatase were assayed using standard techniques (6). Protein content was determined by the method of Lowry et ~2. (7) with bovine serum albumin as the standard. The lipid content was determined by extracting with chloroform methanol (2:1, v/v) and then proceeding as described by Carvalho (8). Total hexoses were assayed by the anthrone reaction (91 and hexosamines by the Elson-Morgan reaction (10). Sialic acid content was determined by thiobarbituric acid (11). Electrophoresis. SDS-polyacrylamide gel electrophoresis of the sarcolemmal proteins was performed in a sulfate-borate discontinuous buffer system using the method of Neville and Glossmann (12). Gels containing 3% (stacking) and 10% (separation) gels were prepared from a stock solution of 30% by weight of acrylamide and 0.1% sodium dodecyl sulfate and were polymerized chemically by the addition of 0.025% by volume of tetramethylethylenediamine and ammonium persulfate. Five-centimeter gels were prepared in glass tubes of a total length of 7.5 cm with an internal diameter of 6 mm. The stacking gel length was 1 cm. The buffers used were: upper reservoir, 0.04 M boric acid-O.041 M Tris (pH 8.6); stacking gel, 0.0267 M H,S04-0.0541 M Tris (pH 6.1); separating gel and lower reservoir, 0.0208 M HCl-0.4244 M Tris (pH 9.18). The membranes were suspended in a total volume of 0.2 ml containing the following: 0.625 M Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue. Electrophoresis was carried out at a current of 2 mA/tube until the tracking dye had reached a mark about 0.5 cm from the end of the gel. After electrophoresis, the gels were stained with 1% amido black in methanol:acetic acid:water (50:7:42, v/v/v). Destaining was carried out with three to five changes of methanol:acetic acid:water for 24 to 36 h. The gels were then sliced into l-mmthick slices which were solubilized in dimethyl sulfoxide prior to recording absorbancies at 660 nm. ‘Abbreviations used: SDS, sodium dodecyl sulfate; ECDQ, 2-ethoxy-N-carbethoxy-1,2-dihydroquinoline carbodiimide; TCA, trichloroacetic acid; EGTA, ethylene glycol bis(P-aminoethyl ether)N,N’-tetraacetic acid.
SARCOLEMMA
303
Molecular weights were estimated by using a calibration curve prepared from standard proteins, including @-galactosidase, ovalbumin, avidin, and transferrin. Calcium binding. Calcium binding by the sarcolemma was studied using the Millipore filtration method. Membrane protein (5-100 pg) was incubated in a medium containing 100 mM KCl, 10 mM MgCl,, 20 rnM Tris-maleate buffer, pH 7.4, and 0.1 mM 45CaC1, with or without 0.5 mM ATP. The reaction was started with the addition of ATP and was terminated at various intervals by filtering 0.8 ml through a Millipore filter (0.45 pm, 25 mm) under light suction. The filter was washed with 5 ml of 100 mM Tris-maleate buffer, pH 7.4, dried at 70°C in a scintillation vial, covered with 10 ml of tolueneLiquifluor, and counted in a liquid scintillation counter. Controls containing no ATP, no MgCl*, or no protein were also used. Calcium binding was estimated from the specific activity of the added Wa and the activity retained by the membrane protein. Separation of lipid- and protein-bound Ca*+. Sarcolemmal membranes were incubated as described above with Wa2+ for 20 min; the reaction was then stopped by adding 5 ml of ice-cold 0.5 mM histidineimidazole buffer, pH 7.0. The membranes were then centrifuged at 6OOOg for 15 min, resuspended in buffer, and centrifuged again. The pellet containing &Ca was extracted three times with chlorofornnmethanol (l:l, v/v) by the method of Burger et al. (131 and the insoluble protein precipitate was removed by centrifugation. The lipid extract was combined with chloroform and water to give chloroform:methanol:water proportions of 10:5:1 (by volume). After centrifugation at 1085g for 10 min, an aqueous upper phase and an organic phase formed. The organic phase contained the phospholipidbound Ca*+, the precipitate contained the proteinbound and the aqueous phase, the unbound Ca2+. Enzyme incubation. Sarcolemmal membrane preparations (0.2-0.3 mg of protein) were suspended in 1.0 ml of a solution containing 100 mM KCl, 10 mM MgCl,, 20 mM Tris-maleate buffer, pH 7.4, and were preincubated with the appropriate amount of the enzyme for 1 h. They were then centrifuged at 1500g for 20 min and the pellets were resuspended in 1.0 ml of 20 mM Tris-maleate buffer, pH 7.4. Aliquots (100-200 ~11 of the suspension were used to determine Wa binding as previously described. For the carbodiimide reaction, a solution containing the sarcolemma (1 mg/ml), 1.33 M glycine methyl ester, and 7.5 M urea was kept at 25°C and pH 4.74. A solution of 0.4 M carbodiimide (2-ethoxyN-carbethoxy-1,2-dihydroquinoline) dissolved in 7.5 M urea was then added to the mixture at a final concentration of 0.1 M. The pH was maintained by automatic titration with 0.5 M HCl. Aliquots (0.5 ml) were removed periodically and added to 5 ml of 1.0 M acetate buffer at pH 4.75 to quench the reac-
304
CONSTANTINOS
tion with the diimide. After standing for a few minutes at 25”C, the solutions were dialyzed for 36 h at 0°C against three changes of 20 liters each for 0.001 M HCl. A suitable portion of the dialysate was then hydrolyzed for 16 h at 110°C in 6 M HCl and analyzed for amino acids. RESULTS
Myocardial sarcolemma isolated by the procedure described was essentially free of contaminating mitochondria or sarcoplasmic reticulum as shown by the relative distribution of “marker” enzymes (Table I). The chemical composition of the sarcolemma is shown in Table II; proteins constituted 79.4% of dry weight and lipid, 13.8%. Sarcolemmal preparations bound Ca2+ to a degree dependent on its concentration in the incubation mixture. Figure 1 gives a Scatchard plot of the Cd2+-binding data; this can be resolved into two curves corresponding to two classes of binding sites with different affinities for calcium. The extrapolated intercepts of the linear segments of the plot can be used to calculate the number of binding sites and the corresponding association constants. The higher affinity sites for calcium with an association constant of 2.0 x lo5 M-l accommodated 17 + 2 (SD) nmol of Ca2+/mg of protein. The lower affinity sites had an association constant of 5.6 x lo2 M-’ and accomodated 680 t 16 (SD) nmol of Ca2+/ mg of protein. Binding of Ca2+ as a function of membrane concentration was studied (Fig. 2); binding increased linearly over a concentration range of 10 to 60 pg of sarcolemmal protein/ml. In the presence of ATP and MgCl*, sarcolemmal proteins are phosphorylated to an extent proportional to the protein concentration in the assay mixture TABLE SUBCELLULAR
DISTRIBUTION
Sarcolemma
JOHN
LIMAS
(Fig. 3). This phosphorylation is sustained only to a minimal degree by other nucleoside triphosphates (Table III) and is due to the presence in the sarcolemmal preparation of cyclic AMP-dependent and -independent protein kinases. Its regulation and relation to Ca2+ binding are detailed in a separate report. Less than 1% of the ATP is retained in the Millipore filter when sarcolemmal protein is omitted from the incubation medium. 45Ca binding also increased rapidly with time, reaching maximal saturation after 2 min of incubation (Fig. 4). There was a marked influence of pH on 45Ca binding with an optimum between 7.4 and 7.8 (Fig. 5). ATP stimulated calcium binding by both highTABLE CHEIOZAL
II
COMPOSITION OF RAT MYOCARDIAL SARCOLEMXA
Percent of dry weight 79.4” 13.8 3.9 5.1
Proteins Lipids Hexoses Hexosamines
’ Each value represents the mean of six separate determinations.
