THE JOURN.<L OF B~~OGICAL CKEMISTRY

Vol. 248, No. 12, Issue of June 25, pp. 4150-4157, 1973

Printed in U.S.A.

Calcium Binding by Skeletal Muscle Sarcolemma*

(Received for publication, December 4, 1072)

PRAKASH V. SULAKHE,$ GEORGE I. DRUMMOND, AND DAVID C. NG

From the Department of Pharmacology, School of Medicine, University of British Columbia, Vancouver 8, Canada

SUMMARY

Plasma membranes isolated from rabbit muscle possessed the ability to bind and accumulate calcium ions; these proc- esses required ATP and Mg*+. Binding of Ca2+ was rapid, reaching a maximum within 30 s and was greatly increased in the presence of oxalate or phosphate. The concentrations of Ca2+, Mg2+, and ATP required for half-maximal ATP-de- pendent calcium binding were 20 KM, 150 PM, and 20 to 30 pM, respectively. In the presence of oxalate, half-maximal calcium accumulation occurred at 120 PM ATP and 500 pM

Mg2+. Both binding and accumulation were highly sensitive to pH, the maximum being at pH 5.5 to 6.0. Bound calcium could be released by depleting ATP from the medium. So- dium or potassium (100 mM) and high concentrations of Mg2f promoted, while La3+ up to 6 mM inhibited the release process. Rapidity of the binding and release of Ca2+ by the sarcolemma suggests its possible involvement in the ex- citation-contraction coupling mechanism in muscle.

The role of Ca2+ in excitation-contraction coupling in skeletal muscle is -well established (I). Depolarization of the membrane causes release of Ca2f from membrane sites, increasing the con- centration of this cation in the vicinity of the myofibrils. Cal- cium then binds to troponin relieving the inhibitory influence of the troponin-tropomyosin complex on actin-myosin interaction. Contraction of muscle fibers ensues (1, 2). The sarcotubular system surrounding the myofibrils possesses a membrane bound calcium “pump” which is considered to regulate Ca2f levels in the cytoplasm (2). Numerous studies have served to characterize the calcium binding and release systems of muscle sarcoplasmic reticulum (3-5). Muscle mitochondria also possess calcium sequestering activity (6-8)) but their participation in the regula- tion of cytoplasmic Ca2+ is uncertain (6). Several workers have suggested the presence of a calcium transport system in skeletal muscle plasma membrane (9) but difficulties have been encoun- tered in demonstrating this. A major problem has been the iso- lation of sarcolemma free of other intracellular membranes. Recently we described the isolation of sarcolemma from rabbit muscle which contained a good yield of adenylate cyclase (lo), an enzyme found mainly in the plasma membranes of most cells and tissues (11). We found that these preparations possessed

* This work was supported by grants from the Medical Research Council of Canada and the British Columbia Heart Foundation.

t Medical Research Council of Canada Postdoctoral Fellow.

the ability to bind Ca2+; binding of this cation was iucrcascd by the addition of Mg2+ and ATP. In addition the membranes possessed MgATPase and CaATPase activities. In this report the calcium binding and accumulating activities of rabbit muscle sarcolemma are described in detail and compared with that of sarcoplasmic reticulum. A preliminary report of some of thcsc experiments has appeared (12). A succeeding paper (13) do- scribes the properties of the ATPase system of the sarcolemmal preparation.

EXPERIMENTAL PROCEDURE

Materials

4”CaCla (20 mCi per mg) was obtained from New England Nuclear. Acetyl phosphate was purchased from Worthington Biochemicals; p-nitrophenylphosphate from J. T. Eaker Chemical Co.; and nucleoside mono-, di-, and triphosphates from cithor Sigma or Calbiochem.

Methods

Isolation 0.f Sarcolemma-The procedure was essentially that described previously (10) except that the number of washings of each sediment were increased. All procedures were carried out at 4”. Hind leg muscle of a rabbit was cleared of fat and con- nective tissue, minced with scissors, and homogenized in 5 vol- umes of 50 mM CaClz in a Sorvall Omni-Mixer for 15 s at maximal velocity. The homogenate, after filtration through a nylon sieve (pore size 1 mm2) was centrifuged at 2,000 X g for 10 min. The sediment was washed by suspending in 10 volumes of 10 InM Tris-HCl, pH 8.0 (based on initial tissue weight) and centri- fuged at 2,000 X g for 10 min. This washing was repeated thrco more times and served to remove superficially bound calcium as well as heavy microsomes and mitochondria sedimented during the initial centrifugation. The washed particles were suspended in 5 volumes of 10 mM Tris-HCl, pH 8.0 (based on initial tissue weight) and 4.0 M LiBr was added to produce a final concentra- tion of 0.4 M. The suspension was stirred gently for 4 to 5 hours. The resulting viscous suspension was diluted 4-fold with 10 IllM

Tris-HCl, pH 8.0 and centrifuged at 2,000 X g for 10 min. The sediment was washed with 10 volumes of this buffer (based on initial tissue weight) followed by centrifugation at 2,000 X g for 10 min. The LiBr-extracted particles were then suspended in 8 volumes of 25% KBr containing 10 InM Tris-HCl, pH 7.5 (based on initial tissue weight) and centrifuged at 25,000 x g for 30 min. More recently we have employed centrifugation at 7000 x g for 30 min. Unextracted fibers remained in the supernatant. The pellet, was washed with 8 volumes of 10 mM Tris-HCl, pH

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8.0 (based on initial tissue weight) and centrifuged at 2,000 x g for 10 min. This washing was repeated once more. The final sediment (sarcolemma) was suspended by use of a Potter- Elvehjem homogenizer in deionized water or 10 mM Tris-maleate, pH 6.0 to a protein concentration of 2 to 5 mg per ml. The preparations were used immediately for calcium binding and ATPase studies.

