The Regulation of Ca2' Transport by Fast Skeletal Muscle Sarcoplasmic Reticulum ROLE OF CALMODULIN AND OF THE 53,000-DALTON GLYCOPROTEIN*

Ca2+ uptake and Ca2+-dependent ATP hydrolysis of fast skeletal muscle sarcoplasmic reticulum (SR) are strongly inhibited by trifluoperazine ('I"). Inhibition, which is Ca2+-dependent, is 90% with 14 p~ TFP and 0.2 p~ Ca2+. TFP interacts strongly, in a Ca2+-dependent way, with two SR proteins, calmodulin and the 53,000-dalton glycoprotein. The two proteins were purified by TFP affinity chromatography. The inhibition of SR activity by TFP was correlated with the interaction of the drug with the glycoprotein, rather than with calmodulin. The main effect was a shift of the (Ca2'-M&')- ATPase from a high to a low affinity form. Calmodulin-dependent phosphorylation of three proteins (M, = 57,000, 35,000, and 20,000) of the SR mem- brane of fast skeletal muscle was also demonstrated. Phosphorylation of these three proteins plays no role in the regulation of the active Caz+-uptake reaction. The effects of CAMP and calmodulin on Ca2+ transport in cardiac SR' are mediated by specific protein kinases that phosphorylate a minor hydrophobic protein component of the SR membrane, phospholamban Erythrocyte (5) and sarcolemmal (6) Ca2'-ATPases, on the other hand, are directly stimulated by the binding of calmodulin to high affinity sites on the enzyme.

Ca2+ uptake and Ca2+-dependent ATP hydrolysis of fast skeletal muscle sarcoplasmic reticulum (SR) are strongly inhibited by trifluoperazine ('I"). Inhibition, which is Ca2+-dependent, is 90% with 14 p~ TFP and 0.2 p~ Ca2+. TFP interacts strongly, in a Ca2+-dependent way, with two SR proteins, calmodulin and the 53,000dalton glycoprotein. The two proteins were purified by TFP affinity chromatography. The inhibition of SR activity by TFP was correlated with the interaction of the drug with the glycoprotein, rather than with calmodulin. The main effect was a shift of the (Ca2'-M&')-ATPase from a high t o a low affinity form.
Calmodulin-dependent phosphorylation of three proteins (M, = 57,000, 35,000, and 20,000) of the SR membrane of fast skeletal muscle was also demonstrated. Phosphorylation of these three proteins plays no role in the regulation of the active Caz+-uptake reaction.
The effects of CAMP and calmodulin on Ca2+ transport in cardiac SR' are mediated by specific protein kinases that phosphorylate a minor hydrophobic protein component of the SR membrane, phospholamban (1-4). Erythrocyte (5) and sarcolemmal (6) Ca2'-ATPases, on the other hand, are directly stimulated by the binding of calmodulin to high affinity sites on the enzyme.
The mechanism of Caz+ uptake by fast skeletal muscle SR preparations, due to their relatively simple protein composition and to the high level of specialization in transport function, has been intensively studied. Little is known, however, of the physiological mechanism(s) for the regulation of this powerful Ca2+ pump, in addition to the obvious regulation induced by the occupation of ATP-, M e -, Ca2+-binding sites. Several minor protein components (calsequestrin, a high affinity Ca2'-binding protein, a 53,000-dalton glycoprotein, and proteolipids) have been found associated with SR vesicles of fast muscles. The physiological role of these components is still largely a matter for speculation, but it is conceivable that some of these components are involved in a regulatory process. Recent work on the 53,000-dalton glycoprotein has shown it to be present in a constant ratio with the ATPase protein in all SR preparations tested (from light to heavy SR fractions) * This work was supported by Grant 3,634-0.80 from the Swiss Nationalfonds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
(7). For this reason, the proposal was made that this glycoprotein, in analogy to the subunit glycoprotein of the plasma membrane (Na'-K+)-ATPase complex might be involved in the regulation of the SR Ca2'-ATPase (7). Alternatively, it was suggested that it might represent an anion carrier (8). In this report, the investigation of some of the minor protein components of fast skeletal muscle SR was extended, and their possible role in the regulation of the Ca2+ pump was explored. In particular, the 53,000-dalton glycoprotein could be isolated in a pure state by affinity chromatography. The results indicate that the transport activity of fast SR is regulated by a glycoprotein-dependent process. They also demonstrate that calmodulin stimulates the phosphorylation of a number of protein components of SR membranes via an endogenous protein kinase.

