Components of purified sarcolemma from porcine skeletal muscle

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 242, No. 1, October, pp. 112-126, 1985

Components

of Purified Sarcolemma JAMES

Department

R. MICKELSON2

from Porcine Skeletal Muscle’

AND

CHARLES

F. LOUIS

of Vekknmy

Biology, University of Minnesota, 295 Animal Science/ Veterinary Medicine Building, 1988 Fitch Avenue, St. Paul, Minnesota 55108 Received

January

15, 1985, and in revised

form

May

13, 1985

Sarcolemmal membranes were isolated from porcine skeletal muscle by modifications of a LiBr-extraction technique. Latency determinations of acetylcholinesterase, ouabainsensitive p-nitrophenylphosphatase, [3H]ouabain binding, and (Na+ + K+)-ATPase activities indicated that 6576% of the membranes were sealed inside-out vesicles. The preparations were enriched in cholesterol and phospholipid, and demonstrated adenylate cyclase activity and both CAMP and cGMP phosphodiesterase activities. An indication of the purity of this fraction was that the Ca2+-ATPase activity (0.13 pmol Pi mg-’ min-’ at 37°C) was 3.8% of that of porcine skeletal muscle sarcoplasmic reticulum preparations. Pertussis toxin specifically catalyzed the ADP-ribosylation of a iV, 41,000 sarcolemmal protein, indicating the presence of the inhibitory guanine nucleotide regulatory protein of adenylate cyclase, Ni. An endogenous ADP-ribosyltransferase activity, with several membrane protein substrates, was also demonstrated. The addition of exogenous CAMP-dependent protein kinase or calmodulin promoted the phosphorylation of a number of sarcolemmal proteins. The calmodulin-dependent phosphorylation exhibited an approximate Klj2 for Ca2+ of 0.5 PM, and an approximate affinity labeling of the sarcolemma, Kl12 for calmodulin of 0.1 PM. ‘%I-Calmodulin using dithiobis(succinimidy1 propionate), demonstrated the presence of M, 160,000 and 280,000 calmodulin-binding components in these membranes. These results demonstrate that this porcine preparation will be valuable in the study of skeletal muscle sarcolemmal ion transport, protein and hormonal receptors, and protein kinasecatalyzed phosphorylation. o 1985 Academic press, I~C.

The plasma membrane of cells performs many functions, including the generation and maintenance of ion gradients, the

transport of metabolites, and the transduction of hormonal signals. Many of these activities, however, have not been well characterized in the sarcolemma (SL),3 or

i This work was funded, in part, by grants from the Muscular Dystrophy Association, the Minnesota Swine Center, and NIH GM-31382. A preliminary report of some of this material has already appeared [J. R. Mickelson, E. M. Gallant, and C. F. Louis (1984) Biophys J. 45, 79a]. * Postdoctoral Fellow of the Muscular Dystrophy Association. To whom correspondence should be addressed.

0003-9861/85 Copyright All rights

$3.00

0 1985 by Academic Press. Inc. of reproduction in any form reserved.

3 Abbreviations used: SDS, sodium dodecyl sulfate; EGTA, ethylene glycol bis (@-aminoethyl ether)N,N’-tetracetic acid; Gpp(NH)p, 5’guanylyl imidodiphosphate; DSP, dithiobis(succinimidy1 propionate); CAMP-PK, CAMP-dependent protein kinase; SR, sarcoplasmic reticulum; LiBr-SR, LiBr-treated SR, SL, sarcolemma; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid.

112

COMPONENTS

OF

SKELETAL

surface membranes of skeletal muscle cells. One reason for this lack of information appears to be the difficulty in obtaining purified, sealed SL vesicles that possess the necessary sidedness properties to be useful for transport studies. The most frequently used technique for skeletal muscle SL isolation is based on the initial extraction of broken fibers with a LiBr solution, followed eventually with final purification by sucrose gradient centrifugation (l-5). SL membranes prepared in this manner have been very useful for the study of (Na+ + K+)-ATPase (l-6) and adenylate cyclase activities (7-g), and the characterization of the acetylcholine receptor (3, 5, 10) and the sodium channel (11, 12). However, as yet the orientation of these vesicles, and their usefulness for transport studies has not been determined. We report here the use of a modification of the LiBr-extraction technique for the isolation of porcine skeletal muscle SL vesicles. The preparation is characterized with regard to its purity, composition, and vesicular orientation, and we demonstrate its suitability for enzymatic, ion transport, and phosphorylation studies that require highly purified SL preparations. EXPERIMENTAL

PROCEDURES

Materials. All pigs were obtained from the University of Minnesota Experimental Farm, and all reagents were of reagent grade or of the highest purity available from Sigma Chemical Company. 45CaCl,, NamI, HeeaPO,, and [aH]ouabain were from New England Nuclear. ol-=P- and y-=P-labeled nucleoside triphosphates and q-labeled cyclic nucleotides were prepared by the methods of Walseth and Johnson (13). [32PlNAD was prepared from [a-q@TP by the method of Cassel and Pfeuffer (14). Calmodulin was prepared from bovine testes by the method of Gopalakrishna and Anderson (15), and was radioiodinated with Enzymobeads from Bio-Rad as described by Louis and Jarvis (16). Purified toxin from Bordate& pertussis was the generous gift of Dr. Lubert Stryer and Dr. Greg Yamanaka, Stanford University School of Medicine, and alamethicin was the gift of Dr. Joseph Grady, The Upjohn Company. CAMPdependent protein kinase (Beef Heart) was purchased from Sigma, and DSP was obtained from Pierce.