Sarcolemma were also isolated by the methods of Rosenthal et al. (14), McCollester (15), Thorpe and Seeman (IS), and Sulakhe ef al. (17).

Preparation of SarcGplasmic Reticulum-The procedure used was a modification of that described by Nakamaru and Schwartz (5). Rabbit muscle was homogenized in 6 volumes of 10 mM NaHC03 in a Sorvall Omni-Mixer for 30 s. The homogenate was filtered through a nylon sieve (pore size 1 mm2) and centri- fuged at 1,000 x g for 10 min. The sediment was washed by suspending in 4 volumes of 10 mM NaHC03 (based on initial tissue weight) and centrifuged at 1000 X g for 10 min. This was repeated once more. The combined supernatant fluid was centrifuged at 8000 X g for 20 min. The sediment was dis- carded. To the supernatant fluid, solid KC1 was added to pro- duce a concentration of 0.6 M. The mixture was left at 4” for 30 min with occasional stirring, then centrifuged at 40,000 x g for 1 hour. The sediment was suspended in deionized water or 10 mM Tris-maleate, pH 6.0 to a protein concentration of 2 to 5 mg per ml. The material was used immediately. In some instances the suspension was divided into small volumes and stored at -80” for up to 2 weeks.

Treatment of Muscle with Phospholipase C-Rabbit leg muscle (16 to 20 g) was very finely minced, divided equally into two flasks, and incubated in 12 ml of oxygenated (95% 02, 5% C02) Krebs-Ringer bicarbonate at 37” for 45 min. To one flask were added 6 mg of phospholipase C. The muscle from each flask was then washed thoroughly with Krebs-Ringer bicarbonate and sarcolemma and sarcoplasmic reticulum were isolated from both preparations simultaneously by the procedures described above. The preparations were assayed immediately for calcium binding and ATPase activity.

Assay of Calcium Binding or Accumulation--Membrane pro- tein (100 to 300 pg per ml) was incubated in a medium con- taining 50 mM Tris-maleate, pH 6.0, 5 mM MgC12, and 0.1 mM 45CaC12 (3,000 to 20,000 cpm per nmole) in the absence and presence of 0.5 or 1 mM ATP at 37” in a final volume of 1 or 2 ml. The reaction was started by the addition of ATP and was ter- minated by filtering a 0.5-ml aliquot through a Millipore filter (0.45 p, 25 mm) under light suction (18). The filter was washed with 5 ml of 100 mM Tris-maleate, pH 6.0, dried at 70” in a scin- tillation vial, covered with 8 ml of fluid containing 4 g of 2,5-di- phenyloxazole and 50 mg of 1,4-bis[2-(5-phenyloxazolyl)lben- zene per liter of toluene, and radioactivity was determined by liquid scintillation spectrometry. Appropriate controls con- taining no ATP, no MgC&, or no protein were included. Cal- cium binding was calculated from the specific activity of the added 4%aC12, and the activity retained by the membrane pro- tein.

For measurement of calcium accumulation, membrane pro- tein (about 135 pg per ml) was incubated in the medium de- scribed above containing either 5 mM potassium oxalate or phos- phate. Calcium binding and accumulation by sarcoplasmic reticulum was determined similarly.

.Ileasurement sf Calcium Release-Sarcolemma (about 500 pg of protein) was incubated at 37” in a medium containing 50 mM Tris-maleate, pH 6.0, 5 mM MgC12, 0.1 mM 45CaC12 (4500

cpm per nmole), and 50 PM ATP (final volume 1 or 2 ml). The reaction was started by the addition of ATP. At 30 s, 300 ~1 of the incubation mixture were filtered and washed with 3 ml of 100 mM Tris-maleate, pH 6.0. At 1 min, 100 ~1 of deionized Hz0 or agent dissolved in Hz0 were added. Additional ali- quots were then withdrawn at required intervals, filtered, and washed as above. At least 90% of the added ATP was hy- drolyzed after 1.5 min. The release of 4SCa was considered due to lack of ATP to support binding (see “Results”). Release of calcium from sarcoplasmic reticulum was studied using similar conditions except that ATP was 25 PM and 700 pg per ml of protein were used.

ATPase Assay-MgATPase, MgCaATPase, and CaATPase activities were determined by measuring liberation of inorganic phosphate following incubation of membranes with ATP in the presence of appropriate cations (13). Inorganic phosphate was determined by the method of Taussky and Shorr (19). Protein was determined by the method of Lowry et ~2. (20).