MATERIALS AND METHODS
TFP and CAPP were generous g&s of Dr. Carl Kaiser of Smith, Kline and French Laboratories. CNBr-activated Sepharose 4B was obtained from Phannacia, Uppsala, Sweden. All other reagents were of the highest purity available. Bovine brain (9) and testis (IO) calmodulin were prepared as described.
Membrane Preparations-SR was isolated from rabbit white leg muscles according to the procedure described in Ref. 11 and from Canadian lobster tails as described in Ref. 12. Erythrocyte ghosts depleted of calmodulin were isolated as described in Ref. 5. All preparations were stored at -80 "C, where they were stable for several weeks.
Affinity Chromatography Columns-Calmodulin was conjugated to CNBr-activated Sepharose 4B by the procedure described previously (5). CAPP-Sepharose 4B resin was prepared basically as described by Jamieson and Vanaman (13).
Assays-Active Ca2+ uptake by SR vesicles was determined using the Millipore filtration technique (14) and radioactively labelled "CaC12. 20-50 pg of SR protein/ml were incubated for 2 min at room temperature in the presence of 20 mM MOPS, 7.0,80 mM KCI, 5 mM MgC12, 0.5 mM ''CaCl2, and EGTA to yield the desired free Ca" concentration. The uptake reaction was started with 1 mM ATP, and aliquots were filtered and washed with a cold medium containing 10 m~ EGTA as described (15).
The ATPase activity was tested at 37 "C either by measuring the rate of Pi liberation as reported by Stewart (16) or by the coupled enzyme assay (17). The basic reaction mixture contained 20 mM MOPS, pH 7.0, 80 mM KCI, 5 mM MgC12, and Ca-EGTA buffers. When Pi liberation was measured, the assay medium contained 20-50 pg of SR protein/ml and 5 mM oxalate. Otherwise, the reaction mixture was supplemented with 2 p~ A23187, 0.5 RIM phosphoenolpyruvate, 1 IU/ml of pyruvate kinase, 1 IU/ml of lactate dehydrogenase, 0.2 m~ NADH, and 5-10 pg of SR protein/ml. The reaction was started with 1 mM ATP. When required, SH vesicles were preincubated in the reaction mixture for 2 min with TFP before the addition of ATP.
The passive permeability characteristics of SR were tested after overnight incubation of the vesicles in a medium containing 200 mM KCl, 5 mM MgCI2,20 mM MOPS, pH 7.0, and either 5 mM 45CaCl? or 10 mM ["Cloxalate. The protein concentration was 10-15 mg/ml. Efflux rates were measured with the filtration technique after a 20fold dilution of the suspension at room temperature in 200 mM KCI, 5 mM MgCL, 20 mM MOPS, pH 7.0. When measuring oxalate efflux rates, the dilution medium was supplemented with either 10 p~ CaCL or 100 p~ EGTA. When required, SR vesicles were preincubated for 2 min with 28 p~ TFP and then diluted in a medium also containing 28 p~ TFP. The phosphoenzyme intermediate steady state level was measured as previously described (15). Essentially, 100-150 pg of SR protein/ ml were incubated at 0 "C in 80 mM KCI, 5 mM MgC12,20 mM MOPS, pH 7.0, and Ca-EGTA buffers to yield the desired free Ca2+ concentration. When required, the reaction mixture contained 20 PM TFP. Phosphorylation was started by the addition of 50 p~ [y-:'"P]ATP and stopped with trichloroacetic acid after 2 s. The denatured protein was centrifuged and resuspended in acid several times and finally tested for protein concentration and bound radioactivity. The Ca"-dependent formation of phosphoenzyme was calculated after subtraction of the basal level, obtained in the absence of CaZ' ions (1 mM EGTA).
