The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. II. The influence of membrane potential

Na3VO4 promotes the crystallization of Ca2+-ATPase in sarcoplasmic reticulum vesicles. The rate of vanadate-induced crystallization is dramatically increased by inside positive membrane potential generated through ion substitution. Negative potential caused the transient disruption of preformed Ca2+-ATPase crystals, followed by slower reappearance of the lattice after the potential was dissipated. We propose that positive transmembrane potential alters the conformation of the Ca2+-ATPase molecules in a manner that favors ATPase-ATPase interactions, while negative potential would have the opposite effect. Changes in enzyme conformation caused by potential changes during the contraction-relaxation cycle could regulate ATPase interactions in a similar manner in vivo, with effects upon the Ca2+ transport activity and permeability of the sarcoplasmic reticulum.     

Membrane crystals of Ca2+-ATPase in sarcoplasmic reticulum of fast and slow skeletal and cardiac muscles

Crystalline arrays of Ca2+ transport ATPase develop in sarcoplasmic reticulum membranes after treatment with Na3VO4 in a calcium-free medium [ Dux , L. and Martonosi , A. (1983) J. Biol. Chem. 258, 2599-2603]. The proportion of vesicles containing Ca2+-ATPase crystals in microsome preparations isolated from rat muscle of different fiber types (semimembranosus, levator ani, extensor digitorum longus, diaphragm, soleus, and heart) correlates well with the Ca2+-ATPase content and Ca2+-modulated ATPase activity. This implies that the concentration of Ca2+-ATPase in sarcoplasmic reticulum membranes of fast and slow skeletal or cardiac muscles differs only slightly, and the low Ca2+ transport activity of 'sarcoplasmic reticulum' preparations isolated from slow-twitch skeletal and cardiac muscles is due to the presence of large amount of non-sarcoplasmic-reticulum membrane elements. This is in accord with the relatively small differences in the density of 8.5-nm intramembranous particles seen by freeze-etch electron microscopy in sarcoplasmic reticulum of red and white muscles. The dimensions of the Ca2+-ATPase crystal lattice are similar in sarcoplasmic reticulum membranes of different fiber types; therefore if structural differences exist between 'isoenzymes' of Ca2+-ATPase, these are not reflected in the crystal-lattice.     

Mechanism of the stimulation of cardiac sarcoplasmic reticulum calcium pump by calmodulin

Calmodulin has been shown to stimulate the initial rates of Ca2+-uptake and Ca2+-ATPase in cardiac sarcoplasmic reticulum, when it is present in the reaction assay media for these activities. To determine whether the stimulatory effect of calmodulin is mediated directly through its interaction with the Ca2+-ATPase, or indirectly through phosphorylation of phospholamban by an endogenous protein kinase, two approaches were taken in the present study. In the first approach, the effects of calmodulin were studied on a Ca2+-ATPase preparation, isolated from cardiac sarcoplasmic reticulum, which was essentially free of phospholamban. The enzyme was preincubated with various concentrations of calmodulin at 0 degrees C and 37 degrees C, but there was no effect on the Ca2+-ATPase activity assayed over a wide range of [Ca2+] (0.1-10 microM). In the second approach, cardiac sarcoplasmic reticulum vesicles were prephosphorylated by an endogenous protein kinase in the presence of calmodulin. Phosphorylation occurred predominantly on phospholamban, an oligomeric proteolipid. The sarcoplasmic reticulum vesicles were washed prior to assaying for Ca2+ uptake and Ca2+-ATPase activity in order to remove the added calmodulin. Phosphorylation of phospholamban enhanced the initial rates of Ca2+-uptake and Ca2+-ATPase, and this stimulation was associated with an increase in the affinity of the Ca2+-pump for calcium. The EC50 values for calcium activation of Ca2+-uptake and Ca2+-ATPase were 0.96 +/- 0.03 microM and 0.96 +/- 0.1 microM calcium by control vesicles, respectively. Phosphorylation decreased these values to 0.64 +/- 0.12 microM calcium for Ca2+-uptake and 0.62 +/- 0.11 microM calcium for Ca2+-ATPase. The stimulatory effect was associated with increases in the apparent initial rates of formation and decomposition of the phosphorylated intermediate of the Ca2+-ATPase. These findings suggest that calmodulin regulates cardiac sarcoplasmic reticulum function by protein kinase-mediated phosphorylation of phospholamban.     