MUSCLE

SARCOLEMMA

113

Isolation of skeletal muscle membranes. Animals were anesthetized with intravenous infusion of sodium thiamylal, after which the muscles were rapidly excised and placed in ice. SL was then prepared from the longissimus dorsi muscle (SO-90% Type II fibers) (1’7) by a modification of the methods of Schapira et al (2) and Barchi et al (5). All procedures were performed at 0-4°C. One hundred grams of muscle was minced with scissors and homogenized in 500 ml of 250 mM sucrose, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, by three 5-s bursts in a Waring blender. The homogenate was centrifuged at 2OOOg for 10 min and the resulting pellet was resuspended in 500 ml of 0.4 M LiBr, 20 mM Tris-HCI, pH 8.5. The pH was readjusted to 8.5 and the suspension was stirred slowly for 16 h. This viscous slurry was centrifuged at 1OOOg for 15 min, followed by centrifugation of the resultant supernatant at 100,OOOg for 30 min to yield a crude membrane pellet. The pellet was then resuspended in 50 ml 0.6 M KCI, 20 mM Tris-HCI, pH 8.0, by eight strokes in a loose-fitting Dounce homogenizer and centrifuged at 10,OOOg for 10 min. This supernatant was retained while the pellet was then extracted twice more with the KCITris medium. This repeated washing of the crude membrane fraction increased the final yield of purified membranes threefold. The three resultant supernatants were combined and centrifuged at 100,OOOg for 30 min. The pellet was resuspended in 0.6~ KCl, 20 mM Tris-HCl, pH 8.0, and further purified by differential centrifugation between 10,OOOg (10 min) and 100,OOOg (30 min). The final 100,OOOg pellet was resuspended in distilled water and centrifuged at 100,OOOg for 30 min. This pellet was resuspended in a small volume of water, the volume was measured, and an equal volume of 57% sucrose (w/v), 20 mM histidine, pH 7.0, was slowly added. This suspension, transferred to centrifuge tubes and overlaid with 15% sucrose, 10 mM histidine, pH 7.0, was centrifuged at 150,000g for 90 min in an anglehead rotor. The band which formed at the 28.5-15% sucrose interface was collected, diluted with 4 vol of water, and centrifuged at 100,OOOg for 30 min. This pellet was resuspended in a minimal volume of 10% sucrose, aliquoted into small vials, frozen in liquid nitrogen, and stored at -70°C. Sarcoplasmic reticulum was prepared from the longissimus dorsi by the method of Kirehberger et al. (18). Chemical determinations on membranes. Protein was measured according to Lowry et al (19). Phospholipids were extracted by the method of Bligh and Dyer (20), and the total phosphorus was measured by the procedure of Chen et aL (21). Cholesterol and its esters were determined enzymatically (22) with a Sigma Chemical Company total cholesterol kit. Enzymatic actititti. Total (Na+ + K+)-ATPase was

114

MICKELSON

measured in 100 mM NaCl, 10 mM KCl, 40 mM histidine, 0.1 mM EGTA, 5 mM MgCla, 5 mrd ATP, pH 7.0, at 37°C. Ouabain-sensitive (Na+ + K+)ATPase was defined as that activity inhibitable by 1 mM ouabain. (Caa+ + Mga+)-ATPase activity was measured in 120 mbi KCl, 40 mM histidine, pH 7.0, 5 mM MgCla, 5 mM ATP, 0.1 rnre CaCla, at 37’C. Mg+ATPase activity was defined as that activity obtained in the absence of CaClc and the presence of 1 mM EGTA. Car+-ATPase activity was defined as the difference between the two activities. Acetylcholinesterase was determined at 22°C by the spectrophotometric method of Steck and Kant (23), using acetylthiocholine as substrate. K+-activated pnitrophenylphosphatase activity was determined at 37°C by the method of Bers (24), and is reported as that activity inhibitable by 1 mM ouabain. Ouabain binding, both (Mga+ + Pi)-dependent (overnight equilibration) and ATP dependent, were measured by the technique of Mitchell et al. (25). To determine enzyme latency and vesicle sidedness sarcolemmal membranes (0.3 mg/ml) were preincubated in varying concentrations of SDS for 20 min at room temperature, in 20 mM histidine, pH 7.0. Adenylate cyclase activity was measured in 40 mM Tris, pH 7.5, 10 mM KCl, 5 mM MgCla, 0.2 mM ATP (containing 200,000 cpm [cy-q]ATP), 1 mM isobutylmethylxanthine, 1 mM dithiothreitol, 1 mM CAMP, 2 mM creatine phosphate, 0.1 mg creatine kinase/ml, 25 fig myokinase/ml, 30 pg membrane protein, in a volume of 100 pl at 37°C. Gpp(NH)p-activated activities increased with incubation time [see also Ref. (26)]; therefore, a single time point of 30 min was chosen. The [aaP]cAMP produced was purified from the reaction mixture by the method of Jakobs et al. (27). Cyclic nucleotide phosphodiesterase activity was determined at 37°C in 3 mM MgCle, 2 mM dithiothreitol, 20 mM Tris, pH 8.0, at substrate concentrations of 1 or 50 PM, containing =P-labeled cyclic nucleotides. 5’-Nucleotidase was used to convert the q-labeled nucleoside monophosphate product to “Pi, and charcoal was used to remove remaining unhydrolyzed cyclic nucleotides (13). Membrane phosphorylationa Membranes (100 pg protein) were phosphorylated in 20 mM Hepes, pH 7.0, 0.1 mM ATP (containing 10 &i [T-~P]ATP) in a final volume of 100 ~1. Concentrations of MgCla, CaCle, EGTA, CAMP, CAMP-dependent protein kinase, and calmodulin are described in the figure legends. Reactions proceeded for 1 min at 20°C and were terminated by the addition of 1% SDS. Pertussis taxin-catalyzed ADP-m’bosylatim The transfer of the [=P]ADP ribose moity from [=PwAD to sarcolemmal proteins was determined by modifications of previous methods (28,29). A 50-~1 reaction