Brain calmodulin stimulation of the Ca"-dependent ATPase of erythrocyte ghosts was measured with the coupled enzyme assay as described above. A curve was obtained by plotting the level of stimulation versus calmodulin concentration. This calibration curve was used to quantify the amount of calmodulin present in boiled SR extracts, according to their stimulation effect.
Hydroxylamine-resistant phosphorylation of SR proteins was studied basically as reported by Tada et al. (22). The experiments were carried out at room temperature in a medium containing 80 mM KCI, 5 mM MgCI,, 20 mM MOPS, pH 7.0, and 1.0-1.5 mg of protein/ml. 1 mM EGTA or 50 p~ CaC12 was present when basal or Ca"-dependent phosphorylation was studied. When required, 0.5 pg of calmodulin/30 pl or 80 p~ TFP was added to the standard reaction mixture. Reactions were started by the addition of 100 p~ [y-"'P]ATP (5-10 Ci/ mmol) and stopped after 1 min with SDS-solubilizing buffer. Proteins were characterized by gel electrophoresis (10-1210 polyacrylamide) as described below. Gels were stained for proteins, dried, and placed on x-ray-sensitive film (Kodak XS-5) with an intensifying screen (KYOKKO H5) and autoradiographed at -70 "C for 2-5 days. The phosphoproteins detected by this procedure were hydroxylamineresistant.
Gel electrophoresis was carried out basically as described by Laemmli (18), using 10-12% acrylamide in the separating gels. Slab gels were polymerized in the presence of 0.5% polyacrylamide. When required, gels and electrode buffer contained 500 p~ CaC12 or 8 mM EDTA. Proteins were stained with Coomassie brillant blue. Glycoproteins were coupled to the fluorescent probe dansylhydrazine by the procedure of Eckhardt et al. (19) as modified in Ref. 20.
Protein concentration was measured according to Lowry et al. (21) using bovine serum albumin as a standard.

Isolation of Calmodulin from Fast Skeletal Muscle SR-
The presence of calmodulin has been demonstrated in several membrane systems. In particular, it was recently reported Details are described under "Materials and Methods." SR vesicles were boiled, and the supernatant obtained after centrifugation (boiled supernatant) was chromatographed on a CAPP-SEPHAHOSI.: 4B column. The column was equilibrated with 300 mM KCI, 1 mM mercaptoethanol, 1 mM CaC12, and 50 mM Tris-CI, pH 6.6. The Ca-ELUATE was obtained after elution of the column in the same medium. The EGTA-ELUATE was obtained after substitution of 1 mM CaCl? with 10 mM EGTA in the elution medium. Electrophoresis of the various fractions on 12% polyacrylamide gels w&q carried out in the presence of EDTA as described under "Materials and Methods." that SR preparations isolated from lobster muscle contain a heat-stable factor with properties similar to those of calmodulin (24). The investigation has now been extended to SR isolated from mammalian fast skeletal muscle. Analysis of the protein composition of this SR preparation after electrophoretic separation has revealed the presence of protein material which co-migrated with isolated brain (or testis) bovine calmodulin in gel systems containing EDTA (Fig. l). However, only a fraction of the protein band migrating at M, = 17,000 co-migrated with exogenous calmodulin on gels containing Ca2' ions (not shown). Under these conditions, the electrophoretic mobility of calmodulin is increased (25), and the band corresponding to calmodulin is clearly resolved from the bulk of the M, = 17,000 stain.
If microsomes were boiled for 5 min (at 10 mg/ml in 1 M sucrose, 0.6 M KCl, 20 mM MOPS, pH 7.0) and subjected to centrifugation, several proteins, including a M , = 17,000 component, were recovered in the supernatant. The 17,000-dalton protein could be identified as calmodulin, since it was selectively bound to a CAPP affinity chromatography column (see "Materials and Methods"). Phenothiazines are known to interact strongly with calmodulin in the presence of Ca'+ ions (26). Fig. 1 shows the purification steps of SR calmodulin. It can be seen that the CAPP-Sepharose 4B column retains the M, = 17,000 component in a Ca"-dependent way and removes it quantitatively from the supernatant of boiled microsomes.