The role of creatine phosphokinase in supplying energy for the calcium pump system of heart sarcoplasmic reticulum.

An investigation of isolated and purified heart sarcoplasmic reticulum performed in the current study indicates the presence of significant creatine phosphokinase (CPK) activity in this preparation. The localization of CPK on the membrane of sarcoplasmic reticulum has been revealed also by an electron microscopic histochemical method. Under the conditions of the Ca(2+)-ATPase reaction in the presence of creatine phosphate, the release of creatine into the reaction medium is observed, the rate of the latter process being dependent on the MgATP concentration in accordance with the kinetic parameters of the Ca2+-ATPase reaction. CPK localized on the reticular membrane is able to maintain the high rate of calcium consumption by the sarcoplasmic reticulum vesicles. The results obtained demonstrate the close functional coupling between CPK and Ca2+-ATPase in the membrane of sarcoplasmic reticulum and indicate the important functional role of CPK in supplying energy for the Ca(2+)-ATPase reaction and ion transport across the membrane of heart sarcoplasmic reticulum.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

Mechanism of the stimulation of Ca2+-dependent ATPase of skeletal muscle sarcoplasmic reticulum by protein kinase

Sarcoplasmic reticulum isolated from moderately fast rabbit skeletal muscle contains intrinsic adenosine 3',5'-monophosphate (cAMP)-independent protein kinase activity and a substrate of 100 000 Mr. Phosphorylation of skeletal sarcoplasmic reticulum by either endogenous membrane bound or exogenous cAMP-dependent protein kinase results in stimulation of the initial rates of Ca2+ transport and Ca2+-ATPase activity. To determine the molecular mechanism by which protein kinase-dependent phosphorylation regulates the calcium pump in skeletal sarcoplasmic reticulum, we examined the effects of protein kinase on the individual steps of the Ca2+-ATPase reaction sequence. Skeletal sarcoplasmic reticulum vesicles were preincubated with cAMP and cAMP-dependent protein kinase in the presence (phosphorylated sarcoplasmic reticulum) and absence (control sarcoplasmic reticulum) of adenosine 5'-triphosphate (ATP). Control and phosphorylated sarcoplasmic reticulum were subsequently assayed for formation (5-100 ms) and decomposition (0-73 ms) of the acid-stable phosphorylated enzyme (E approximately P) of Ca2+-ATPase. Protein kinase mediated phosphorylation of skeletal sarcoplasmic reticulum resulted in pronounced stimulation of initial rates and levels of E approximately P in sarcoplasmic reticulum preincubated with either ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) prior to assay (Ca2+-free sarcoplasmic reticulum), or with calcium/EGTA buffer (Ca2+-bound sarcoplasmic reticulum). These effects were evident within a wide range of ionized Ca2+. Phosphorylation of skeletal sarcoplasmic reticulum by protein kinase also increased the initial rate of E approximately P decomposition. These findings suggest that protein kinase-dependent phosphorylation of skeletal sarcoplasmic reticulum regulates several steps in the Ca2+-ATPase reaction sequence which result in an overall stimulation of the active calcium transport observed at steady state.     

Inhibition of calcium-dependent ATPase from sarcoplasmic reticulum by a new class of indolizidine alkaloids, pumiliotoxins A, B, and 251D

Pumiliotoxins (PTX) A, B, and 251D, members of a new class of indolizidine alkaloids isolated from the skin of poison frogs of the family Dendrobatidae, inhibit Ca2+-ATPase activity in sarcoplasmic reticulum vesicles from frog and rat hind-limb muscles. PTX-B and PTX-A appear to be relatively specific inhibitors of Ca2+-ATPase; PTX-A is much less potent than PTX-B. PTX-251D is a potent inhibitor of Ca2+-ATPase, and was also found to inhibit Na+, K+, and Mg2+-ATPases in rat brain synaptosomes. Caffeine and verapamil, two drugs known to affect calcium translocation, are very weak inhibitors of the Ca2+-ATPase. The Ki values for inhibition of the Ca2+-ATPase of rat and frog sarcoplasmic reticulum by PTX-B were comparable and ranged between 22 and 36 microM. Inhibition of calcium-dependent ATPase in sarcoplasmic reticulum by pumiliotoxin-B is noncompetitive with calcium and is not readily reversible. Based on structure-activity profiles, it is concluded that inhibition of Ca2+-ATPase by the indolizidine alkaloids is responsible for the alkaloid-elicited prolongation of twitch in intact muscle.     