AND

LOUIS

volume containing 50 wg membrane protein, 50 mM Tris, pH 7.5, 100 mM NaCl, 20 mM KCI, 1 mM dithiothreitol, 2 mM EDTA, 10 pM NAD (containing 10 PCi [32PjNAD), 10 mM thymidine, 1 mM ATP, and 1 pg pertussis toxin was incubated at 30°C; the reactions were terminated by the addition of 1% SDS. The pertussis toxin was activated by incubation for 30 min in 50 mM dithiothreitol, 50 mM Tris, pH 7.5, at 30°C immediately prior to use. A$inity lob&w with mI-calwudulin The technique of Louis and Jarvis (16), utilizing DSP, was used to chemically crosslink calmodulin to membrane receptor proteins. The reaction mixture contained 20 mM Hepes, pH 7.0, 1 mg membrane protein/ml, 0.4 pM ‘251-calmodulin, and varying concentrations of EGTA, CaC12, and MgClc. The reaction was initiated by the addition of 0.2 mM DSP in dimethyl sulfoxide (1% final), and was incubated for 30 min at 20°C before termination with 1 ml of 0.1 M glycine, pH 7.0. To remove unreacted ‘%I-calmodulin, the samples were centrifuged at 100,000~ for 20 min. The pellets were then resuspended in 1% SDS prior to electrophoretic analysis. Electrophoretic analysis. Samples were electrophoresed on 5-20% gradient polyacrylamide gels in the presence of 0.1% w/v SDS (30). Gels were stained with Coomassie blue and dried, and the location of radioactively labeled proteins was identified by autoradiography. Electron microscopy. Membrane pellets were fixed in 2.5% glutaraldehyde, 100 mM sodium phosphate, pH 7.4, on ice overnight. The pellet was then stained with 1% OsO*, 50 mM sodium phosphate, pH 7.4, for 2 h, after which it was dehydrated and embedded. Negative staining of intact vesicles was by the procedure of Lampe et al. (31). Samples were placed on carbon-coated grids which had been treated by glow discharge. They were then stained with 1.5% uranyl acetate. RESULTS

Characterization of Skeletal Muscle Sarcolemma Preparations: Enzymatic Activities, Vesicle Sidedness, and Lipid Content When the unmodified LiBr-extraction technique was applied to the isolation of SL from porcine skeletal muscle, we achieved only limited success and very low yields. However, with the modifications to this procedure that we have now adopted (Experimental Procedures), it has proven possible to obtain very reproducible

COMPONENTS

OF

SKELETAL

preparations, with yields of 2-4 mg SL protein/100 g muscle. To confirm that the isolated porcine muscle SL membranes are in vesicular form, an electron microscopic examination of glutaraldehyde-fixed samples was performed. Figure 1A shows that in a glutaraldehyde-fixed membrane pellet the preparation is relatively homogeneous, consisting mainly of closed-vesicular membranes. With negative staining of intact preparations the vesicular nature of the SL preparations is also readily apparent (Fig. 1B). The mean diameter of the vesicles, as calculated from micrographs of the negatively-stained preparations, was 0.20 + 0.04 pm (SE). Although Fig. 1 suggests that the membranes are present in vesicular form, it does not indicate their sidedness or extent of sealing. To assess the relative proportion of inside-out, right-side-out, and leaky vesicles in our preparations, we determined the latency of several activities following preincubation of the vesicles with varying concentrations of SDS (25, 32-35). SDS preincubation renders sealed membranes permeant, thus allowing pas-

FIG. 1. Electron micrographs of porcine prepared as described under Experimental (B) negative staining of intact vesicles.

MUSCLE

SARCOLEMMA

115

sage of previously impermeant substrates through the disrupted membrane. Figure 2A shows that when either acetylcholinesterase or ouabain-sensitive K+activated p-nitrophenylphosphatase activity was determined at increasing levels of SDS during preincubation, latent activity was unmasked, reaching a maximum in the range of 0.40-0.50 mg SDS/mg protein, and declining thereafter. Figures 2B and C show a similar effect of SDS preincubation on phosphorylation-dependent ouabain binding and on (Naf + K+)ATPase activity, respectively. The latter figure also shows that the ouabain-insensitive, or Me-ATPase, activity was insensitive to SDS preincubation. As utilized previously by other investigators (25,32-35), our estimates of vesicle sidedness are based on various assumptions. First, that the maximal or total activity, expressed upon SDS preincubation, is the sum of all inside-out, rightside-out, and leaky vesicles. Second, that although SL acetylcholinesterase is clustered at the motor end plates (less than 1% of the total SL area), the entire surface membrane contains acetylcholinesterase

skeletal muscle sareolemma Procedures. (A) Section

membranes. Samples were through a membrane pellet;

116

MICKELSON

AND

LOUIS

FIG. 2. Latency of sarcolemmal activities. Varying concentrations of SDS were added to sarcolemma suspensions (0.3 mg protein/ml) that were then incubated at room temperature for 20 min. Different activities were then measured as described under Experimental Procedures. Bars represent fl SD from the mean of six different preparations. (A) 0, ouabain-sensitive K+activated p-nitrophenylphosphatase; W, acetylcholinesterase. (B) (Mga+ + Pi)-dependent ouabain binding. (C) (Na+ + K+)-ATPase: n , total (Na+ + K+)-ATPase; 0, Mga+-ATPase (in the presence of 1 mM ouabain).

activity, the majority of which faces the extracellular space (36, 37). Third, the ATP hydrolytic site of the (Na+ + K+)ATPase faces the cytosol, while the K+phosphatase (controversial at present) and ouabain-binding sites face the extracellular space (38, 39). Therefore, without prior SDS preincubation, acetylcholinesterase and ouabain-sensitive p-nitrophenylphosphatase activities are an indication of combined right-side-out and leaky vesicles. Also, without detergent treatment,

ouabain-inhibition of (Na+ + K+)-ATPase activity would mainly be a result of leaky membranes, as the ATP or ouabain requirement on either side of the membrane could not be met with sealed vesicles. In all cases the percentage of inside-out vesicles is obtained by subtracting the values for right-side-out plus leaky percentages from the total latent activity expressed upon SDS preincubation. Mitchell et al. (25) have described a technique whereby ouabain binding, which