The functional activity of calmodulin from fast skeletal muscle SR was tested on the Ca"-ATPase activity of calmodulin-depleted erythrocyte ghosts and found to be comparable to that of isolated brain calmodulin. 1 pg of calmodulin/ml produced a 5-8-fold stimulation of the Ca"-dependent hydro-lytic activity at the saturating free Ca2' concentration of 10 w * T o quantify the amount of calmodulin associated with the isolated SR membranes, the level of stimulation of erythrocyte ghosts ATPase by the supernatant of boiled preparations was compared with the stimulation induced by known amounts of brain calmodulin. It could be established that the total level of calmodulin present in the starting SR preparations was of the order of 0.5-1.0 pg/mg of protein.
By washing the vesicles with alkaline media in the presence of EGTA and by combining the washings with osmotic shocks, only up to 40-50% of the endogenous calmodulin could be removed. This portion of extractable calmodulin was probably associated with glycogenolytic enzymes contaminating the SR preparations, since the same amount of activator protein could be removed upon incubation of the vesicles with a-amylase, which quantitatively removes the glycogen particles normally attached to the SR membranes (27, 28). T o obtain an essentially complete removal of calmodulin, detergent treatments, applied under conditions that solubilize many proteins from the SR membrane, were required. It is clear, then, that a significant amount of calmodulin is strongly associated with the SR vesicles. It is reasonable to suggest that it may be bound to (a) hydrophobic component(s) of the membrane.
Isolation of the 53,000-dalton Glycoprotein from SR Membranes-If SR membranes were applied to a CAPP-Sepharose column after their partial solubilization with Triton X-100, a glycoprotein with a molecular weight of 53,000 was selectively bound to the phenothiazine. Fig. 2 shows the protein composition of the various fractions obtained from the CAPP column. During elution with a Ca"containing buffer, two peaks containing membranous material could be resolved (Ca-eluates I and 2). The most important difference in the protein composition of the fractions pooled in the two peaks was in the relative amount of 53,000-dalton glycoprotein, which was partially retained only in Ca-eluate 2 (see glycoprotein staining in Fig. 2 ) . After elution of the column with an EGTA-containing buffer, the glycoprotein was eluted in an almost pure form. Thus, the interaction of the glycoprotein with the CAPP columns was Ca"-dependent. It is probable that the partial retention of the Ca-eluate 2 on the CAPP-column was due to the presence of glycoprotein that was not fully solubilized. It is remarkable that, under these conditions, no calmodulin was retained by the column. The possibility that the presence of Triton prevented the binding of calmodulin to phenothiazine could be excluded, since purified calmodulin, chromatographed under the same conditions, was selectively retained by the column in a Ca"dependent way. On the other hand, it was possible that Triton, at the concentration used (0.2%), did not extract the calmodulin-target complex from the SR membrane. As a result, the interaction with the CAPP-Sepharose column might have been inadequate to retain, during the extensive washing with Ca" buffer, the whole membranous particulate matter to which calmodulin was attached. This possibility was tested by using a detergent different from Triton X-100.
When CAPP-Sepharose chromatography was carried out with SR membrane partially solubilized with 0.1% desoxycholate, calmodulin, the 53,000-dalton glycoprotein, and three or four contaminating proteins were retained in a Ca2'-dependent manner. The presence of these contaminants, however, was not due to a specific interaction with the phenothiazine, but rather to the carbohydrate moiety of the column, since the same proteins were isolated under the same conditions using a Sepharose 4B column.