Silver ions trigger Ca2+ release by interaction with the (Ca2+-Mg2+)-ATPase in reconstituted systems

It has been suggested that vesicles derived from the sarcoplasmic reticulum of skeletal muscle contain Ca2+ channels which can be opened by interaction with sulfhydryl reagents such as Ag+ or Hg2+. We show that, in reconstituted vesicles containing the (Ca2+-Mg2+)-ATPase purified from sarcoplasmic reticulum as the only protein, the ATPase can act as a pathway for Ca2+ efflux and that Ag+ induces a rapid release of Ca2+ from such reconstituted vesicles. We also show that Ag+ has a marked inhibitory effect on the ATPase activity of the purified ATPase. We suggest that the (Ca2+-Mg2+)-ATPase can act as a pathway for rapid Ca2+ release from sarcoplasmic reticulum.     

Effect of chemical modification on the crystallization of Ca2+-ATPase in sarcoplasmic reticulum

The influence of chemical modification on the morphology of crystalline ATPase aggregates was analyzed in sarcoplasmic reticulum (SR) vesicles. The Ca²⁺-ATPase forms monomer-type (P1) type crystals in the E₁ and dimer-type (P2) crystals in the E₂ conformation. The P1 type crystals are induced by Ca²⁺ or lanthanides; P2 type crystals are observed in Ca²⁺-free media in the presence of vanadate or inorganic phosphate. P1- and P2-type Ca²⁺-ATPase crystals do not coexist in significant amounts in native sarcoplasmic reticulum membrane. The crystallization of Ca²⁺-ATPase in the E₂ conformation is inhibited by guanidino-group reagents (2,3-butanedione and phenylglyoxal), SH-group reagents, phospholipases C or A₂, and detergents, together with inhibition of ATPase activity. Amino-group reagents (fluorescein 5′-isothiocyanate, pyridoxal phosphate and fluorescamine) inhibit ATPase activity but do not interfere with the crystallization of Ca²⁺-ATPase induced by vanadate. In fluorescamine-treated sarcoplasmic reticulum the vanadate-induced crystals contain significant P1-type regions in addition to the dominant P2 form.     

Critical evaluation of cardiac Ca2+-ATPase phosphorylation on serine 38 using a phosphorylation site-specific antibody

The phosphorylation of the cardiac muscle isoform of the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA2a) on serine 38 has been described as a regulatory event capable of very significant enhancement of enzyme activity (Hawkins, C., Xu, A., and Narayanan, N. (1994) J. Biol. Chem. 269, 31198-31206). Independent confirmation of these observations has not been forthcoming. This study has utilized a polyclonal antibody specific for the phosphorylated serine 38 epitope on the Ca(2+)-ATPase to evaluate the phosphorylation of SERCA2a in isolated sarcoplasmic reticulum vesicles and isolated rat ventricular myocytes. A quantitative Western blot approach failed to detect serine 38-phosphorylated Ca(2+)-ATPase in either kinase-treated sarcoplasmic reticulum vesicles or suitably stimulated cardiac myocytes. Calibration standards confirmed that the detection sensitivity of assays was adequate to detect Ser-38 phosphorylation if it occurred on at least 1% of Ca(2+)-ATPase molecules in SR vesicle experiments or on at least 0.1% of Ca(2+)-ATPase molecules in cardiac myocytes. The failure to detect a phosphorylated form of the Ca(2+)-ATPase in either preparation (isolated myocyte, purified sarcoplasmic reticulum vesicles) suggests that Ser-38 phosphorylation of the Ca(2+)-ATPase is not a significant regulatory feature of cardiac Ca(2+) homeostasis.     

The effects of membrane potential and lanthanides on the conformation of the Ca2+-transport ATPase in sarcoplasmic reticulum.