COMPONENTS OF SKELETAL MUSCLE SARCOLEMMA

requires (Naf + K+)-ATPase phosphoenzyme formation, can be used to distinguish between right-side-out and leaky vesicles. When ATP is used to phosphorylate the enzyme (ATP-dependent), patent ouabainbinding is referable only to leaky membranes, where both sides are accessible to the substrates during the assay. However, by overnight equilibration with Mg2f and Pi, phosphoenzyme is formed from Pi regardless of membrane orientation. In this case, patent ouabain-binding measures combined right-side-out and leaky vesicles. Therefore, the percentage of right-side-out vesicles would be equal to the patent (MS + Pi)-dependent ouabain binding minus the ATP-dependent ouabain binding, as a fraction of the total binding capacity in the presence of SDS (25). Table I provides the necessary data from which to estimate the sidedness of the porcine SL preparations, showing that the results obtained by all four methods are very similar. The table indicates that 65-76s of the membranes are sealed inside-out vesicles, with the remainder being leaky or unsealed. The equivalence of the patent ATP-dependent and (MS+ + Pi)dependent ouabain-binding data (Table I), and the lack of alamethicin stimulation of adenylate cyclase activity (see below), indicate the absence of right-side-out SL vesicles (25). Monensin and valinomycin, which dissipate the ion gradients formed by the (Na+ + Kf)-pump, stimulate the (Na+ + K+)-ATPase activity of sealed SL (33) and transverse tubule vesicles (25). The addition of either 1 pM monensin or 1 pM valinomycin also stimulated the ouabainsensitive (Na+ + K+)-ATPase of the porcine SL preparations 26 and 210%) respectively. These data further indicate the existence of sealed vesicles which are capable of generating ion gradients. Table II compares the lipid content of the SL and SR preparations obtained from porcine skeletal muscle. It is evident that the SL contains significantly more cholesterol (17 times) and phospholipid (3.5 times) per milligram of protein than does

117

the SR. The high cholesterol content of the SL relative to the SR is reflected also in the 4.5-fold difference in the mole ratio of cholesterol to phospholipid. Electrophoretic Analysis, Ca’+-ATPase Activity, and an Estimation of SR Contamination of Sarcolemmal Preparations

The major potential contaminant in our SL preparation was likely to be the SR. Figure 3 shows a comparison of electrophoretically fractionated SL and SR proteins, indicating that the protein compositions of these membranes are quite dissimilar. The most prominent protein in the SR is the 102-kDa Ca’+-ATPase (40). A band of similar relative mobility is present in the porcine muscle SL, albeit in greatly reduced proportions. The extent of SR contamination of the SL preparations was determined by measuring Ca’+-ATPase activity. The SR preparations used for comparison were a crude SR fraction, sedimenting between 10,000 and lOO,OOOg, which was extracted with 0.6 M KC1 (18), and LiBr-treated SR, which was prepared from crude SR, processed according to an SL isolation procedure (Experimental Procedures). The sucrose gradient centrifugation step for LiBr-SR was omitted, however, because preliminary experiments demonstrated the lack of any SR or LiBr-SR material banding at the 28.5-15s sucrose interface (as SL does). Table III shows that whereas the Ca’+-ATPase of SR was 3.45 pmol Pi mg-l min-l, the value for SL was only 0.13 pmol Pi mg-’ min-‘; LiBr-treated SR retained nearly 50% of its Ca2+-ATPase. If the Ca2+-ATPase present in the SL fraction were due entirely to SR contamination, it can be calculated that at most SR would represent 0.13 f 3.45, or 3.8% of the total protein. Similarly with the LiBr-SR value, 0.13 t 1.62, or 8.0% of the protein would be contaminant. This represents a maximal estimate of SR contamination and does not account for any Ca2+-ATPase which was endogenous to the SL.

118

MICKELSON

AND TABLE

LOUIS

I

ACTIVITIES OF PORCINE SKELETAL MUSCLE SARCOLEMMA' Percentage Patent activity

Total activity

IS0

L + RSO

L

(Na* + K+)-ATPase* No addition + Ouabain Ouabain sensitive

18.1 e3.4 3.6 fl.O 14.7 +3.6

48.8 f 9.1 3.5 k 1.2 45.8 + 8.2

66.3 f 4.7

33.7 f 4.7

-

Acetylcholinesterase”

62 +ll

186 f40

65.0 f 6.0

35.0 + 6.0

-

12.1 f 1.7

50.6 + 1.8

75.7 e4.0

24.3 k4.0

19.7 k3.6 19.9 + 5.9

59.9 2 8.2

70.7 k 7.2

29.3 27.3

Ouabain-sensitive phosphatased

K+-

Ouabain binding” ATP-dependent (Mgz+ + Pi)-dependent

30.0 k 3.3

‘Patent activity was determined without SDS preincubation, while total activity was the maximal activity following SDS preincubation. IS0 (inside out) is the percentage of the total activity unmasked by SDS, or (total-patent)/total; L + RSO (leaky + right side out) is equal to patent/total. “Means + SD of 10 preparations, expressed in micromoles Pi formed per milligram protein per hour at 3’7°C. Activity was determined in the presence or absence of 1 mre ouabain; ouabain-sensitive (Na+ + K+)ATPase was the difference between the two activities, for each preparation. ‘Means + SD of six preparations, expressed in nanomoles acetylthiocholine hydrolyzed per milligram protein per minute at 22°C. d Means + SD of six preparations, expressed in nanomoles pnitrophenol formed per milligram protein per minute at 37°C. e Means +- SD of six preparations, expressed in picomoles ouabain bound per milligram protein at 37°C.