In looking for an explanation to the finding that the 53,000dalton glycoprotein specifically bound to a drug known to interact with calmodulin, the possibility was considered that the 53,000-dalton glycoprotein was actually a complex composed of calmodulin and a glycoprotein with a molecular weight lower than 53,000 (SR preparations contain, indeed, minor glycoproteins in the M, = 30,000 range). This possibility was made unlikely by the fact that the complex was undissociable in SDS during electrophoresis conditions, and by the fact that extensive boiling of the isolated 53,000-dalton glycoprotein released no calmodulin into the supernatant. On the other hand, the glycoprotein itself could interact strongly with calmodulin to form a complex, the calmodulin part of which could, somehow, remain bound to the CAPP column. However, (a) the molar content of glycoprotein in the tested SR preparations is at least 10 times higher than that of calmodulin; (b) in the presence of Triton X-100, no calmodulin was eluted from the CAPP-Sepharose column in the fraction containing the isolated glycoprotein (EGTA eluate); and (c) under various experimental conditions (i.e. use of Triton X-100, deoxycholate, or Lubrol), no glycoprotein was specifically retained by a calmodulin-Sepharose 4B column. Therefore, a logical explanation for the results is that the 53,000-dalton glycoprotein and calmodulin interact independently with the phenothiazine in a specific way.
Physiological Role of the Endogenous SR Calmodulin a n d the 53,000-dalton Glycoprotein-The presence of calmodulin in SR vesicles of fast skeletal muscle raises the possibility of a calmodulin-dependent regulation of their functional activity. Exogenously added calmodulin did not significantly affect the Ca"-uptake activity of fast SR vesicles, probably because endogenous calmodulin had to be extracted. As described above, however, it was impossible to completely remove the tightly bound calmodulin without damage of the vesicular structure.
Thus the physiological role of calmodulin in SR membranes was started with a 20 X dilution of the suspension with the same medium, but without CaClz at room temperature. When required, Caloaded vesicles were preincubated with 28 p~ TFP before adding to dilution medium (also containing 28 p~ TFP). 0, control; 0, 28 PM TFP. B, passive oxalate efflux. Experiments were carried out as described for A but in the presence of 10 m~ ['4C]oxalate in the could only be tested by selectively inhibiting the effect by the endogenous activator on the intact membranes with anticalmodulin drugs (phenothiazines). It was found that, under specific experimental conditions, the steady state rate of Ca'+ accumulation by fast SR vesicles ( a s measured in the presence of oxalate) was strongly inhibited by TFP (80-90% inhibition by 14 p~ TFP). The observed inhibition of Ca2+ uptake could be due to the alteration of permeability characteristics of the SR membrane to Ca2+ (ie. Ca2+ leakage) or oxalate ions (ie. blockade of the anion carrier). Therefore, the effect of TFP on the role of passive efflux of radioactively labeled Ca2* and oxalate ions from SR vesicles was studied. Fig. 3 shows that drug concentrations as high as 28 p~ induced no change in the permeability characteristics of the system.
In the course of this study, however, it was observed that Ca2+ ions themselves, in the micromolar range, strongly inhibited the oxalate efflux rate (Fig. 3 B under these experimental conditions, no Ca-oxalate crystals are formed). A similar modulation of the permeability of SR membranes to monovalent cations by Ca2+ has been recently observed (44).
The analysis of the Ca2+ dependence of the TFP inhibition of Ca2+ uptake has provided strong support for the specific nature of the drug interaction. From the results presented in Fig. 4, it is evident that the mechanism of the TFP inhibition implies a shift in the Ca2+ affinity of the ATPase, while the Vmax of the reaction is relatively less affected. In the presence of low TFP concentrations (14 p~) , the apparent K,,, increases The Ca*+-dependent ("extra") ATPase activity of the microsomes was qualitatively inhibited in a very similar way by TFP. Quantitation of the inhibition pattern as a function of the free Ca2+, however, was experimentally difkult, since at the low free Ca2+ concentrations required to observe a marked inhibition, the basal (Ca'+-independent) rate of ATP hydrolysis became predominant.