20
40
60
SO
100
120
140
Bound Co++ hmoleslmg proteInI
FIG. 1. Scatchard plot of calcium binding by rat, myocardial sarcolemma in the presence (0) and absence (0) of 1 mM ATP.
I OF MARKER
ENZYME@
Sarcoplasmic reticulum
10.2 + 3.7 0.05 + 0.001 Na+, K+-ATPaseb 24 k 6.2 79 f 6.70 Cytochrome oxidase” 1.32 2 0.45 Glucose 6-phosphatasd 0.18 k 0.04 0.06 -e 0.01 0.01 c 0.04 Acid phosphatasd a Results represent means k SEM from six separate determinations. L Micromoles of inorganic phosphate per milligram of protein per hour at 37°C. c Nanomoles of cytochrome oxidized per milligram of protein per minute at 27°C.
Mitochondria 0.04 k 0.001 1247 + 56 0.14 * 0.04 0.20 2 0.03
CALCIUM-BINDING
SITES IN RAT MYOCARDIAL
305
SARCOLEMMA
enzyme incubations (Table V): Whereas trypsin and Pronase pretreatment resulted in a 43 to 49% decrease in 45Ca binding, phospholipases and neuraminidase were much less effective even though phospholipid Pi content and sialic acid content fell TABLE
III
EFWCT OF NUCLEOSIDE TRIPHOSPHATES SARCOLEMMAL PHOSPHOBYWTIO~~
Frc. 2. Calcium binding as a function of sarcolemma1 protein concentration. The incubation mixture consisted of 100 mM KCI, 10 mM MgCl,, 20 mM Tris-maleate buffer, pH 7.4, 0.1 mM WaCl,, and varying amounts of sarcolemmal membrane in a final volume of 1.0 ml. Incubation was carried out at 37°C for 10 min and calcium binding was determined by the Millipore filtration method as detailed in the text.
Protein
Concentration
ON
Nucleoside triphospate
[3ZPlPhosphate incorporated (pmol/pg of protein)
None GTP UTP CTP ATP
13.1 11.8 10.2 11.6 160.8
B The phosphorylation reaction was carried out as described in the legend to Fig. 3, using 0.5 mM of the appropriate 32P-labeled nucleoside triphosphate.
(pg)
FIG. 3. Phosphorylation of sarcolemmal proteins. The reaction was carried out at 37°C in 0.3 ml containing 40 mM histidine buffer (pH 6.Q 2.5 mM EGTA, 0.5 mM ATz2P, 2 mM MgC12, and variable amounts of sarcolemmal proteins. After 10 min of incubation, the reaction was terminated by the addition of 3.0 ml of unlabeled ATP, followed by 3.3 ml of cold 10% TCA-3% sodium pyrophosphate prior to filtration through nitrocellulose filters presoaked in 1 mM ATP. Each filter was washed twice with 5 ml of cold 5% TCA-1.5% sodium pyrophosphate, ovendried, and counted in 5 ml of toluene-based scintillation fluid.
and low-affinity sites (Fig. 11, while AMP and ADP were ineffective. Ruthenium red decreased Ca2+ binding in a nonspecific manner, whereas lanthanum was effective only on the low-affinity sites (Fig. 6). Table IV shows the distribution of 45Ca binding among the protein and lipid phases. It is evident that binding is predominantly a function of protein fraction. This is also borne out by the effects of
Minutes
FIG. 4. Time course of Wa binding by sarcolemma. The incubation mixtute contained 100 mM KCl, 10 mM MgC12, 20 mM Tris-maleate buffer, pH 7.4, 0.1 mM WaCl,, and 60 pg of sarcolemmal protein in a final volume of 1.0 ml. After incubation for l-20 min at 37”C, the samples were analyzed for 45Ca binding as detailed in the text.
O-
5.0
6.0
PH
FIG. 5. The effect of pH on calcium binding.
306
CONSTANTINOS
FIG. 6. Scatchard plots of the effects of lanthanum and ruthenium red on calcium binding by myocardial sarcolemma.
DISTRIBUTION OF PROTEINS, LIPID,
Fraction
TABLE IV Wa BINDING AND Aqueous
Amount recovered No ATP
Protein Lipid Aqueous
AMONG THE FRACTION
nmol/g 49.1 ? 2.3 9.2 2 1.6 2.9 +- 0.7
ATP added % 80.0 15.2 4.8
nmol/g 164.2 2 5.6 18.7 + 1.8 12.1 2 0.9
% 84.2 9.6 6.2
by 60 and 75%, respectively, following incubation. The possibility that changes in the distribution of 45Ca were partly secondary to the chloroform-methanol extraction procedure was considered. The following calculations, however, make it unlikely that migration of Wa from the lipid to the protein fraction contributed significantly to the results. Assuming that Ca2+ can only bind to acidic phospholipids, the total concentration of Ca2+-binding lipid in 1 g dry weight is 0.095 g (i.e., 1 g dry weight sarcolemma contains 13.8% lipid, of which 40% is in the form of phosphatidyl-ethanolamine, phosphatidylserine, and phosphoinositides). If the average molecular weight of phospholipid is taken as 750, 0.095 g equals 121 pm01 of phospholipid. Assuming that each Ca2+ ion is bound via two phosphate groups at the maximum, then the phospholipids contained in 1 g dry weight could bind a maximum of 60 pm01 of Ca2+. Under the binding conditions used in the extraction experiments (260 pm01 of Cd+/g weight) this represents 23% of the total Ca2+ bound to the whole membrane. From the chloroform-methanol extraction
JOHN
LIMAS
studies only 15.2% of the total bound Ca2+ was recovered in the lipid phase. Thus, if migration of Ca2+ from phospholipid to protein had occurred during the extraction, the extent would be no greater than 23 - 15.2 = about 8% of the total Ca2+ bound. SDS-polyacrylamide gel electrophoresis of the sarcolemmal proteins showed multiple bands corresponding to proteins with molecular weights of 45,000 to 150,000. SDS-polyacrylamide gel electrophoresis was also performed following incubation of sarcolemmal proteins with 45CaC12to characterize the distribution of Ca2+-binding proteins. A parallel gel with WaCl, but no protein was also run as control; under these conditions, Va incorporation was uniform throughout the gel and always less than 5% of that obtained in the presence of proteins. The 45Ca electrophoretic pattern was obtained following subtraction of the control counts from the experimental gel. Omission of sodium dodecyl sulfate resulted in a 10 to 15% increase in total counts, but no change in the incorporation pattern. Electrophoresis of 45Caloaded membranes resulted in one welldefined band near the origin with several smaller peaks of lower molecular weights (Fig. 7). Electrophoresis of sarcolenunal membranes in the presence of 20 PM LaCl, TABLE EFFECT
V
OF ENZYME INCUBATIONS OF *%a BINDING BY MYOCARDIAL SARCOLEMMA~
Enzyme
Percentage decrease in %!a binding
Neuraminidase 8.2 Pronase 43.2 Trypsin 49.6 Phospholipase A, 4.8 Phospholipase C 3.4 Phospholipase D 6.4 LIMembranes (0.2-0.3 mg of protein) suspended in 1.0 ml of 20 mr.i Tris-maleate buffer, pH 7.4,lOO miu KCl, 10 miu MgCl, were preincubated with 10 units of neuraminidase, 100 pg of Pronase or trypsin or 200 pg ofphospholipase (A,, C, D) for 60 min at 37°C. Control samples not containing enzymes were run concurrently. After incubation, the membranes were centrifuged and suspended in 2.0 ml of 20 mM Tris-maleate buffer, pH 7.4. Aliquots were then taken for Wa binding as described in the text.