Adenylate Cyclase Activity Presence of Ni

and the

Table IV indicates the presence of a high-activity adenylate cyclase in the porcine skeletal muscle SL preparation. Basal adenylate cyclase activity was stimulated 3-fold by the nonhydrolyzable GTP analog, Gpp(NH)p. The activation is further enhanced (2.5-fold) by the simultaneous addition of the ,8-adrenergic agonist, isoproterenol, which by itself stimulated adenylate cyclase only slightly. Sodium fluoride also greatly stimulated basal adenylate cyclase activity (approximately 8fold), as did forskolin, although to a lesser extent (approximately 5-fold). Alamethicin has been shown to stimulate adenylate cyclase activity of cardiac

muscle SL preparations by promoting entry of substrates into sealed vesicles (35). No enhancement of adenylate cyclase acTABLE LIPID

II

CONTENT OF PORCINE SKELETAL MUSCLE MEMBRANES’ SL

Cholesterol (bgug/mgprotein) Phospholipid (pg P/mg protein) Cholesterol/ phospholipid (mole ratio) ‘Mean

SR

254 f25

15 2 10

78.1 + 7.8

21.5 + 3.0

0.261 + 0.025

0.057 f 0.029

f SD of 10 SL and 4 SR preparations.

COMPONENTS SR

SL

SR

SL

IO 20

25 50

25 50

x) 100

4’

OF

SKELETAL

ilitJ MOLECULAR .a* em WEIGHT -205

_

K

-

116K 97K

-

66K

-

45

-

29K

-

20K

K

14 K

FIG. 3. Electrophoretic analysis of porcine skeletal muscle membranes. SDS-polyacrylamide gel electrophoresis and Coomassie blue staining were as described under Experimental Procedures. The sample size, in micrograms of protein applied, is given above each lane. The positions of molecular weight markers are indicated on the right.

tivity was seen when alamethicin was preincubated with skeletal muscle SL in the range O-l.5 mg/mg protein (data not shown). This result may reflect the absence of sealed right-side-out vesicles in the preparations. The ability of pertussis toxin to catalyze the ADP-ribosylation of the a! subunit of the inhibitory guanine nucleotide regulatory protein of adenylate cyclase, Ni, has been used to demonstrate the presence of this component in plasma membranes (29, 41,42). Figure 4 shows that when skeletal muscle SL membranes are incubated with [=P]NAD in the absence of pertussis toxin, several proteins were ADP-ribosylated (Lane 2). This basal ADP-ribosylation is probably due to the high endogenous ADPribosyltransferase activity that has been identified in this membrane (43). However, in the presence of pertussis toxin and [32P]NAD, there was a specific labeling of a 41-kDa protein, which increased with time of incubation (Lanes 3-5). This 41-kDa component, which has the same M, as the (Y subunit of Ni in other tissues (29, 41, 42), was correlated with a minor

MUSCLE

119

SARCOLEMMA

Coomassie blue-staining protein in electrophoretograms of SL membranes (data not shown). Phosphodiesterase Sarcolemma

Activities

in

Table IV also shows the presence of various forms of cyclic nucleotide phosphodiesterase activity in the skeletal muscle SL. The data indicate a CAMP phosphodiesterase which was relatively active at 1 PM CAMP. At a substrate concentration of 50 PM, this CAMP hydrolysis was further stimulated by 1 PM cGMP. A cGMP phosphodiesterase activity was also present in the SL preparation. In comparison, the activity at 1 PM cGMP was low, while at 50 PM cGMP it was 1% fold greater. The addition of 1 mM EGTA, or 25 PM CaC12 in the presence or absence of 0.5 PM calmodulin had no effect on any phosphodiesterase activity (data not shown). Protein

Kinase

Substrates in Sarcolemma

A preliminary experiment, in which SL was phosphorylated in the presence of [T-~~P]ATP, demonstrated that in the absence of Me no phosphorylation of SL membrane components was observed (data not shown). Figure 5 shows that when Mg2+ alone (Lane l), or Mg2+ and CAMP TABLE Car+-ATPase

III

ACTIVITY OF PORCINE MUSCLE MEMBRANES ’ SL

Mg=+-ATPase (Mg*+ + Ca’+)ATPase Cal+-ATPase

SR

SKELETAL

LiBr-SR

0.07

kO.04

0.14

kO.09

0.33

f 0.13

0.20 0.13

k 0.06 kO.05

3.59 3.45

kO.85 f0.86

1.96 1.62

kO.53 +0.63

“Mean k SD of 10 SL and 4 SR preparations, expressed in micromoles Pi formed per milligram protein per minute at 37°C. Ca*+-ATPase was defined as the difference between the Mg*+-ATPase and the (Mg*+ + Ca’+)-ATPase activity of each individual preparation.

120

MICKELSON TABLE

ADEN~LATE CYCLASE PHOSPHODIESTERASE

SKELETAL

AND CYCLIC ACTIVITIES

MUSCLE

Adenylate cyclasea Basal activity +50 w GPP(NH)P +10 PM Isoproterenol +50 PM Gpp(NH)p PM Isoproterenol +lO mM NaF +lO PM Forskolin

IV NUCLEOTIDE OF PORCINE

SARCOLEMMA

4.4 + 1.4 13.3 + 3.5 5.6 k2.0 + 10 34.2 34.3 20.8

+ 8.5 + 7.3 + 9.0

CAMP phosphodiesterase* 1 pM CAMP, basal activity +l /AM cGMP 50 pM CAMP, basal activity +l ELM cGMP

0.073 0.071 0.162 0.230

f + + f

cGMP phosphodiesterase* 1 /LM cGMP 50 PM cGMP

0.021 0.382

f 0.002 + 0.026

0.013 0.017 0.037 0.053

“Means + SD of 10 preparations, expressed in nanomoles CAMP formed per milligram protein per 30 minutes. Activity was determined at 37°C in the basal incubation medium as described under Experimental Procedures, or one supplemented with the listed activators. *Means f SD of six preparations, expressed in nanomoles cyclic nucleotide hydrolyzed per milligram protein per minute. Activity was determined at 37°C in the basal incubation medium, containing cyclic nucleotide at either 1 or 50 pM, as described under Experimental Procedures.