The effect of TFP on the steady state level of the phos-from 0.7 to 1.7 pM.  Table I shows that phenothiazine strongly decreased the phosphoenzyme level at low free Ca2+ concentrations, while it had a much less significant effect at saturating Gaff concentrations. This observation further demonstrates that TFP, at the low concentrations used in this study, is not affecting the turnover rate of the transport reaction, but decreases the Ca affinity of the Ca2+-transporting sites of the ATPase. This is precisely what would be expected if TFP interferes with membrane-bound calmodulin. Indeed, the transition of the enzyme from high to low Ca2+ affiiity is the result of calmodulin depletion or inhibition in several other Ca2+-pumping ATPases (6, 29).
The mechanism of the hypothetical calmodulin stimulation of fast skeletal muscle SR ATPase could involve a direct interaction of the activator protein with the pumping enzyme, or a calmodulin-dependent protein kinase, as has been sug-

Ka TABLE I1 Effect of TFP on the Ca2'-dependent ATPase activity of Ca-eluates I and 2
The Ca2+-dependent Pi liberation by SR fractions was measured at room temperature as described (16). The medium contained 80 mM KCI, 5 mM MgC12, 20 m MOPS, pH 7.0, Ca-ECTA buffers to yield the concentrations of free Ca2+ shown, 5 mM K,-oxalate, and 50-70 pg of protein/ml. The reaction was started with 2 mM K2ATP. Caeluate fractions 1 and 2 were obtained from the CAPP-Sepharose 4B column as described in the legend to Fig. 2 The formation of a calmodulin. ATPase complex in SR membrane is very unlikely for many reasons, among them the fact that the SR ATPase is not retained by calmodulin chromatography columns.' Therefore, the possibility that membrane-bound calmodulin was involved in the stimulation of phosphoprotein phosphorylation by a calmodulin-dependent protein kinase was investigated. Using radioactively labeled [y-"P]ATP and autoradiography techniques, it could be observed that several SR proteins became phosphorylated, in analogy with previous reports (30). In addition, however, it was found that the phosphorylation of the three major proteins of M, = 20,000, 35,000, and 57,000 (and to some extent, also, a protein of M, A number of minor protein components are retained, under specitic experimental conditions, by the calmodulin column. Their identities and their relationships to the calmodu1in:dependent regulation system are under study. = 13,000-15,000) is Ca2'-dependent (Fig. 5). Of great interest was the finding that, in the presence of Ca'' ions, TFP inhibited, while exogenous calmodulin greatly stimulated, the phosphorylation of these three proteins. On the other hand, calmodulin and TFP had no effect when the phosphorylation experiments were carried out in the absence of Ca'' ions (Fig.   5). The results demonstrate that the SR preparation used contained a calmodulin-dependent protein kinase which phos-  Effect of TFP on the Ca2'-dependent ATPase activity of deoxycholate-treated SR vesicles The Ca2+-dependent ATPase activity was measured at 37 "C with the coupled enzyme assay, as described under "Materials and Methods." Protein concentrations were 5-10 pg/ml, and K2ATP was 0.5 mM. Data represent mean values (fS.E.) for three experiments. " SR vesicles (3 mg/ml) were extracted at 0 "C in the presence of 1 mM EGTA, 20 mM Tris-C1, pH 8.0. The vesicles were Centrifuged, washed, and resuspended in 1 M sucrose, 20 mM MOPS, pH 7.0. SR vesicles (3 mg/ml) were partially solubilized at 0 "C with deoxycholate (0.8 mg/ml) in 20 mM MOPS, pH 7.4, and 0.5 M sucrose. The vesicles were then centrifuged and washed as described in Footnote a.

A
SR vesicles were partially solubilized as described in Footnote b, in a medium supplemented with 1 M KCL.