CALCIUM-BINDING
SITES IN RAT MYOCARDIAL
SARCOLEMMA
307
quinoline (ECDQ) which is known to modify carboxyl groups (14). As shown in Fig. 9, there was significant, time-dependent inhibition of Ca2+ binding by the carbodiimide . DISCUSSION
FIG. 7. SDS-polyacrylamide gel electrophoresis of Wa-loaded sarcolemmal membranes. Preparations were incubated for 10 min at 37’C in a medium containing 100 mM KCl, 10 mu MgCl*, 20 mM Trismaleate buffer, pH 7.4, and 0.1 mM WaC12 and were then collected by centrifugation and solubilized in 0.2 ml of 0.1 M sodium phosphate buffer, pH 7.4, and 0.1% SDS prior to electrophoresis. I
I
FIG. 8. Effects of lanthanum (20 PM) and hydroxylamine (2 M) on the electrophoretic pattern of Wa-loaded sarcolemmal membranes.
showed only one peak at a molecular weight of about 100,000 (Fig. 8) and sharp decrease Va incorporation in the remaining protein bands. Hydroxylamine resulted in almost complete decreases of this peak* In order to define better the molecular groups responsible for Ca2+ binding, we interacted the sarcolemmal membranes with p-mercuribenzoate, iodoacetamide, and maleic anhydride. Neither of these agents significantly modified the Ca2+binding ability of the sarcolemma, suggesting that sulfhydryl, amino, and thiol groups are not necessary. In view of the relatively high concentration of acidic amino acids in sarcolemma, the importance of carboxyl groups in calcium binding was investigated using the carbodiimide 2-ethoxy-N-carbethoxy-1,2-dihydro-
Following depolarization of the muscle membrane, an increase in the concentration of intracellular calcium occurs and leads to the interaction of the contractile proteins, which is manifested as tension development. This increase in intracellular calcium may be due to a change in the permeability of the cell membrane to Ca2+ so that it enters from extracellular sites or to release of the ion from intracellular stores. In skeletal muscle it seems clear that Ca2+ ions must be maintained primarily in an intracellular store, from which they can be mobilized for repetitive contractions. This conclusion is based primarily on the fact that the skeletal muscle cell can continue to contract and relax when the extracellular calcium concentration is very low. The terminal cisternae of the sarcoplasmic reticulum are the most likely anatomical candidates for this intracellular store (2). The source of the activator calcium in the heart muscle is less clearly defined. Several observations make it likely that both intracellular and superficial sites are important. The rapid decline in tension developed by cardiac muscle when the extracellular Ca2+ concentration is reduced (3) implies that sites rapidly exchanging with all extracellular space are important. Lanthanum, which displaces Ca2+ from extracellular sites but does not enter the in-
Time
(mm)
FIG. 9. Inhibition of Wa binding by S-ethoxy-Ncarbethoxy-1,2-dihydroquinoline carbodiimide.
308
CONSTANTINOS
tracellular space, effectively blocks excitation-contraction coupling in the heart but not skeletal muscle (4). Kinetic studies of calcium exchange in myocardium have yielded fast and slowly exchanging compartments. The size of the fast exchanging compartment, which is freely accessible from the extracellular space, is related to changes in contractile force and is increased by digitalis glycosides (15). The latter compounds also increase the amount of Ca2+ displaceable by lanthanum (15); conversely, doses of verapamil and periods of &hernia which reduce twitch-tension diminish the amount of Ca2+ which is displaceable by La3+ (17). The relative contribution of the two fundamentally different Ca2+ stores to the contractile event has been estimated in the range of 30 to 50% of the activator from the external membrane system and 50 to 70% from the intracellular store (1). The morphological basis for sarcolemma1 calcium binding is not known. Under the electron microscope, the myocardial cell appears enveloped by a relatively thick (300-400 A> lamina externa which communicates with the extracellular space (18, 19). This lamina has staining properties suggestive of high contention mucopolysaccharide-protein complexes. Recent evidence in other cell types (20) suggests that these complexes are responsible for the characteristic selective deposition of La3+ on the cellular surface. Ruthenium red, a stain for negatively charged groups (21) which would bind cations including Ca2+, has similar staining pattern. Although a high content of cell surface material in sialic acid residues, which are capable of binding Ca2+, has been shown in HeLa cells and neuraminidase has been reported to decrease the La3+ binding sites of the cells (20) dramatically, a study of the chemical groups responsible for Ca2+ binding in the myocardium has not been previously undertaken. The results of the present and previous studies (22, 23) suggest the presence of at least two classes of binding sites with low and high affinities for calcium, respectively. Since there was no distinct break in the Scatchard plots, there may be addi-
JOHN LIMAS
tional classes of binding sites with differing affinities for Ca2+. Some of the characteristics of these sites have been described; their total capacity is in the order of 520 nmol/g, which far exceeds the amount of calcium thought to enter the cell following membrane depolarization. Theoretically, then, sarcolemma alone can contribute all of the activator calcium; there is, however, no direct evidence that calcium bound at these sarcolemmal sites is all involved in excitation-contraction coupling. On a quantitative basis, the low affinity sites may be more important than the high a.f finity ones. Both are nonspecifically stimulated by ATP, which increases the apparent affinity of both classes of sites for calcium but does not appreciably alter the total binding capacity of the sarcolemmal preparations. In this regard, the effects of ruthenium red and lanthanum are of interest. Whereas ruthenium red inhibited both the low- and high-affinity sites, lanthanum inhibited only the low-affinity sites. Ruthenium red has been shown to inhibit the mitochonrial and sarcolemmal Ca2+-ATPases (24) while having little or no effect on sarcoplasmic reticulum. Lanthanum and verapamil, on the other hand, specifically inhibit the slow calcium current, curing the plateau of the action potential which is thought to represent, in part, the source of activator calcium. Lowaffinity sites, therefore, may reflect the potential sarcolemmal contribution to the activator calcium pool. The nature of the high affinity sites is less evident. The electrophoretic data in the presence of lanthanum suggest that a protein band with a molecular weight of about 100,000 may be primarily responsible. It is interesting that hydroxylamine abolished the incorporation of 45Ca into this protein fraction; in addition, AT32P incorporation (and its enhancement by Ca2+) occurred in the same electrophoretic position, suggesting that Ca2+-ATPase might be responsible for most of the high-affinity binding. Such ATPases have been described for both cardiac and skeletal muscle sarcolemma although their function in transmembrane Ca2+ fluxes is not known (25., 26). They may cooperate with sarcoplasmic reticu-
CALCIUM-BINDING
SITES
IN
RAT
lum in lowering mycoplasmic Ca*+ levels to effect relaxation. Although sarcolemma exhibits staining characteristics suggestive of acid mucopolysaccharides and has a relatively high sialic acid content, neuraminidase treatment did not effect calcium binding to any significant extent. It is conceivable, however, that the 25% sialic acid residues not accessible to neuraminidase might be functionally more important than the ones that were removed by the enzyme. There are, as yet, no data on the in vivo effects of neuraminidase on the calcium fluxes and contractility of the myocardium. By contrast, phospholipase treatment resulted in a significant decrease of Ca2+ binding, in accordance with other studies supporting a fundamental role of phospholipids in calcium binding (23, 27). This may not necessarily represent a direct effect of phospholipids but, rather, a stabilizing influence of the integrity of cell membranes. The distribution of 45Ca radioactivity supports the conclusion that proteins are a major site of calcium binding in sarcolemma1 preparations. Incubation experiments failed to demonstrate the importance of sulfhydryl, amino, or thiol groups in calcium binding. On the other hand, carbodiimide modification of the sarcolemmal membranes strongly suggested that carboxy1 groups are involved in this process. This is in keeping with the high content of sarcolemma in carboxylic amino acids (28) and the effect of ruthenium red, which depends on the presence of ionizable carboxy1 groups. REFERENCES M., AND GOLDMAN, Y. (1953)Progr. Biophys. 27, 259-312. 2. PAGE, E. (1968) J. Gen. Physiol. 51, 211-220. 1. MORAD,