AND

LOUIS

substrates for CAMP-PK, with many of them having M, similar to SL substrates (26,500, 66,000, 125,000, and 160,000). The major CAMP-PK substrate of the SR, with M, 95,000, was greatly reduced in the SL. The presence of an endogenous calmodulin-dependent protein kinase in the skeletal muscle SL is indicated in Fig. 6. In the presence of Mg2+ alone (Lane l), MS+ plus calmodulin (Lane 2), Mgs+ plus Ca2+ (Lane 3), or Ca2+ plus calmodulin (Lane 5), little phosphorylation of any SL component was apparent. However, Lane 4 demonstrates that when both MS+ and Ca2+ were present, calmodulin stimulated endogenous protein kinase activity such that several SL proteins were phosphorylated. The M, of the major substrates were 21,500, 27,500, 66,000, 102,000, 125,000, 160,000, and 245,000. SR proteins were also phosphorylated in the presence of Ca2+, M$+, and calmodulin (Lanes 7-12). Again, several SR substrates had M, sim-

MOLECULAR WEIGHT

w 205K-

m

rr-

*

Il6K-

+45K36K-

(Lane 2) were present, proteins with iV, of 16,500 and 66,000 were the major phosphorylated components. In the presence of M$+, CAMP, and increasing amounts of CAMP-dependent protein kinase (CAMP-PK) the phosphorylation of proteins with llf, of 26,500, 42,000, 66,000, 125,000,160,000, and 245,000 was increased (Lanes 3-5). Lane 6 indicates that the phosphorylation of these components was reduced, but not abolished, in the absence of added CAMP, possibly due to SL adenylate cyclase activity. Lane 7 demonstrates the autophosphorylation, and position of the added CAMP-PK alone. Skeletal muscle SR (Lanes 8-13) also possessed

123456

em **-

14K-

FIG. 4. Autoradiograph of pertussis toxin-catalyzed ADP-ribosylation of skeletal muscle sarcolemma. Reactions were performed, and samples were analyzed electrophoretically, as described under Experimental Procedures. Lane 1, control, contained pertussis toxin but not SL; 60-min incubation. Lane 2, control, contained SL but not pertussis toxin; 60min incubation. Lane 3, SL and pertussis toxin; 15min incubation. Lane 4, 30-min incubation; Lane 5, 60-min incubation. Lane 6, control, in addition to SL and pertussis toxin contained 300 j&M NAD; 30-min incubation. The positions of molecular weight markers are indicated on the left.

COMPONENTS

OF

2

3

4

56

7

8

9

lOllI

MOLECULAR WEIGHT 205KIl6K96K-

*--a-

45K-

MUSCLE

121

SARCOLEMMA

The effect of varying either the calmodulin concentration (Lanes 1-7) or the free Ca2+ concentration (Lanes 8-X), on the SL calmodulin-dependent protein kinase activity, is shown in Fig. 7. From these data we estimate that in the presence of 10 IIIM MgClz and 0.1 InM total CaClz, O.l0.3 PM calmodulin was sufficient for maximal phosphorylation, while in the presence of 10 mM MgClz and 1 PM calmodulin, 0.48-1.0 yM free Ca2+ was sufficient.

SR

SL I

SKELETAL

29K-

A&nity 14Kh4gC12 CAMP PK

Labeling

of Sarcolemma

with

+

+ + +++c +

+

+

+

+

+

+

+ + +

+

+ I. ++++ +

+

+

+

-I

+

+

FIG. 5. Autoradiograph of CAMP-dependent protein kinase-catalyzed phosphorylation of skeletal muscle membranes. Phosphorylations and electrophoretic analyses were as described under Experimental Procedures. The presence of additional compounds during the reaction is indicated at the bottom of each lane. When present, MgC& was 10 maa; CAMP was 5 pM; SL or SR was 1 mg/ml; CAMP-PK was 0.01 mg/ml (Lanes 3 and lo), 0.05 mg/ml (Lanes 4 and ll), or 0.10 mg/ml (Lanes 5,6, 7,12,13); EGTA was present in all reactions at 1 mM. The positions of molecular weight markers are indicated on the left.

An autoradiogram obtained when either SR or SL were incubated with CaClz, calmodulin, and the M&lz , lzr’I-labeled crosslinking agent, DSP, is shown in Fig. which 8. The presence of ‘%I-calmodulin, remained bound to the membranes but was not crosslinked, was seen at M, 17,000. Lanes l-6 show that calmodulin became

SL 7

MOLECULAR WEIGHT

8 9

101112

*.

205K-

ilar to SL phosphoproteins (M, 21,500, 27,500, 66,000, 102,000, and 245,000). Figure 5 also shows that the relative levels of =P incorporation into SL proteins catalyzed by the Caz+-calmodulin-dependent kinase were much greater than those catalyzed by the CAMP-PK-dependent process (compare Lanes 4 and 6). Table V confirms this finding by reporting the level of phosphate incorporation into individual SL proteins. Basal values ranged from 1 to 17.8 pmol P/mg protein, but were increased, in most cases by three to sixfold, by CAMP-PK or the endogenous calmodulin-dependent protein kinase. Proteins of M, 66,000, 125,000, and 160,000 are of particular interest in that they were major substrates of both kinases, demonstrating approximately additive incorporations when simultaneously phosphorylated by both kinases.