RESULTS

Preliminary

It was expected that 45Ca bound to membrane protein might be rapidly released especially upon depletion of ATP. It was therefore necessary to define precise conditions for washing of the Millipore discs following filtration of the incubation mix- tures. In a test experiment, 500~~1 aliquots of incubation mix- tures (containing 2.6 X lo5 cpm of 45CaC1J were washed (light suction) with 0.5, 2, 5, and 10 ml of 100 mM Tris-maleate, pH 6.0. Incubation mixtures consisted of (a) a control in which no protein was added (oxalate absent); (6) the complete binding system; and (c) the complete accumulating system (5 mM oxa- late). In the control (a), counts retained on the filter were 88, 60, and 43 after washing with 2,5, and 10 ml, respectively. Thus, negligible radioactivity was retained on the filters in the absence of protein. When oxalate was present (c), counts retained on the filter remained constant (2.3 X 104) after washing with 2, 5, or 10 ml of buffer. In the complete binding system (b), counts retained were 8500, 6800, and 5000 after washing with 2, 5, and 10 ml, respectively. Thus, binding in the absence of oxalate was fairly easily reversed and it was necessary to wash under carefully controlled conditions. A constant ratio (10 volumes) of washing buffer to aliquot filtered was maint’ained t,hrough- out. Although some loss was unavoidable, highly reproducible measurements of radioactivity retained on the filters could be obtained. Under the conditions of the assay, the membrane preparation bound significant amounts of Ca2+; binding was in- creased in the presence of ATP and Mg2+ (Table I). Potassium oxalate greatly stimulated Ca2+ uptake.

TABLE I

Calcium binding and accumulation activities of skeletal muscle sarcolemma and sarcoplasmic reticulum

Numbers in parentheses represent the number of preparations assayed.

I I Calcium binding or accumulation

I Sarcolemma Sarcoplasmic reticulum

nmoles ca*/mg prot&/30 s

-ATP.. . . . . . . . . . . . . . 3.10 % 1.8 (11) 6.80 f 0.95 (6) +ATP . . . . . . . . . . . . . . 18.36 f 1.58 (18) 202.55 & 6.65 (20) +ATP + oxalate.. . . . 189.9 f 11.3 (10) 2427 f 162 (8)

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In studies with sarcoplasmic reticulum, increased Ca2+ uptake in the presence of oxalate has been interpreted as accumulation of the cation in vesicular membranes (9). Oxalate is freely permeable and when Ca2+ concentration in the vesicles increases, calcium oxalate precipitates and the system becomes irreversible. The sarcolemmal membranes described here are fragile and aggregate tenaciously at the final step. Homogenization in a Potter-Elvehjem homogenizer with a tight fitting pestle was necessary to obtain a suspension suitable for accurate pipeting. Such preparations lose their microscopic appearance (empty tubular structures). Electron photomicrographs prepared from such suspensions revealed vesicle-like structures. The presence of membrane vesicles might thus account for the increased Ca2+ uptake in the presence of oxalate. In accord with the usual nomenclature we use the terrn “binding” to represent Ca2f up- take in the presence of ATP, and ‘LaccumuIation” to describe uptake when the assay system is supplemented with oxalate. Both energy-dependent binding and accumulation of sarcolemma were much lower than that of sarcoplasmic reticulum (Table

I).

Criteria of Purity of Xarcolemma

Since sarcoplasmic reticulum as well as mitochondria possess energy-linked Ca2f transport systerns, it was necessary to rig- orously examine the possibility that our membranes were con taminated with membrane material arising from these intracel- lular organelles. We reported earlier (10) that cytochrorne oxidase, acid maltase, acid phosphatase, and pyrophosphatase could not be detected in the preparation. The membrane frac- tion contained (Na+-K+)-stimulated ATPase, and acetylcholin- esterase (10) ; these enzymes are known to be present in muscle plasma membranes (17, 21-23). (Na+-II+)-ATPasel and ace- tylcholinesterase are reportedly absent from preparations of muscle sarcoplasmic reticulum (17, 24, 25). Adenylate cyclase, an enzyme found predominantly in plasma membranes of cells (1 I), was present in good yield (10). The lipid composition of the membrane preparation (10) (cholesterol to phospholipid ratio of 0.6) was also consistent with that of sarcolemma (26, 27).

A variety of additional experiments were conducted to examine the possible contamination of the membrane preparation with sarcoplasmic reticulum.

Inactivation of Sarcoplasmic Reticular Calcium Accumulation by Salt Trealment-The procedure for isolating sarcolemma employs extraction of a low gravitational sediment with 0.4 M LiBr. When isolated sarcoplasmic reticulum was similarly treated considerable loss in calcium sequestering activity oc- curred (Table II). Similar observations have been reported by Repke and Katz (28) for cardiac microsomal particles. In addition sedimentation of sarcoplasmic reticulum in 0.4 M LiBr required high gravitational forces (40,000 X g 60 min). No residue was obtained under conditions for sedimenting sarcolemma (2000 X g for 10 min). Furthermore isolated sar- coplasmic reticulum suspended in 25% KBr (density 1.21) could be sedimented only by centrifuging at 40,000 X g for 60 min. The sarcolemmal preparation sedimented easily at 25,000 X g (30 min) or even at 7,000 X g (30 min). KBr treatment of

isolated sarcoplasmic reticulum also led to considerable loss in calcium accumulating ability (Table II). These data would

indicate that any sarcoplasmic reticulum still present in the

1 The abbreviation used is: (Na+-K+)-ATPase, sodium and potassium ion-stimulated adenosine triphosphatase.

TABLE II

Inactivation of isolated sarcoplasmic reticulum bT/ LiRr and Kl1r

Experiment 1-20 mg of sarcoplasmic reticulum (from 10 g of muscle) were suspended in 50 ml of 10 mM Tris-HCl pH 8 and 4 M