3. FLECKENSTEIN, A. (1964) in The Cellular tions of Membrane Transport (Hoftman, ed.), pp. 71-93, Prentice Hall, N. J.
FuncJ. F.,
MYOCARDIAL
SARCOLEMMA
4. SANBORN,
W. G., AND LANGER,
309 G. A. (1970)
J.
Gen. Physiol. 56, 191-217. 5. KAUFMANN, R. L., HOMBURGER, H., AND WIRTH, H. (1971) Circ. Res. 28, 346-357. 6. LIMM, C. J., NOTARGIACOMO, A, V., AND COHN, J. N. (1973) Cardiovasc. Res. 7, 477-481. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. C&m. 193, 265-275. 8. CARVALHO, A. P., AND LEO, B. (1967) J. Gen. Physiol. 50, 1327-1352. 9. &E, J. H. (1955) J. Biol. Chem. 212, 335-354. 10. Boos, N. F. (1953) J. Biol. Chem. 204,553-563. 11. WARREN, L. (1959) J. Biol. Chem. 234, 19711975. 12. NEVILLE, D. M., AND GLOSSMANN, H. (1971) J.
Biol. Chem. 246, 6335-6342. S. P., FUJII, T., AND HANORHAM, (1968) Biochemistry 7, 3682-3700.
13. BURGER, 14. HOARE,
D. G., AND KOSHLAND,
D. J.
D. E., JR. (1967)
J. Biol. Chem. 242, 2447-253. 15. CARRIER, G. O., L~~LLMAN, H., NEUBAUER, L., AND PETERS, T. (1974) J. Mol. Cell Cardiol. 6, 333-347. 16. NAYLER, W. G. (1973) J. Mol. Cell Cardiol. 5, 101-110. 17. NAYLER, W. G., AND MERRILLEES, N. C. R. (1971) in Calcium and the Heart (Harris, P., and Opie, L. H., eds.), Academic Press, New York/London. 18. McNurr, N. W. (1975) Circ. Res. 37, 1-13. 19. SHEA, S. M. (1971) J. Cell Biol. 51, 611-620. 20. BOYD, K., MELNYDOVYCH, G., AND FISKIN, A. M. (1972) J. Cell Biol. 55, 252. 21. LUFT, J. G. (1971)Anat. Rec. 171, 347-362. 22. DUFFY, M. J., AND S~HWARZ, V. (1974) B&him. Biophys. Acta 330, 294-301. 23. SHLATZ, L., AND MARINETTI, G. V. (1972)
Biochim. Biophys. Acta 290, 70-83. 24. MADEIRA, V. M. C., AND ANTUNES-MADEIRA, M. C. (1974) J. Membrane Biol. 17, 41-50. 25. DIETZE, G., AND HEPP, K. D. (1971) Biochm. Biophys. Res. Commun. 44, 1041-1049. 26. SULAKHE, P. V., AND KHALLA, N. S. (1971) Life sci. 10, 185-191. 27. SIJLAKHE, P. V., DRUMMOND, G. I., AND NG, D. C. (1973) J. Biol. Chem. 248, 4150-4157. 28. VANDENBURGH, H. H., SHEFF, M. F., AND ZACKS, S. I. (1974) J. Membrane Biol. 17,1-12. 29. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4414.
OF BIOCHEMISTBY
AND
Calcium-Binding
BIOPIiYSIC&
179,
302-309
Sites in Rat Myocardial CONSTANTINOS
University
of Minnesota
(1977)
JOHN
Sarcolemma
LIMAS
Medical School, Department of Medicine, Cardiovascular Memorial Building, Minneapolis, Minnesota 55455
Division,
Box 83, Mayo
Received October 3, 1976 Myocardial sarcolemmal preparations have the ability to bind Caz+; this binding shows saturation kinetics, has a pH optimum of 7.4 to 7.8, and is stimulated by ATP. Scatchard analysis reveals both high- and low-affinity binding sites. Both classes are inhibited by ruthenium red, whereas only the high-afiinity sites are affected by lanthanum and hydroxylamine. Electrophoresis in the presence of sodium dodecyl sulfate suggests that the high-affinity sites are associated .with a protein peak of molecular weight of about 100,000. Enzyme treatment of the membranes suggested that proteins accounted for most of the %a binding with phospholipids and sialic acids residues playing a secondary role. Interaction with different function group reagents indicated that the carboxyl residues were necessary for the calcium binding, whereas thiol, amino, and sulIhydryl~groups were unimportant.
calcium for binding sites) specifically inhibits excitation-contraction coupling in the heart, while it is much less effective in skeletal muscle (4). All of these observations suggest superficial binding sites as a major source of activator calcium in the heart. In addition, there is accumulating evidence that the major defect in spontaneous or induced heart failure may involve deficient excitation-contraction coupling (5). The present study was undertaken, therefore, to characterize the chemical groups responsible for Ca2+ binding by myocardial sarcolemma.