SR

123456

i

I IbK97K66K-

a

45K-

2

a

-

z 14w -

---

ugaz

+

EGTA

+ +

CtlCl2

COhOd.

CAMP PK

l

+

l +

+

l

+

+

+

l

l

+

+

t

+ +

+ c

+

+

+

+I

+

+ l

l

+ +

FIG. 6. Autoradiograph of protein kinase-catalyzed phosphorylation of skeletal muscle membranes. Phosphorylations and electrophoretic analyses were as described under Experimental Procedures. The presence of additional compounds during the reaction is indicated at the bottom of each lane. When present, MgClz was 10 mM, EGTA was 1 mEd, CaCl, was 0.1 mM, calmodulin was 1 PM, CAMP was 5 PM, and CAMP-PK was 0.10 mg/ml. The positions of molecular weight markers are indicated on the left.

122

MICKELSON

AND TABLE

PHOSPHOPROTEIN

Jfr 340,000 245,000 160,000 125,000 102,000 66,000 49,000 42,000 27,500 26,500 21,500 16,500

Basal 1.4 2.6 9.7 4.7 4.9 10.6 3.6 5.9 5.7 5.7

+0.2 +O.l kO.1 f 0.1 f0.8 + 0.1 +0.5 +0.1 kO.8 f0.8

1.0 20.1 17.8 k4.5

LEVELSIN

3.0 5.5 19.0 15.4 9.0 18.0 5.6 8.8

V

SKELETAL

+5 PM CAMP and 0.1 mg CAMP-PK/ml ic 0.2* f 0.4* f 4.4* *4.1* + 2.5* + 1.8* +0.2* f 1.6*

26.3 + 5.9* 3.3 + 0.8* 25.8 + 7.7

LOUIS

MUSCLE

+O.l

SARCOLEMMA~

mM

CaCIa

and 1 pM calmodulin 12.7 17.6 34.1 14.8 27.4 29.8 13.1 14.8 21.8

+ 2.s**** + 2.8**** f 5.9**** f 2.2* + 6.3*~** + 4.3**** f 0.8**** f 2.5*~** f4.9* -

24.6 + 5.3*!** 23.0 + 7.6

+5 PM CAMP, 0.1 mg CAMP-PK/ml, 0.1 mM CaC&, and 1 UM calmodulin 14.0 19.0 41.2 24.8 29.6 37.4 14.9 16.1

+ + f f + f f f

4.3**** 3.9**** 9.6**** 7.1*,*** 6.7**** 4.2****~*** 1.4**** 3.8*~** -

25.6 f 6.6*~** 29.4 + 10.6

“The mean of determinations on three different SL preparations expressed in picomoles P per milligram SL protein + SD. SL membranes were phosphorylated for 1 min at 20°C, in the basal medium as described under Experimental Procedures with the inclusion of CAMP-PK or calmodulin as listed in the headings. After electrophoretic analysis and exposure of the autoradiogram, the radioactive bands were located and cut out, and the radioactivity was determined by scintillation counting. * Different than basal, P < 0.05. ** Different than CAMP plus CAMP-PK, P < 0.05. *** Different than CaCla plus calmodulin, P < 0.05.

covalently crosslinked to several SL components in a Ca’+-dependent manner. The reaction also required Mg2f, with 10 mM MgClz being more effective than 1 mM MgC& (Lanes 4-6). The il!, of the two major crosslinked calmodulin complexes were 180,000 and 300,000. Subtracting the size of calmodulin gives an estimate for the M, of approximately 160,000 for the most prominent and 280,000 for the less prominent SL calmodulin-binding components. In contrast to the SL, SR (Lanes ‘7-12) showed virtually no labeling under identical conditions. DISCUSSION

The LiBr-extraction technique, with various modifications, has been used by several groups of investigators to obtain SL membranes from the skeletal muscle of various species. These preparations have been appropriate for various types

of studies and a high degree of purity has been documented (1-12). However, the important aspect of vesicle sidedness, as well as the functions of most of the many skeletal muscle SL proteins, have not been determined. A comparison of the properties of SL preparations from other species, that have been obtained in different laboratories, is presented in Table VI. All are based on a LiBr technique except for the method of Seiler and Fleischer (33). When comparing values of other LiBr-derived SL preparations, the patent ouabain-sensitive (Na+ + K+)-ATPase activities (11-29 pmol Pi mg-’ h-l) are comparable to our value (15 pmol Pi mg-’ h-‘, Table II). The high latent (Na+ + K+)-ATPase activity (45.8 pmol Pi mg-’ h-‘) reported for our preparation is the first rigorous determination of the latency of this activity performed on LiBr-SL. Also, our MS+-ATPase activ-

COMPONENTS MOLECULAR WEIGHT

OF

- iwqj -cuim

SKELETAL

g#Z”Jt: I)(rlr*

205K-

JYh

116K97K-

i-#W8

66K45K29K,4K-

--

‘0 DI D3 .I .3 c

CALMOUJUN FM

I 3 +

0 .I3 2aa t

FRE

lin2s34.6100 CALCIUM PM

--,

FIG. 7. Autoradiograph of calmodulin-dependent protein kinase-catalyzed phosphorylation of skeletal muscle sarcolemma. Phosphorylations and electrophoretic analyses were as described under Experimental Procedures. In Lanes l-7, CaCla was 0.1 mM and calmodulin concentrations were as indicated below each lane. In Lanes 8-15, calmodulin was 1 PM and ionized Ca2’ concentrations were as indicated. In all reactions MgCla was 10 mM. The positions of molecular weight markers are indicated on the left.

ity (total minus ouabain-inhibitable, Table VI), of 4 pmol Pi mg-’ h-‘, is for the most part much lower than that reported previously (range of 4-29 prnol Pi mg-’ h-l). This could either reflect the inactivation of this labile activity during the isolation procedure (44, 45), or indicate low levels of T-tubule contamination (46). Seiler and Fleischer (33) have recently reported a different method for the preparation of SL from rabbit skeletal muscle. It did not depend on the LiBr extraction, but instead utilized a 0.75 M KC1 extraction for a shorter length of time. Table VI shows that, in general, the rabbit skeletal muscle SL preparation of Seiler and Fleischer is very similar to the porcine muscle SL, although a greater proportion of the rabbit SL vesicles appear to be sealed [(33), and footnote 8 of Ref. (25)]. The accompanying paper describes our use of the sealed inside-out nature of the majority of vesicles in the porcine preparations (65-76%, Table I) to study aspects of ATP-dependent calcium transport in skeletal muscle SL (48).