LiBr were added to a final concentration of 0.2 or 0.4 M. The suspension was stirred at 4’ for 5 hours. After dilution 4-fold with Tris buffer, the material was sedimented by centrifugation at 40,000 X g for 60 min. The residue was washed with buffer, suspended in deionized water, and assayed for calcium accumula- tion. Experimend 11-15 mg of sarcoplasmic reticulum were SIIR- pended in 60 ml of 25y, KBr in 15 rnM Tris-HCl, pH 7.5, then cen- trifuged at 40,000 X g for 1 hour. Another sample was diluted to 10% KBr with Tris-HCl and centrifuged at 40,000 X g for 1 ho11r (40To of the original protein sedimented). The sediments were washed with Tris-HCl, suspended in deionized wat,er, and assayed for calcium accumlllating activity (oxalate present).

Salt treatment

Experiment I Control. 0.2 M LiBr.. 0.4 M LiBr..

Experiment II Control. . 10% KBr. 25% KBr

-

-

Calcium accumulation

30 s 2 min 5 min

1.87 3.43 4.05 0.90 1.78 2.75 0.70 1.42 2.43

1.59 3.25 3.18 0.35 0.42 0.49 0.18 0.43 0.60

pmozes ca2+/mg grotein

Fraction

Control homogenate

Homogenate plus sarco- plasmic reticulum

Yield

w/g lima 1.88

1.97

TABLE III

Calcium binding of sarcolemma from muscle supplemenled wilh sarcoplasmic reticulum

Isolated sarcoplasmic reticulum from 45 g of mllscle was added to homogenate prepared from 6.7 g of muscle. Another 6.7 g of muscle were homogenized as usual. Sarcolemma were isolated from both homogenates. Standard Ca2+ binding assay was used.

-~.

Calcium bound Assay

conditions 30 s 2 min

~___ nmoles caz+/ mg $roteilG

- MgATP 1.77 1.49 +MgATP 8.32 6.08 - MgATP 1.84 1.56 +MgATP 8.57 6.20

initial sediment after four washings should be effectively re- moved by the LiBr and KBr extractions. Furthermore the calcium accumulating activity of any remaining would be largely inactivated by the salt treatments (Table II).

Isolation of Xarcolemma from Muscle Homogenate Supplemented

with Xarcoplasmic Reticulum-Isolated sarcoplasmic reticulum from 45 g of muscle was added to an initial homogenate prepared from 6.7 g of tissue. Sarcolemma were then isolated from t,his and from a control homogenate prepared from 6.7 g of muscle. The ability of the two preparations to bind Ca2+ in the presence and absence of ATP is shown in Table III, and are seen to be identical. Since sarcoplasmic reticulum possesses high binding activity (200 nmoles per mg of protein per 30 s, Table I), one would have expected a much higher binding capacity in the sarcolemma isolated from the supplemented hornogenatc com- pared with the control if sarcoplasmic reticulum contaminated

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the final preparation. In an additional experiment, sarcoplasmic reticulum was added to a final sarcolemmal preparation. The mixture was sedimented at 2000 x g for 10 min and the residue was washed with Tris-HCl buffer in the usual manner. The pellet was suspended in buffer and this suspension and the com- bined supernatant fluid and washings were assayed for calcium binding and accumulating activity along with the control prep- aration of both sarcolemma and sarcoplasmic reticulum. The specific activity (nmoles of Ca2+ per mg) of the sarcolemma (2000 x g pellet) recovered was similar to that of the starting material (Table IV). Similarly the added sarcoplasmic re- ticulum was quantitatively recovered in the supernatant 5uid. Since the specific activity of the sarcoplasmic reticulum frac- tion added was much greater than that of the sarcolemma, one would have expected an increased specific activity of the latter fraction if contaminating sarcoplasmic reticulum were still present. That this did not occur attests to the effectiveness of the washing procedure and indicates t,hat the sarcolemma at the final stage of purity had no measurable ability to adsorb or entrap sarcoplasmic reticulum.

Effect of Phospholipase C-Martonosi (29) has reported that potassium release was greatly increased in frog sartorius and rat diaphragm after treatment of the muscles with phospholipase C. Calcium accumulation in microsomes isolated from phospholipase C-treated muscle was not different from those isolated from control muscles. The Ca2+ uptake of isolated sarcoplasmic reticulum, however, was greatly decreased after treatment with this enzyme (30, 31). Martonosi concluded that the effect of phospholipase C on potassium release resulted from an action on the surface membrane of the cell. These experiments sug- gested a means of selectively inactivating the Ca2+ accumulating activity of our membrane preparation. Minced rabbit muscle was incubated in the presence and absence of phospholipase C (see “Methods”) and sarcolemma and sarcoplasmic reticulum were isolated from both preparations. Calcium binding and accumulating ability and ATPase activities of these preparations were determined. The ability of sarcolemma from phospho- lipase C-treated muscle to bind and accumulate Ca2+ was reduced

by 75 to 87% (Table V), while activities of sarcoplasmic re- ticulum isolated from phospholipase C-treated muscle were sim- ilar to those from control tissue. In addition, MgATPase and MgCaATPase of the sarcolemma isolated from the phospho-

TABLE IV

Removal of added sarcoplasmie reticulum from isolated sarcolemma by washing

Isolated sarcoplasmic reticulum (5.6 mg) was added to 1 mg of isolated sarcolemma. The mixture was stirred at 4” for 15 min, then centrifuged at 2000 X g for 10 min. The sediment was sus- pended in Tris-HCl and the suspension and combined supernatant and washings were assayed for calcium binding and accumulation along with the initial fractions. Protein recovery of the sarco- lemma and sarcoplasmic reticulum fractions were 95 and 98oj,, respectively.