The interaction of contractile proteins is regulated in the myocardium, as in all types of muscle, by the amounts of calcium ion entering the cytoplasm from extracelMar and intracellular storage sites in response to membrane depolarization. The relative contributions of intra- and extracellular sites are a matter of debate. It seems increasingly evident, however, that a difference exists between skeletal and cardiac muscle; whereas sarcoplasmic reticulum and to some extent mitochondria, appear to play the predominant role as source of activator calcium in the former type of muscle, sarcolemma is more important in the heart (1). Structural and functional observations support this conclusion. The sarcotubular lateral sacs are less developed in the myocardium and may constitute as little as 2% of the whole cell volume. By contrast, the volume of the T system is proportionately increased (2). The force of contraction is rapidly diminished when cardiac muscle is perfused with calcium-free fluid with a half-life of less than 2 min, suggesting that the activator calcium is in rapid equilibrium with the extracellular space (3). Finally, lanthanum (which does not enter the intracellular space and competes effectively with
MATERIALS
AND METHODS
Experiments were conducted on 4- to 6-month-old male Wistar rats, weighing 250 to 350 g. Isolation of sarcolemma. The hearts were rapidly excised, washed in cold 10 mM NaHCO,, minced, and blotted dry. Homogenization was performed in 10 vol of 0.34 M sucrose using a Polytron PT-20 (rheostat setting, 5; duration, 30 9. The homogenate was filtered and centrifuged at 2000g for 10 min. The resultant sediment was suspended in 10 vol of 10 rnr.4 Tris-HCl, pH 6.0 and centrifuged at 2OOOg for 10 min. This step was repeated three times. The final pellet was suspended in 5 vol of 10 mu Tris-HCl, pH 8.0 and 4.0 M LiBr was added to bring the final concentration to 0.4 M. The solution was stirred gently for 4 to 5 h and was then diluted fourfold with 302
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0993-9861
CALCITJM-BINDING
SITES IN RAT MYOCARDIAL
buffer. It was then centrifuged at 2OOOgfor 10 min and washed with 10 vol of buffer, and the centrifugation was repeated. The sediment was washed with 8 vol of buffer and centrifuged at 2000g for 10 min; this step was repeated once and the final pellet was suspende’ddin distilled water at a protein concentration of 2 to 5 mg/ml. Phase contrast microscopy at this level showed predominantly empty sarcolemma1 sacs which had vesicular appearance under the electron microscope. Mitochondria and sarcoplasmic reticulum were also prepared and enzyme activities of Na+, K+-ATPase, adenylate cyclase, cytochrome oxidase, glycose 6-phosphatase, and acid phosphatase were assayed using standard techniques (6). Protein content was determined by the method of Lowry et ~2. (7) with bovine serum albumin as the standard. The lipid content was determined by extracting with chloroform methanol (2:1, v/v) and then proceeding as described by Carvalho (8). Total hexoses were assayed by the anthrone reaction (91 and hexosamines by the Elson-Morgan reaction (10). Sialic acid content was determined by thiobarbituric acid (11). Electrophoresis. SDS-polyacrylamide gel electrophoresis of the sarcolemmal proteins was performed in a sulfate-borate discontinuous buffer system using the method of Neville and Glossmann (12). Gels containing 3% (stacking) and 10% (separation) gels were prepared from a stock solution of 30% by weight of acrylamide and 0.1% sodium dodecyl sulfate and were polymerized chemically by the addition of 0.025% by volume of tetramethylethylenediamine and ammonium persulfate. Five-centimeter gels were prepared in glass tubes of a total length of 7.5 cm with an internal diameter of 6 mm. The stacking gel length was 1 cm. The buffers used were: upper reservoir, 0.04 M boric acid-O.041 M Tris (pH 8.6); stacking gel, 0.0267 M H,S04-0.0541 M Tris (pH 6.1); separating gel and lower reservoir, 0.0208 M HCl-0.4244 M Tris (pH 9.18). The membranes were suspended in a total volume of 0.2 ml containing the following: 0.625 M Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.001% bromphenol blue. Electrophoresis was carried out at a current of 2 mA/tube until the tracking dye had reached a mark about 0.5 cm from the end of the gel. After electrophoresis, the gels were stained with 1% amido black in methanol:acetic acid:water (50:7:42, v/v/v). Destaining was carried out with three to five changes of methanol:acetic acid:water for 24 to 36 h. The gels were then sliced into l-mmthick slices which were solubilized in dimethyl sulfoxide prior to recording absorbancies at 660 nm. ‘Abbreviations used: SDS, sodium dodecyl sulfate; ECDQ, 2-ethoxy-N-carbethoxy-1,2-dihydroquinoline carbodiimide; TCA, trichloroacetic acid; EGTA, ethylene glycol bis(P-aminoethyl ether)N,N’-tetraacetic acid.
SARCOLEMMA
303
Molecular weights were estimated by using a calibration curve prepared from standard proteins, including @-galactosidase, ovalbumin, avidin, and transferrin. Calcium binding. Calcium binding by the sarcolemma was studied using the Millipore filtration method. Membrane protein (5-100 pg) was incubated in a medium containing 100 mM KCl, 10 mM MgCl,, 20 rnM Tris-maleate buffer, pH 7.4, and 0.1 mM 45CaC1, with or without 0.5 mM ATP. The reaction was started with the addition of ATP and was terminated at various intervals by filtering 0.8 ml through a Millipore filter (0.45 pm, 25 mm) under light suction. The filter was washed with 5 ml of 100 mM Tris-maleate buffer, pH 7.4, dried at 70°C in a scintillation vial, covered with 10 ml of tolueneLiquifluor, and counted in a liquid scintillation counter. Controls containing no ATP, no MgCl*, or no protein were also used. Calcium binding was estimated from the specific activity of the added Wa and the activity retained by the membrane protein. Separation of lipid- and protein-bound Ca*+. Sarcolemmal membranes were incubated as described above with Wa2+ for 20 min; the reaction was then stopped by adding 5 ml of ice-cold 0.5 mM histidineimidazole buffer, pH 7.0. The membranes were then centrifuged at 6OOOg for 15 min, resuspended in buffer, and centrifuged again. The pellet containing &Ca was extracted three times with chlorofornnmethanol (l:l, v/v) by the method of Burger et al. (131 and the insoluble protein precipitate was removed by centrifugation. The lipid extract was combined with chloroform and water to give chloroform:methanol:water proportions of 10:5:1 (by volume). After centrifugation at 1085g for 10 min, an aqueous upper phase and an organic phase formed. The organic phase contained the phospholipidbound Ca*+, the precipitate contained the proteinbound and the aqueous phase, the unbound Ca2+. Enzyme incubation. Sarcolemmal membrane preparations (0.2-0.3 mg of protein) were suspended in 1.0 ml of a solution containing 100 mM KCl, 10 mM MgCl,, 20 mM Tris-maleate buffer, pH 7.4, and were preincubated with the appropriate amount of the enzyme for 1 h. They were then centrifuged at 1500g for 20 min and the pellets were resuspended in 1.0 ml of 20 mM Tris-maleate buffer, pH 7.4. Aliquots (100-200 ~11 of the suspension were used to determine Wa binding as previously described. For the carbodiimide reaction, a solution containing the sarcolemma (1 mg/ml), 1.33 M glycine methyl ester, and 7.5 M urea was kept at 25°C and pH 4.74. A solution of 0.4 M carbodiimide (2-ethoxyN-carbethoxy-1,2-dihydroquinoline) dissolved in 7.5 M urea was then added to the mixture at a final concentration of 0.1 M. The pH was maintained by automatic titration with 0.5 M HCl. Aliquots (0.5 ml) were removed periodically and added to 5 ml of 1.0 M acetate buffer at pH 4.75 to quench the reac-