phorylates several protein substrates.' The physiological role of the calmodulin-dependent phosphorylation and the nature of the phosphorylation products is under study. The possibility that the M, = 20,000 component is phospholamban is considered unlikely, since this protein fails to become phosphorylated upon incubation with CAMP and a CAMP-dependent protein kinase (1-4). It is clear from these experiments, however, that the SR membranes are not saturated with calmodulin, since exogenously added activator stimulates severalfold the phosphorylation level of these proteins. It seems unlikely, therefore, that calmodulin is involved in the regulation of CaZ+ uptake activity, since a corresponding stimulation of transport or ATPase activity by exogenous calmodulin was never observed. Therefore, the possibility was considered a t this point that the TFP inhibition of the Ca" pump was due to the specific interaction of the phenothiazine with the 53,000-dalton glycoprotein, and the effect of TFP on SR preparations depleted of the 53,000-dalton glycoprotein was investigated. As can be seen in Fig. 2, the glycoprotein is absent from the fraction designated Ca-eluate 1. Table I1 shows that TFP inhibited the Ca"-dependent ATPase activity of this preparation much less than that of Ca-eluate 2, which contained the glycoprotein. The glycoprotein could also be removed by extracting the SR vesicles with low concentrations of deoxycholate at high ionic strengh (8). Fig. 6  "In a very recent abstract (43), fast skeletal muscle SR was also shown to contain a calmodulin-dependent regulation system. A substrate of molecular weight 6 0 , O O O appears to be phosphorylated by this system. concentrations. On the other hand, no significant Ca"-dependent inhibition could be observed in SR preparations containing no glycoprotein, and, as expected, the fraction partially depleted of glycoprotein showed an intermediary behavior. To further substantiate the hypothesis that the 53,000-dalton glycoprotein is involved in the regulation of SR activity, SR from lobster muscle was prepared. As already

Modulation of Ca2+ Movements in Fast Skeletal Muscle SR
reported (24), this preparation contains calmodulin but is completely free of the 53,000-dalton glycoprotein (Fig. 7). Therefore, the effect of TFP on the Ca'+-uptake activity of two SR preparations differing in glycoprotein content could be compared. The results presented in Fig. 8 show that the strong Ca"-dependent inhibition of Ca'+ uptake observed in the fast skeletal muscle preparation was completely absent in lobster SR vesicles.

DISCUSSION
Plasma membranes, like cardiac sarcolemma and erythrocyte ghosts, possess a Ca'+-transporting system, which is regulated (stimulated) by calmodulin. The stimulation depends on the direct, Ca*'-dependent binding of calmodulin to high affinity sites on the pumping enzyme (5, 6). The observations presented here, while demonstrating a role for calmodulin in fast skeletal muscle SR, rule out the possibility of a similar regulation mechanism for its Ca'+-ATPase. The phosphorylation studies presented demonstrate that SR vesicles contain a calmodulin-dependent protein kinase, in analogy with previous findings on cardiac muscle SR. In the latter system, a stimulation of Ca"-uptake activity by exogenous calmodulin has been observed (3, 4), and the stimulation has been correlated to the calmodulin-dependent phosphorylation of phospholamban, a 22,000-dalton integral protein of cardiac SR (4). The present work has shown that calmodulin promotes the phosphorylation of three SR proteins having molecular weights of 20,000,35,000, and 57,000. A the moment, however, no conclusion can be reached on the physiological role of the phosphorylation of these three proteins, nor on their identity.
Two points can be made here: the presence of a phospholamban-like protein in fast skeletal muscle SR has never been demonstrated, and the evident stimulation of the hydroxylamine-resistant phosphorylation of the three proteins mentioned by exogenous calmodulin is not accompanied by a parallel stimulation of the Ca'+-transport activity.
At this point, one could thus consider the possibility that a calmodulin-dependent system in fast skeletal muscle SR might be involved in the long term regulation of Ca' ' release during excitation. It may be relevant, in this context, that calmodulin has been localized in fast skeletal muscle by immunocytochemical techniques mainly at the level of the Ibands, which are rich in /3-glycogen particles, and in the terminal cisternae (32). Several lines of evidence show that the cisternal compartments of the SR network are the sites which are primarily involved in excitation-contraction coupling and in the release of the activating Ca'+ (31). The recent finding that the phosphorylation of the 57,000-dalton protein, which we found to be calmodulin-dependent, is inhibited by the specific inhibitor of Ca'+ release in skeletal muscle, Na-Dantroliene (30), may also be relevant. The possibility of a calmodulin regulation of the release of Ca'+ implies that the response of SR membranes to the electrical or chemical signal which triggers the release of Ca'+ may be modulated by a phosphorylation system. The proposal of a similar, CAMPdependent modulation of Ca2+ release by cardiac SR has already been made (33, 34).