Calcium binding or accumulation

SaKOleUUIlt3 Sarcoplasmic reticulum

-0xa1ate +ox&Lte --O.-date +oxa1ate

?wdes C&/,g protein nmoles C&/mg potein

Initial.. 6.43 433 57 2423 Final. 5.78 462 46 2163

lipase C-treated muscle was reduced to 50% that of the control. These activities were not altered, however, in the sarcoplasmic reticulum fraction from phospholipase-treated tissue. These results, which are in agreement with those of Martonosi (29) suggested that the action of phospholipase C was selective for surface membranes and did not penetrate the cell to affect in- tracellular membranes. In additional experiments we observed that marked losses in calcium binding and accumulating ac- tivities of both isolated sarcolemma and sarcoplasmic reticulum occurred as a result of incubation with phospholipse C. These studies provide further evidence that our membrane preparation is of surface origin. All these studies together with those pre- viously reported (10) lead us to conclude that the membrane preparation consists of sarcolemma in a high degree of purity.

Several workers (32-34) have failed to observe energy-linked calcium binding by sarcolemmal fractions of skeletal muscle isolated by a variety of procedures. We have isolated sar- colemma from rabbit muscle by several methods (14-17) and have examined calcium binding and accumulation in these prep- arations (Table VI). The methods of McCollester (15) and

TABLE V Calcium binding and accumulating activity of sarcolemma and

sarcoplasmic reticulum isolated from control and phospholipase C-treated muscle

Phospholipase C treatment and isolation of membranes is de- scribed under “Methods.” Standard assay conditions were used.

I Calcium bound or accumulated

Assay conditions I

Sarcolemma Sarcoplasmic reticulum

+ATP. ................ +ATP + oxalate .......

twnoles/mg/30 s qo

180 2,000 I 1,900 i

150 13 5

Method of isolation Assay conditions

Rosenthal et al. (14)

McCollester (15)

Thorpe & Seeman (16)

Sulakhe et al. (17)

TABLE VI Calcium binding and accumulation by sarcolemma isolated by

various methods

Calcium binding or accumulation

- ATP + ATP

+ATP + Pi + ATP + oxalate

30 s I 2 min I 5 min

nndes C&+/mg protein

1.60 1.20 1.30 5.40 5.50 4.80 5.85 8.70 10.40

70.00 152.00233.00

- ATP 0.91 0.64 0.54 + ATP 1.84 1.97 1.53 + ATP + Pj 1.98 1.73 1.51 + ATP + oxalate 24.00 59.48 92.20

- ATP 1.68 1.35 1.55 + ATP 4.76 4.15 4.10 + ATP + Pi 7.80 6.24 5.20 + ATP + oxalate 18.00306.00476.00

- ATP 5.76 5.26 4.90 + ATP 6.23 5.30 5.20 + ATP + Pi 6.15 6.30 6.10 + ATP + oxalate 12.77 13.50 15.20

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Sulakhe et al. (17) yielded preparations with very low calcium frozen muscle (- 80” for 24 hours to 8 days) was only one-tenth accumulating activity, while those of Rosenthal et al. (14) and to one-fifth that of fresh muscle. Because of this, fresh mus- Thorpe and Seeman (16) were active, but less so than our present cle was routinely employed and all membrane preparations were preparation (see Table I). Long periods of extraction in the used immediately. In contrast, Ca2+ binding activity of sar- presence of high salt concentrations as well as differences in the coplasmic reticulum isolated from frozen tissue is fully as active methods for initial homogenization may be the reason for these as that from fresh tissue, and isolated sarcoplasmic reticulum preparations possessing low activity. can be stored at -80” for months with only negligible loss in

binding capacity (35).

Binding and accumulation of Cazq in sarcolemma isolated from

Properties 05 Sarcolemmal Ca2+ Binding System

Xtability-The calcium binding and accumulating activity of muscle sarcolemma. were labile. Preparations were stored at

4”, -2O”, and -80” for 24 hours and the activities were com- pared to those obtained immediately after preparation. The loss in Cazf binding activity on storage at 4”, -2O”, and -80” was 60, 50, and 40%, respectively; the loss in Ca2f accumulating activity (oxalate oresent) was 90, 72. and 55% respectively.

-

Effect of Time on Ca

assay method used had distinct limitations.