304
CONSTANTINOS
tion with the diimide. After standing for a few minutes at 25”C, the solutions were dialyzed for 36 h at 0°C against three changes of 20 liters each for 0.001 M HCl. A suitable portion of the dialysate was then hydrolyzed for 16 h at 110°C in 6 M HCl and analyzed for amino acids. RESULTS
Myocardial sarcolemma isolated by the procedure described was essentially free of contaminating mitochondria or sarcoplasmic reticulum as shown by the relative distribution of “marker” enzymes (Table I). The chemical composition of the sarcolemma is shown in Table II; proteins constituted 79.4% of dry weight and lipid, 13.8%. Sarcolemmal preparations bound Ca2+ to a degree dependent on its concentration in the incubation mixture. Figure 1 gives a Scatchard plot of the Cd2+-binding data; this can be resolved into two curves corresponding to two classes of binding sites with different affinities for calcium. The extrapolated intercepts of the linear segments of the plot can be used to calculate the number of binding sites and the corresponding association constants. The higher affinity sites for calcium with an association constant of 2.0 x lo5 M-l accommodated 17 + 2 (SD) nmol of Ca2+/mg of protein. The lower affinity sites had an association constant of 5.6 x lo2 M-’ and accomodated 680 t 16 (SD) nmol of Ca2+/ mg of protein. Binding of Ca2+ as a function of membrane concentration was studied (Fig. 2); binding increased linearly over a concentration range of 10 to 60 pg of sarcolemmal protein/ml. In the presence of ATP and MgCl*, sarcolemmal proteins are phosphorylated to an extent proportional to the protein concentration in the assay mixture TABLE SUBCELLULAR
DISTRIBUTION
Sarcolemma
JOHN
LIMAS
(Fig. 3). This phosphorylation is sustained only to a minimal degree by other nucleoside triphosphates (Table III) and is due to the presence in the sarcolemmal preparation of cyclic AMP-dependent and -independent protein kinases. Its regulation and relation to Ca2+ binding are detailed in a separate report. Less than 1% of the ATP is retained in the Millipore filter when sarcolemmal protein is omitted from the incubation medium. 45Ca binding also increased rapidly with time, reaching maximal saturation after 2 min of incubation (Fig. 4). There was a marked influence of pH on 45Ca binding with an optimum between 7.4 and 7.8 (Fig. 5). ATP stimulated calcium binding by both highTABLE CHEIOZAL
II
COMPOSITION OF RAT MYOCARDIAL SARCOLEMXA
Percent of dry weight 79.4” 13.8 3.9 5.1
Proteins Lipids Hexoses Hexosamines
’ Each value represents the mean of six separate determinations.
20
40
60
SO
100
120
140
Bound Co++ hmoleslmg proteInI
FIG. 1. Scatchard plot of calcium binding by rat, myocardial sarcolemma in the presence (0) and absence (0) of 1 mM ATP.
I OF MARKER
ENZYME@
Sarcoplasmic reticulum
10.2 + 3.7 0.05 + 0.001 Na+, K+-ATPaseb 24 k 6.2 79 f 6.70 Cytochrome oxidase” 1.32 2 0.45 Glucose 6-phosphatasd 0.18 k 0.04 0.06 -e 0.01 0.01 c 0.04 Acid phosphatasd a Results represent means k SEM from six separate determinations. L Micromoles of inorganic phosphate per milligram of protein per hour at 37°C. c Nanomoles of cytochrome oxidized per milligram of protein per minute at 27°C.
Mitochondria 0.04 k 0.001 1247 + 56 0.14 * 0.04 0.20 2 0.03
CALCIUM-BINDING
SITES IN RAT MYOCARDIAL
305
SARCOLEMMA
enzyme incubations (Table V): Whereas trypsin and Pronase pretreatment resulted in a 43 to 49% decrease in 45Ca binding, phospholipases and neuraminidase were much less effective even though phospholipid Pi content and sialic acid content fell TABLE
III
EFWCT OF NUCLEOSIDE TRIPHOSPHATES SARCOLEMMAL PHOSPHOBYWTIO~~
Frc. 2. Calcium binding as a function of sarcolemma1 protein concentration. The incubation mixture consisted of 100 mM KCI, 10 mM MgCl,, 20 mM Tris-maleate buffer, pH 7.4, 0.1 mM WaCl,, and varying amounts of sarcolemmal membrane in a final volume of 1.0 ml. Incubation was carried out at 37°C for 10 min and calcium binding was determined by the Millipore filtration method as detailed in the text.
Protein
Concentration
ON
Nucleoside triphospate
[3ZPlPhosphate incorporated (pmol/pg of protein)
None GTP UTP CTP ATP
13.1 11.8 10.2 11.6 160.8
B The phosphorylation reaction was carried out as described in the legend to Fig. 3, using 0.5 mM of the appropriate 32P-labeled nucleoside triphosphate.
(pg)
FIG. 3. Phosphorylation of sarcolemmal proteins. The reaction was carried out at 37°C in 0.3 ml containing 40 mM histidine buffer (pH 6.Q 2.5 mM EGTA, 0.5 mM ATz2P, 2 mM MgC12, and variable amounts of sarcolemmal proteins. After 10 min of incubation, the reaction was terminated by the addition of 3.0 ml of unlabeled ATP, followed by 3.3 ml of cold 10% TCA-3% sodium pyrophosphate prior to filtration through nitrocellulose filters presoaked in 1 mM ATP. Each filter was washed twice with 5 ml of cold 5% TCA-1.5% sodium pyrophosphate, ovendried, and counted in 5 ml of toluene-based scintillation fluid.
and low-affinity sites (Fig. 11, while AMP and ADP were ineffective. Ruthenium red decreased Ca2+ binding in a nonspecific manner, whereas lanthanum was effective only on the low-affinity sites (Fig. 6). Table IV shows the distribution of 45Ca binding among the protein and lipid phases. It is evident that binding is predominantly a function of protein fraction. This is also borne out by the effects of
Minutes
FIG. 4. Time course of Wa binding by sarcolemma. The incubation mixtute contained 100 mM KCl, 10 mM MgC12, 20 mM Tris-maleate buffer, pH 7.4, 0.1 mM WaCl,, and 60 pg of sarcolemmal protein in a final volume of 1.0 ml. After incubation for l-20 min at 37”C, the samples were analyzed for 45Ca binding as detailed in the text.
O-
5.0
6.0
PH
FIG. 5. The effect of pH on calcium binding.
306
CONSTANTINOS
FIG. 6. Scatchard plots of the effects of lanthanum and ruthenium red on calcium binding by myocardial sarcolemma.