MUSCLE

123

SARCOLEMMA

Electrophoretic analysis of porcine muscle SL demonstrates the presence of a great variety of proteins, in contrast to SR (Fig. 3). It also indicates, however, that, as in other reports (4, 8, 33, 46), a protein with a similar M, to the SR Ca’+ATPase is present in surface membrane fractions. The identity of this component in SL is still unknown, but it is not likely to be simply an indication of SR contamination. Actual measurements of Ca’+ATPase activity in the SL fraction demonstrates that if all the Ca2+-ATPase is assumed to be due to SR, then the maximal SR contamination represents only 3-B% of the total protein, depending on the SR fraction used for comparison (Table III). Recently, the presence of the stimulatory guanine nucleotide regulatory complex of adenylate cyclase, N,, was demonstrated in skeletal muscle SL. This was done by directly labeling the 45-kDa (Y subunit with the use of [32P]NAD and cholera toxin (49). We have utilized the ability of pertussis toxin to specifically

SL MOLECULAR WElGhT

SR

123456

7

s

9

IO II

12

89 9

205K-

“d

Il6K97K-

17KCoCl2

m

__^^

(mM)

0

0

0 0.1 0.1 01

yc12(InM)

0

I

10

0

I

IO

0 0

0 I

0 0.1 0.1 0.1 IO 0 I IO

FIG. 8. Autoradiograph of skeletal muscle membranes affinity-labeled with ‘2SI-calmodulin. Crosslinking conditions and electrophoretic analyses were as described under Experimental Procedures. The concentrations of CaCl, and MgCla present in each reaction are indicated below each lane; when CaCl, was 0, EGTA was present at 1 mM. The positions of molecular weight markers are indicated on the left.

MICKELSON

AND

LOUIS

catalyze the ADP-ribosylation of the 41kDa (Y subunit of Ni (28, 29, 41, 42) to demonstrate the presence of this inhibitory guanine nucleotide regulatory protein in the skeletal muscle SL (Fig. 4). The specific role Ni plays in the regulation of muscle adenylate cyclase activity is unknown, however, for as yet neither an inhibitory receptor nor inhibitory agonists have been identified. An interesting aspect of these experiments was the confirmation of a high endogenous ADP-ribosyltransferase activity, also of unknown function, in the SL preparations (43). In this study we have reported molecular weights of individual protein substrates for exogenous CAMP-dependent protein kinase (Fig. 5), and an endogenous calmodulin-dependent protein kinase (Figs. 6 and 7). Although phosphorylations of SL membranes have been demonstrated in previous studies (50-52), usually only an increase in total 32P incorporation has been determined. We have shown that the SL calmodulin-dependent kinase requires micromolar Ca” concentrations, and nanomolar levels of added calmodulin (Fig. 7), similar to the calmodulin-dependent protein kinase activity present in cardiac muscle membranes (53,54). However, also of interest is the report of the association of phosphorylase kinase with rabbit muscle T-tubules (54). The phosphorylation of membrane proteins by this endogenous kinase was Ca2+ plus calmodulin stimulatable but, in contrast to the SL, was not absolutely dependent on calmodulin. It is possible, though, that the ikf, 125,000 SL substrate for protein kinases is actually the (Y subunit of phosphorylase kinase (55), and that phosphorylase kinase is the calmodulin-dependent protein kinase present in the SL preparations. Furthermore, SL proteins of M, approximately 27,000 and 66,000 were major substrates for protein kinases, similar to SR [(54-59), and Figs. 5 and 61. Clearly, further work is required to examine the similarity and significance of the distribution of protein kinase substrates among the different muscle membrane preparations.

COMPONENTS

OF

SKELETAL

Chemical crosslinking of ‘%I-labeled calmodulin to SL membranes with DSP demonstrated the presence of calmodulinbinding proteins with M, of 160,000 and 280,000 (Fig. 8). The formation of these components, which were not present in SR, was dependent on the presence of both Ca2+ and M$+. The possibility that the M, 160,000 component is the plasma membrane Ca’+-ATPase, which in erythrocytes has a M, of approximately 140,000 (60), is a topic of ongoing investigation in our laboratory. We conclude that highly purified SL vesicles, of predominantly inside-out orientation, can be obtained from porcine skeletal muscle by modifications of the LiBr-extraction technique. These preparations have made possible the demonstration of SL proteins which are substrates for CAMP-dependent and calmodulin-dependent protein kinases, as well as ADP-ribosyltransferases. The identification of the Ni complex cyclic nucleotide phosphodiesterases, and calmodulin-binding components confirms the usefulness of these membranes in the study of SL structure and function. ACKNOWLEDGMENTS The authors thank Dr. Tim Walseth and Dr. Nelson Goldberg ^. for invaluable assistance in the preparation of q-labeled nueleotide; Dr. Esther Gallant for constructive advice throughout the course of this work, Dr. Paul Lampe and Dr. Ross Johnson for assistance with electron microscopy; and Dr. Bill Rempel for providing experimental animals. We also wish to thank Linda Inman, Jackie Johnson, and Jane Sprangers for their excellent typing services.

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MUSCLE

SARCOLEMMA

125

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