2i Binding and Accumulation-Calcium

Even

binding by sarcolemma was a rapid process. It is seen in Fig. 1 that binding was complete in 30 s at 23”. In this experiment ATP was 5 mM, a concentration which reduced maximal bind- ing (compare with Table I). Addition of precipitating agents such as phosphate and especially oxalate greatly increased CaZf uptake and this accumulation did not reach maximal levels for up to 5 min. Because of the rapidity of the binding process, the

though

I 2 3 4 5% LJ Mlll”kS ATP jmh4)

FIG. 1 (top). Effect of time on calcium binding and accumula- tion. The reaction was carried out directly in the Millipore filter assembly at 23’; sarcolemmal suspension (190 fig of protein) was used and the ATP concentration was 5 mM, final volume 0.5 ml; otherwise standard assay conditions were used: 0, no ATP; l , + ATP; A, + ATP + 5 mM potassium phosphate; A, + ATP + 5 ITIM potassium-oxalate. The final pH in each case was 6.0. At the times indicated suction was applied; filtration was completed in 2 s.

FIG. 2 (bottom left). Effect of ATP on Ca* binding and accumula- tion. A, standard binding assay conditions were used (540 rg of protein) except the final volume was 3 ml and ATP concentration (mm) was varied as indicated by the numbers on each curve. At the times indicated 500-&l aliquots were filtered. B, standard

1 co2+ Mx@ Mg2+(mMl

SARCOLEMMA SARCOLEMMA

binding (0) and accumulation (+ oxalate) (0) conditions were used except ATP concentration was varied. For sarcoplasmic reticulum, 100 and 10 pg of protein were used in the absence and presence of oxalate, respectively, For sarcolemma, 480 and 240 pg of protein, respectively, were used.

FIG. 3 (bottom right). Effect of Ca2+ and Mgz+ concentration on Ca* binding and accumulation. A, standard binding assay was employed (450 pg of protein) except that WaC12 was varied as shown and ATP was present at the concentrations (mM) indicated by the numbers on each curve. Values are corrected for binding in the absence of ATP at each Ca* concentration. B, standard binding (0) and accumulation (5 mM oxalate) (0) conditions were used except that Mg2+ was varied.

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filtration of the sample required only 2 s (excluding washing), it was not possible to perform velocity measurements with pre- cision. However, the maximal amount of Ca2+ bound remained constant for several minutes, or declined only slightly (see Fig. 2), so that total binding could be determined accurately. Rou- tinely, an incubation period of 30 s at 37” was employed, and when accumulation was measured, incubation periods were extended to several minutes.

E$ect of ATP, Ca2+, and Mg2+-The effect of several con- centrations of ATP on Ca2+ binding is shown in Fig. 2A. At all concentrations tested, binding was complete in 30 s, thereafter, the amount of bound cation decreased slightly. This decline was especially noticeable at 170 pM ATP, probably a reflection of substrate depletion. Calcium binding increased with in- creasing ATP concentration up to about 1.7 mM. Higher con- centrations (8.33 InM) were inhibitory. This could be due to chelation of Ca2+ by ATP or to a direct effect on the calcium binding system. An attempt to estimate the ATP concentra- tion for half-maximal binding and accumulation is given in Fig. 2B, and compared with that of isolated sarcoplasmic reticulum. Isolated sarcolemma and sarcoplasmic reticulum possessed sim- ilar dependency on ATP for Ca2+ binding, and accumulation, the concentration for half-maximal binding for sarcoplasmic reticulum tended to be lower (about 5 PM) than that for sar- colemma which was approximately 20 /.hM.

The effect of Ca2+ concentration on binding in the presence of several fixed concentrations of ATP is shown in Fig. 3A. At each ATP concentration half-maximal binding occurred at a Ca2f concentration of 20 pM. Although ATP affected total binding (high concentrations (5 mM) significantly reduced the maximum, see also Fig. l), affinity for binding sites remained the same. Concentrations of Ca2+ above 500 PM greatly re- duced binding. Energy-dependent binding and accumulation were increased by Mg2+ (Fig. 3B). Half-maximal binding and accumulation by sarcolemma occurred at about 0.15 and 0.5 mM

I SARCOLEMMA

2

SARCOPLASMIC RETICULUM 1

Incubation (Min.)

FIG. 4. Effect of phosphate donors on Ca* binding and accu- mulation. Standard assay conditions were used except that ATP (grey bars) was replaced by acetylphosphate (hatched bars) and p- nitrophenylphosphate (solid bars). In the absence of oxalate, sarcolemma (309 pg of protein) bound 10.2 nmoles Ca* per mg at 30 s, while in the presence of oxalate, the values were 481 and 794 nmoles per mg at 2 and 10 min, respectively. Sarcoplasmic retic- ulum (250 rg of protein) bound 210 nmoles Ca* per mg of protein; in the presence of oxalate (50 pg of protein) 780 and 2109 nmoles per mg were bound at 2 and 20 min, respectively.

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Mg2+, respectively. Half-maximal binding and accumulation in sarcoplasmic reticulum occurred at Mg2+ concentrations of 20 PM and 0.15 mM, respectively. In sarcolemma, Mg2f could be replaced by Mn2+ or Co2+, but these ions were only 40 to 70% as effective.

E$ect of Phosphate Donors Other than A TP-When substituted for ATP, acetyl phosphate or p-nitrophenyl phosphate sup- ported calcium binding by sarcolemma very poorly (Fig. 4). In contrast, these high energy phosphate compounds could partially replace ATP to support Ca2+ binding and accumula- tion in sarcoplasmic reticulum. This latter result is in agree- ment with other workers (36-38). Both sarcolemma and sar- coplasmic reticulum possess p-nitrophenylphosphatase activity which is stimulated by low Ca2+ as well as by K+ (13).