DISTRIBUTION OF PROTEINS, LIPID,
Fraction
TABLE IV Wa BINDING AND Aqueous
Amount recovered No ATP
Protein Lipid Aqueous
AMONG THE FRACTION
nmol/g 49.1 ? 2.3 9.2 2 1.6 2.9 +- 0.7
ATP added % 80.0 15.2 4.8
nmol/g 164.2 2 5.6 18.7 + 1.8 12.1 2 0.9
% 84.2 9.6 6.2
by 60 and 75%, respectively, following incubation. The possibility that changes in the distribution of 45Ca were partly secondary to the chloroform-methanol extraction procedure was considered. The following calculations, however, make it unlikely that migration of Wa from the lipid to the protein fraction contributed significantly to the results. Assuming that Ca2+ can only bind to acidic phospholipids, the total concentration of Ca2+-binding lipid in 1 g dry weight is 0.095 g (i.e., 1 g dry weight sarcolemma contains 13.8% lipid, of which 40% is in the form of phosphatidyl-ethanolamine, phosphatidylserine, and phosphoinositides). If the average molecular weight of phospholipid is taken as 750, 0.095 g equals 121 pm01 of phospholipid. Assuming that each Ca2+ ion is bound via two phosphate groups at the maximum, then the phospholipids contained in 1 g dry weight could bind a maximum of 60 pm01 of Ca2+. Under the binding conditions used in the extraction experiments (260 pm01 of Cd+/g weight) this represents 23% of the total Ca2+ bound to the whole membrane. From the chloroform-methanol extraction
JOHN
LIMAS
studies only 15.2% of the total bound Ca2+ was recovered in the lipid phase. Thus, if migration of Ca2+ from phospholipid to protein had occurred during the extraction, the extent would be no greater than 23 - 15.2 = about 8% of the total Ca2+ bound. SDS-polyacrylamide gel electrophoresis of the sarcolemmal proteins showed multiple bands corresponding to proteins with molecular weights of 45,000 to 150,000. SDS-polyacrylamide gel electrophoresis was also performed following incubation of sarcolemmal proteins with 45CaC12to characterize the distribution of Ca2+-binding proteins. A parallel gel with WaCl, but no protein was also run as control; under these conditions, Va incorporation was uniform throughout the gel and always less than 5% of that obtained in the presence of proteins. The 45Ca electrophoretic pattern was obtained following subtraction of the control counts from the experimental gel. Omission of sodium dodecyl sulfate resulted in a 10 to 15% increase in total counts, but no change in the incorporation pattern. Electrophoresis of 45Caloaded membranes resulted in one welldefined band near the origin with several smaller peaks of lower molecular weights (Fig. 7). Electrophoresis of sarcolenunal membranes in the presence of 20 PM LaCl, TABLE EFFECT
V
OF ENZYME INCUBATIONS OF *%a BINDING BY MYOCARDIAL SARCOLEMMA~
Enzyme
Percentage decrease in %!a binding
Neuraminidase 8.2 Pronase 43.2 Trypsin 49.6 Phospholipase A, 4.8 Phospholipase C 3.4 Phospholipase D 6.4 LIMembranes (0.2-0.3 mg of protein) suspended in 1.0 ml of 20 mr.i Tris-maleate buffer, pH 7.4,lOO miu KCl, 10 miu MgCl, were preincubated with 10 units of neuraminidase, 100 pg of Pronase or trypsin or 200 pg ofphospholipase (A,, C, D) for 60 min at 37°C. Control samples not containing enzymes were run concurrently. After incubation, the membranes were centrifuged and suspended in 2.0 ml of 20 mM Tris-maleate buffer, pH 7.4. Aliquots were then taken for Wa binding as described in the text.
CALCIUM-BINDING
SITES IN RAT MYOCARDIAL
SARCOLEMMA
307
quinoline (ECDQ) which is known to modify carboxyl groups (14). As shown in Fig. 9, there was significant, time-dependent inhibition of Ca2+ binding by the carbodiimide . DISCUSSION
FIG. 7. SDS-polyacrylamide gel electrophoresis of Wa-loaded sarcolemmal membranes. Preparations were incubated for 10 min at 37’C in a medium containing 100 mM KCl, 10 mu MgCl*, 20 mM Trismaleate buffer, pH 7.4, and 0.1 mM WaC12 and were then collected by centrifugation and solubilized in 0.2 ml of 0.1 M sodium phosphate buffer, pH 7.4, and 0.1% SDS prior to electrophoresis. I
I
FIG. 8. Effects of lanthanum (20 PM) and hydroxylamine (2 M) on the electrophoretic pattern of Wa-loaded sarcolemmal membranes.
showed only one peak at a molecular weight of about 100,000 (Fig. 8) and sharp decrease Va incorporation in the remaining protein bands. Hydroxylamine resulted in almost complete decreases of this peak* In order to define better the molecular groups responsible for Ca2+ binding, we interacted the sarcolemmal membranes with p-mercuribenzoate, iodoacetamide, and maleic anhydride. Neither of these agents significantly modified the Ca2+binding ability of the sarcolemma, suggesting that sulfhydryl, amino, and thiol groups are not necessary. In view of the relatively high concentration of acidic amino acids in sarcolemma, the importance of carboxyl groups in calcium binding was investigated using the carbodiimide 2-ethoxy-N-carbethoxy-1,2-dihydro-
Following depolarization of the muscle membrane, an increase in the concentration of intracellular calcium occurs and leads to the interaction of the contractile proteins, which is manifested as tension development. This increase in intracellular calcium may be due to a change in the permeability of the cell membrane to Ca2+ so that it enters from extracellular sites or to release of the ion from intracellular stores. In skeletal muscle it seems clear that Ca2+ ions must be maintained primarily in an intracellular store, from which they can be mobilized for repetitive contractions. This conclusion is based primarily on the fact that the skeletal muscle cell can continue to contract and relax when the extracellular calcium concentration is very low. The terminal cisternae of the sarcoplasmic reticulum are the most likely anatomical candidates for this intracellular store (2). The source of the activator calcium in the heart muscle is less clearly defined. Several observations make it likely that both intracellular and superficial sites are important. The rapid decline in tension developed by cardiac muscle when the extracellular Ca2+ concentration is reduced (3) implies that sites rapidly exchanging with all extracellular space are important. Lanthanum, which displaces Ca2+ from extracellular sites but does not enter the in-
Time
(mm)
FIG. 9. Inhibition of Wa binding by S-ethoxy-Ncarbethoxy-1,2-dihydroquinoline carbodiimide.