The calmodulin affinity chromatography approach failed to identify clearly (2) calmodulin target protein(s) in the SR membrane. No protein was bound by the calmodulin column in the presence of Triton X-100 or Lubrol. When deoxycholate was used to solubilize SR membranes, some minor proteins were retained by the column in a Ca2+-dependent way. The very low yield of these proteins and the fact that some of them were bound also by uncoupled Sepharose 4B suggest caution in interpreting these experiments. At the moment, it is possible to rule out the Ca2'-ATPase, calsequestrin, and the 53,000-dalton glycoprotein as possible targets of calmodulin. In fact, the isolated glycoprotein and calsequestrin did not interact with the calmodulin column, nor did they form Ca2+dependent complexes with calmodulin when subjected to coelectrophoresis under nondenaturing conditions (i.e. in the absence of SDS). The identification and the isolation of the hypothetical calmodulin-dependent kinase will require further work.
On the other hand, the strong interaction between phenothiazines and the 53,000-dalton glycoprotein is likely to be responsible for the observed TFP inhibition of Ca2+ uptake by SR vesicles. This is further supported by the finding that the TFP inhibition of Ca' ' uptake is less pronounced in glycoprotein-poor vesicles and is completely absent in lobster SR, which does not contain the 53,000-dalton glycoprotein.
The rationale for the Ca2+-dependent binding of the glycoprotein to the phenothiazine column is not clear. In analogy with what has been suggested for the calmodulin.TFP complex (35), it is likely that the glycoprotein interacts hydrophobically with TFP in a way which depends on whether or not Ca2+ ions are bound to the protein.
The concept that the 53,000-dalton glycoprotein might stimulate fast skeletal muscle SR activity is not novel (7, 8).
Recent observations have unraveled many interesting similarities between the Ca2+-pumping ATPase of SR and the (Na+-K+)-ATPase complex of plasmalemma, which is also known to contain a glycoprotein (36, 37). The similarities of the glycoprotein subunit of the (Na'-K')-ATPase with the SR 53,000-dalton glycoprotein are striking, and range from the molecular weight to the amino acid and sugar compositions (38). Recent studies have shown that the glycoprotein is present in light, intermediate, and heavy S R vesicles in a constant molar ratio with the Ca2+-ATPase (7). It has also been observed that reconstituted ATPase vesicles containing the glycoprotein take up Ca' ' more efficiently than vesicles deprived of it (8). This evidence seems to suggest that the functional Ca2+ pump of SR includes the Ca"-ATPase and the glycoprotein. At variance with the Na+-K+-pumping complex of sarcolemma, however, the present work has shown that the SR ATPase can be easily separated from the glycoprotein, and that the glycoprotein is not absolutely required for the Ca2+-pumping activity.
Our characterization of the TFP-induced inhibition of the S R ATPase has provided useful information on the role of the glycoprotein in the stimulation of Ca2+ uptake. Although the molecular mechanism of the stimulation is still obscure, it is apparent that the main effect on the transport kinetics is a decrease of the K , for Ca2+ of the ATPase. This shift in K , takes place in a Ca' ' concentration range which is crucial for the activation of the pump under physiological conditions. On the other hand, TFP is not affecting the passive permeability of SR to oxalate, and the inhibition of Ca2' uptake by TFP is not reversed by ionophores such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (not shown). It is unlikely, therefore, that the glycoprotein represents the anions or the monovalent cation (H', K' ) channel, whose activity is required for optimal Ca2+ transport (39-41).
A final comment that may be made is that this work has permitted the isolation, by a rapid and nondenaturing procedure, of the glycoprotein component of the Ca2+-ATPase system. Thanks to this procedure, this protein, which may play a very important role in the overall process of Ca2+ transport by SR vesicles, will now be easily available for further study.