E$ect of pH--Sarcolemmal Ca 2f binding and accumulation possessed a pH optimum of 6.0 (Fig. 5). Binding and accumula- tion decreased precipitously at pH values above this, and at pH 8.0 were barely measurable. This pH profile was very similar to that for Ca2+ binding in sarcoplasmic reticulum (Fig. 5) (5, 39).

Action c$ La3+ on Calcium Binding and Accumulation-Several investigators have used Las+ to displace bound Ca2+ in a variety of systems (4&42). Entman et al. (43) reported that La3+ did not affect Ca2f binding by cardiac microsomes. As shown in Fig. 6, Las+ was a powerful inhibitor of ATP-dependent binding in sarcolemma at concentrations greater than 100 PM. At 0.5 mM La3f calcium binding was inhibited 90%. Calcium accumu- lation was significantly inhibited at, 10 PM La3+ and was reduced by 60% at 1 mM La3+.

Release of Cu2+-Calcium release from the loaded sarcolemmal membranes was studied in an assay system containing 50 PM ATP (see “Methods”). Under these conditions it was found

A.

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FIG. 5. Effect of pH on Ca2f binding and accumulation in sar- colemma and Ca* binding in sarcoplasmic reticulum. Standard assav conditions were used except the pH (Tris-maleate buffer) was “varied as shown. Ordinate 2, Ca*- binding in sarcolemma: comolete svstems (0): -ATP (0). Ordinate B, Ca* binding in &k~pla.&ic reti&&: complete system (0); LATP (m). -Or- dinate C, Ca* accumulation (+ oxalate) in sarcolemma (A).

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FIG. 6 (left). Inhibition of Ca* binding and accumulation by Las+. Standard assay conditions were used and La3+ was added at the concentrations indicated (387 fig of protein). Complete binding system (0); -ATP (0); accumulating system (+ oxa- late) (A).

FIG. 7 (right). Demonstration of Cazf release from sarcolemma. Assay conditions are described under “Methods” (final volume of 3 ml). Initial ATP concentration was 50 pM. At the arrows, 100 ~1 of water or ATP (0.37 InM) final concentration were added. Ad- ditional aliquots (300 11) were then filtered at the times indicated.

that Ca2f binding measured at 30 s rapidly decreased during the next 4 to 6 min (Fig. 7). R.elease from bound sites was con- sidered due to depletion of energy source (ATP) since addition of extra ATP (arrows, Fig. 7) reversed the reaction and delayed the decrease. It was found that several agents altered the rate of release. For example: Na+, K+, and Mg2f significantly in- creased the rate (Fig. 8A). In contrast, Las+ inhibited the relea,se of bound Ca2+ in a concentration dependent fashion (Fig. 8C). Maximum inhibition occurred at 0.5 to 1.0 mM and was actually observable at 7 FM La”+. Calcium release from sarcoplasmic reticulum was similarly affected by these ma- terials; thus, Na+, K+, and Mg zf increased the rate (Fig. 8B). Caffeine and procaine increased Ca2+ release at 11 mM. Lan- thanum up to 1.25 rnM was inhibitory. Sucrose up to 500 rnnt had no significant effect on Ca2+ release.

DISCUSSION

The study provides evidence for the presence of an BTP- dependent Ca2+ binding system in muscle plasma membranes. Marker enzyme activities, lipid composition, and phase con- trast microscopic studies reported earlier (10) suggested that the membrane preparation constituted plasma membrane in a high degree of purity. The present work supports this view. Addi- tion of excess sarcoplasmic reticulum to homogenates did not alter the yield or specific activity of Ca2+ binding activity of the isolated sa.rcolemma. Isolated sarcolemma was easily re- covered after mixing with excess sarcoplasmic reticulum. Such experiments demonstrated the effectiveness of the extraction and washing procedures used. Phospholipase C treatment of the muscle homogenate provided further evidence that the mem- branes isolated were surface in origin. Unfortunately no reli- able enzyme marker for sarcolemma is available. 5’-Nucleo- tidase, which has been used as a plasma membrane marker in liver was almost exclusively soluble in rabbit muscle homogenates (10). No evidence exists that (Na+-K+)hTPase is confined to plasma membrane, and accurate determination of this activity in crude muscle homogenates is made difficult by the fact that K+ and Na+ produce a relatively small stimulation above the relatively large MgATPase activity. Precise determination of the over-all yield is therefore difficult. Isolation of sarcolemma containing an active Ca*+ binding system and adenylate cyclase and seemingly free of sarcoplasmic reticulum is probably de-