308
CONSTANTINOS
tracellular space, effectively blocks excitation-contraction coupling in the heart but not skeletal muscle (4). Kinetic studies of calcium exchange in myocardium have yielded fast and slowly exchanging compartments. The size of the fast exchanging compartment, which is freely accessible from the extracellular space, is related to changes in contractile force and is increased by digitalis glycosides (15). The latter compounds also increase the amount of Ca2+ displaceable by lanthanum (15); conversely, doses of verapamil and periods of &hernia which reduce twitch-tension diminish the amount of Ca2+ which is displaceable by La3+ (17). The relative contribution of the two fundamentally different Ca2+ stores to the contractile event has been estimated in the range of 30 to 50% of the activator from the external membrane system and 50 to 70% from the intracellular store (1). The morphological basis for sarcolemma1 calcium binding is not known. Under the electron microscope, the myocardial cell appears enveloped by a relatively thick (300-400 A> lamina externa which communicates with the extracellular space (18, 19). This lamina has staining properties suggestive of high contention mucopolysaccharide-protein complexes. Recent evidence in other cell types (20) suggests that these complexes are responsible for the characteristic selective deposition of La3+ on the cellular surface. Ruthenium red, a stain for negatively charged groups (21) which would bind cations including Ca2+, has similar staining pattern. Although a high content of cell surface material in sialic acid residues, which are capable of binding Ca2+, has been shown in HeLa cells and neuraminidase has been reported to decrease the La3+ binding sites of the cells (20) dramatically, a study of the chemical groups responsible for Ca2+ binding in the myocardium has not been previously undertaken. The results of the present and previous studies (22, 23) suggest the presence of at least two classes of binding sites with low and high affinities for calcium, respectively. Since there was no distinct break in the Scatchard plots, there may be addi-
JOHN LIMAS
tional classes of binding sites with differing affinities for Ca2+. Some of the characteristics of these sites have been described; their total capacity is in the order of 520 nmol/g, which far exceeds the amount of calcium thought to enter the cell following membrane depolarization. Theoretically, then, sarcolemma alone can contribute all of the activator calcium; there is, however, no direct evidence that calcium bound at these sarcolemmal sites is all involved in excitation-contraction coupling. On a quantitative basis, the low affinity sites may be more important than the high a.f finity ones. Both are nonspecifically stimulated by ATP, which increases the apparent affinity of both classes of sites for calcium but does not appreciably alter the total binding capacity of the sarcolemmal preparations. In this regard, the effects of ruthenium red and lanthanum are of interest. Whereas ruthenium red inhibited both the low- and high-affinity sites, lanthanum inhibited only the low-affinity sites. Ruthenium red has been shown to inhibit the mitochonrial and sarcolemmal Ca2+-ATPases (24) while having little or no effect on sarcoplasmic reticulum. Lanthanum and verapamil, on the other hand, specifically inhibit the slow calcium current, curing the plateau of the action potential which is thought to represent, in part, the source of activator calcium. Lowaffinity sites, therefore, may reflect the potential sarcolemmal contribution to the activator calcium pool. The nature of the high affinity sites is less evident. The electrophoretic data in the presence of lanthanum suggest that a protein band with a molecular weight of about 100,000 may be primarily responsible. It is interesting that hydroxylamine abolished the incorporation of 45Ca into this protein fraction; in addition, AT32P incorporation (and its enhancement by Ca2+) occurred in the same electrophoretic position, suggesting that Ca2+-ATPase might be responsible for most of the high-affinity binding. Such ATPases have been described for both cardiac and skeletal muscle sarcolemma although their function in transmembrane Ca2+ fluxes is not known (25., 26). They may cooperate with sarcoplasmic reticu-
CALCIUM-BINDING
SITES
IN
RAT
lum in lowering mycoplasmic Ca*+ levels to effect relaxation. Although sarcolemma exhibits staining characteristics suggestive of acid mucopolysaccharides and has a relatively high sialic acid content, neuraminidase treatment did not effect calcium binding to any significant extent. It is conceivable, however, that the 25% sialic acid residues not accessible to neuraminidase might be functionally more important than the ones that were removed by the enzyme. There are, as yet, no data on the in vivo effects of neuraminidase on the calcium fluxes and contractility of the myocardium. By contrast, phospholipase treatment resulted in a significant decrease of Ca2+ binding, in accordance with other studies supporting a fundamental role of phospholipids in calcium binding (23, 27). This may not necessarily represent a direct effect of phospholipids but, rather, a stabilizing influence of the integrity of cell membranes. The distribution of 45Ca radioactivity supports the conclusion that proteins are a major site of calcium binding in sarcolemma1 preparations. Incubation experiments failed to demonstrate the importance of sulfhydryl, amino, or thiol groups in calcium binding. On the other hand, carbodiimide modification of the sarcolemmal membranes strongly suggested that carboxy1 groups are involved in this process. This is in keeping with the high content of sarcolemma in carboxylic amino acids (28) and the effect of ruthenium red, which depends on the presence of ionizable carboxy1 groups. REFERENCES M., AND GOLDMAN, Y. (1953)Progr. Biophys. 27, 259-312. 2. PAGE, E. (1968) J. Gen. Physiol. 51, 211-220. 1. MORAD,
3. FLECKENSTEIN, A. (1964) in The Cellular tions of Membrane Transport (Hoftman, ed.), pp. 71-93, Prentice Hall, N. J.
FuncJ. F.,
MYOCARDIAL
SARCOLEMMA
4. SANBORN,
W. G., AND LANGER,
309 G. A. (1970)
J.
Gen. Physiol. 56, 191-217. 5. KAUFMANN, R. L., HOMBURGER, H., AND WIRTH, H. (1971) Circ. Res. 28, 346-357. 6. LIMM, C. J., NOTARGIACOMO, A, V., AND COHN, J. N. (1973) Cardiovasc. Res. 7, 477-481. 7. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. C&m. 193, 265-275. 8. CARVALHO, A. P., AND LEO, B. (1967) J. Gen. Physiol. 50, 1327-1352. 9. &E, J. H. (1955) J. Biol. Chem. 212, 335-354. 10. Boos, N. F. (1953) J. Biol. Chem. 204,553-563. 11. WARREN, L. (1959) J. Biol. Chem. 234, 19711975. 12. NEVILLE, D. M., AND GLOSSMANN, H. (1971) J.
Biol. Chem. 246, 6335-6342. S. P., FUJII, T., AND HANORHAM, (1968) Biochemistry 7, 3682-3700.
13. BURGER, 14. HOARE,
D. G., AND KOSHLAND,
D. J.
D. E., JR. (1967)
J. Biol. Chem. 242, 2447-253. 15. CARRIER, G. O., L~~LLMAN, H., NEUBAUER, L., AND PETERS, T. (1974) J. Mol. Cell Cardiol. 6, 333-347. 16. NAYLER, W. G. (1973) J. Mol. Cell Cardiol. 5, 101-110. 17. NAYLER, W. G., AND MERRILLEES, N. C. R. (1971) in Calcium and the Heart (Harris, P., and Opie, L. H., eds.), Academic Press, New York/London. 18. McNurr, N. W. (1975) Circ. Res. 37, 1-13. 19. SHEA, S. M. (1971) J. Cell Biol. 51, 611-620. 20. BOYD, K., MELNYDOVYCH, G., AND FISKIN, A. M. (1972) J. Cell Biol. 55, 252. 21. LUFT, J. G. (1971)Anat. Rec. 171, 347-362. 22. DUFFY, M. J., AND S~HWARZ, V. (1974) B&him. Biophys. Acta 330, 294-301. 23. SHLATZ, L., AND MARINETTI, G. V. (1972)
Biochim. Biophys. Acta 290, 70-83. 24. MADEIRA, V. M. C., AND ANTUNES-MADEIRA, M. C. (1974) J. Membrane Biol. 17, 41-50. 25. DIETZE, G., AND HEPP, K. D. (1971) Biochm. Biophys. Res. Commun. 44, 1041-1049. 26. SULAKHE, P. V., AND KHALLA, N. S. (1971) Life sci. 10, 185-191. 27. SIJLAKHE, P. V., DRUMMOND, G. I., AND NG, D. C. (1973) J. Biol. Chem. 248, 4150-4157. 28. VANDENBURGH, H. H., SHEFF, M. F., AND ZACKS, S. I. (1974) J. Membrane Biol. 17,1-12. 29. WEBER, K., AND OSBORN, M. (1969) J. Biol. Chem. 244, 4406-4414.