. . . &CL SARCOLEMMA 8. SARCOLEMMA

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FIG. 8. Effect of various agents on release of Ca* from sar- colemma and sarcoplasmic reticulum. A, sarcolemma (442 pg of protein) was incubated in the standard release assay. Reaction was started by the addition of ATP, 50 PM. At 30 s, a 0.2-ml ali- quot was filtered and washed with 2 ml of buffer. At 1 min, 0.1 ml of Hz0 or agent in Hz0 was added. At the times indicated 0.2-ml aliquots were filtered and washed. Ca* binding at 30 s was taken as maximum (16 nmoles Ca* per mg of protein). After 1.5 min, 9570 of the added ATP was hydrolyzed. Additions were: I-I20 (0); caffeine or procaine, 7.4 mM (0); KCl, 74 mM (0); NaCI, 74 mM (m) ; MgC12,37 mM (a); and ethylene glycol his@-amino ethyl- ether)-N,N’-tetraacetic acid, 3.7 mM, (A). B, sarcoplasmic reticular protein (770 pg of protein) was used. Conditions were as for A, except ATP concentration was 25 KM. Symbols are t,he same as for A; caffeine and procaine were 11 InM, NaCl and KCl, 110 mM, MgClz, 56 mM, and ethylene glycol his@-amino ethyl- ether)-N,N’-tetraacetic acid, 0.56 mM. C, Experiment 1 (---), sarcolemmal prot,ein (550 rg) was treated as in A at 1 min, 0.1 ml of Hz0 or La3+ at the final concentration indicated was added. Aliquots were filtered at 2 min (0) and at 5 min (A). Experiment 2 (- - -), sarcolemmal protein (1.32 mg) was used. Aliquots were filtered at 2 min (0 ) and at 5 min (A). CaZC bound at 30 s was taken as 100% and represented 16 and 9 nmoles of Ca*+ per mg of protein in Experiment 1 and Experiment 2, respectively.

pendent mainly on the following: (a) relatively short homogeni- zation in 50 mM CaClz (4”), preventing irreversible contracturc

of the fibers (15) ; (b) thorough washing of the low gravitational sediment with hypotonic buffer aiding in removal of Ca2f and emptying of the fibers; cc) extraction with LiBr at low tcm- peratures for short periods of time; and (d) the use of low gravita- tional forces throughout the procedure to separate the mcn- branes from other particulate material.

Although there are some differences between the C’a2+ binding systems of sarcolemma and sarcoplasmic reticulum such as sta- bility, and specificity with respect to phosphate donor, the two systems are similar in many respects. Similarities iucludc r(l- sponse to oxalate or Pi, PI-I dependency, pattern of Ca2+ release, affinities for ATP and Ca2f, and MgZ+ and Ca2f dependency of ATPase (13). Quantitatively, the sarcoplasmic reticular cal- cium pump is much more active than that of sarcolemma. Wc cannot rule out the presence of transverse tubules in our mem- brane fraction; nothing is known about these structures in trrms of biochemical activities. In studies not reported in detail wc have estimated that sarcoplasmic reticulum, mitochondria, and sarcolemma comprise about 80, 10, and lo%, respectively, of the total calcium accumulating capacity of a muscle homogcnatc. These estimations were based on certain assumptions: azide-

sensitive Ca2+ accumulation was considered due to mitochoudria

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(9), while oxalate was considered to increase accumulation in both sarcoplasmic reticulum and sarcolemma (Table I) (2). Total Ca2+ accumulation by myofibrils (80 mg per g of muscle) accounted for about 1% of the total homogenate capacity; oxa- late was considered to have no effect on myofibrillar Ca2+ bind- ing. Briggs and co-workers (44) have used a similar approach to estimate the content of sarcoplasmic reticulum in frog mus- cle. We feel that the rapidity of binding and release of Ca2+ as well as the affinity for this cation are in accord with a phys- iological role of sarcolemma in calcium transport during excita- tion-contraction coupling. The presence of ATPase and its stimulation by low concentrations of Ca2+ point to a possible link between calcium transport and ATP hydrolysis analogous to that of sarcoplasmic reticulum (3).

Among the actions of cations examined, those of La3+ are noteworthy. Numerous investigators have observed that this cation inhibit,s calcium efflux from a variety of muscle prepara- tions (41, 42, 45-47). Our studies suggest a possible mechanism for these effects. Perhaps La 3+ binds to the sites occupied by Ca*+ rather tightly, thereby blocking the channels for Ca2+ permeability or exchangeability. Furthermore, La3+ can be

recognized by Ca2f binding sites since we have observed that La3+ can substitute for Ca2+ to activate MgATPase of sarcolemma and sarcoplasmic reticulum (13). Caffeine is known to produce contracture of muscle and this is believed to be due to an effect on Ca2+ release from sarcoplasmic reticulum (1). Similarly, local anesthetics produce contracture and high con- centrations caused release of Ca2+ from muscle. Recently, Thorpe and Seeman (48) have observed release of Ca2+ from sarcolemma by procaine and Ca 2+ displacement, by local an- esthetics has been reported by Feinstein and Paimre (49). It is not clear whether or not these agents interfere with the Ca2+ binding system in sarcoplasmic reticulum or sarcolemma or both. In t’he present study these agents had a greater effect on Ca2+ rcleasc from sarcoplasmic reticulum.

The presence of a Ca 2+ binding system in plasma membrane is not unique to skeletal muscle. Energy-dependent Ca2f trans- port mechanisms have been recently described in plasma mem- branes from red cells (47, 50), fibroblasts (51), and intestinal smooth muscle (52). Whether or not this represents a general property of plasma membranes or a special control site in the process of excitation contraction coupling remains to be elucidated. The studies support the view that sarcolemmal membranes possess active transport systems for Ca2+ as well as for Xa+ and K+. With the presence of adenylate cyclase, transport ATPase, and acetylcholinesterase, the sarcolemma is further recognized as a dynamic structure in cellular metabolism.

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Prakash V. Sulakhe, George I. Drummond and David C. NgCalcium Binding by Skeletal Muscle Sarcolemma

1973, 248:4150-4157.J. Biol. Chem. 

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