The effects of Ca2+, lanthanide ions (Gd3+, La3+ and Pr3+) and membrane potential on the fluorescence of tryptophan and covalently bound fluorescein were analysed in native and fluorescein isothiocyanate (FITC)-labelled sarcoplasmic reticulum vesicles. The binding of Ca2+ and lanthanides to the Ca2+-ATPase increases the fluorescence intensity of tryptophan and decreases the fluorescence intensity of FITC; the dependence of these effects on cation concentration is consistent with the involvement of the high-affinity Ca2+-binding sites of the Ca2+-ATPase in the cation-induced fluorescence changes. The fluorescence of FITC-labelled sarcoplasmic reticulum vesicles is also influenced by membrane potential changes induced by ion substitution. Inside positive potential increases, while inside negative potential decreases, the fluorescence of bound FITC. Smaller potential-dependent changes in tryptophan fluorescence were also observed. The effects of Ca2+, lanthanides and membrane potential on the fluorescence of tryptophan and FITC are discussed in terms of the two major conformations of the Ca2+-ATPase (E1 and E2), that are assumed to alternate during Ca2+ transport. The observations support the suggestion [Dux, Taylor, Ting-Beall & Martonosi (1985) J. Biol. Chem. 260, 11730-11743] that the vanadate-induced crystals of Ca2+-ATPase represent the E2, while the Ca2+ and lanthanide-induced crystals the E1, conformation of the enzyme.     

The binding of monoclonal and polyclonal antibodies to the Ca2(+)-ATPase of sarcoplasmic reticulum: effects on interactions between ATPase molecules.

We analyzed the interaction of 14 monoclonal and 5 polyclonal anti-ATPase antibodies with the Ca2(+)-ATPase of rabbit sarcoplasmic reticulum and correlated the location of their epitopes with their effects on ATPase-ATPase interactions and Ca2+ transport activity. All antibodies were found to bind with high affinity to the denatured Ca2(+)-ATPase, but the binding to the native enzyme showed significant differences, depending on the location of antigenic sites within the ATPase molecule. Of the seven monoclonal antibodies directed against epitopes on the B tryptic fragment of the Ca2(+)-ATPase, all except one (VIE8) reacted with the enzyme in native sarcoplasmic reticulum vesicles in both the E1 and E2V conformations. Therefore these regions of the Ca2(+)-ATPase molecule are freely accessible in the native enzyme. The monoclonal antibody VIE8 bound with high affinity to the Ca2(+)-ATPase only in the E1 conformation stabilized by 0.5 mM Ca2+ but not in the E2V conformation stabilized by 0.5 mM EGTA and 5 mM vanadate. Several antibodies that reacted with the B fragment interfered with the crystallization of Ca2(+)-ATPase in the presence of EGTA and vanadate and at least two of them destabilized preformed Ca2(+)-ATPase crystals, suggesting inhibition of interactions between ATPase molecules. Of five monoclonal antibodies with epitopes on the A1 tryptic fragment of the Ca2(+)-ATPase only one gave strong reaction with the native enzyme, and none interfered with ATPase-ATPase interactions as measured by the polarization of fluorescence of FITC-labeled Ca2(+)-ATPase. Therefore the regions of the molecule containing these epitopes are relatively inaccessible in the native structure. Partial tryptic cleavage of the Ca2(+)-ATPase into the A1, A2 and B fragments did not promote the reaction of anti-A1 antibodies with sarcoplasmic reticulum vesicles, but solubilization of the membrane with C12E8 rendered the antigenic site fully accessible to several of them, suggesting that their epitopes are located in areas of contacts between ATPase molecules. Two monoclonal anti-B antibodies that interfered with ATPase-ATPase interactions, produced close to 50% inhibition of the rate of ATP-dependent Ca2+ transport, with significant inhibition of ATPase; this may suggest a role for ATPase oligomers in the regulation of Ca2+ transport. The other antibodies that interact with the native Ca2(+)-ATPase produced no significant inhibition of ATPase activity even at saturating concentrations; therefore their antigenic sites do not undergo major movements during Ca2+ transport.     

The binding of vanadium (V) oligoanions to sarcoplasmic reticulum

The binding of monovanadate and decavanadate anions to sarcoplasmic reticulum vesicles was measured by equilibrium sedimentation. The affinity of vanadate binding and the molar amount of vanadium (V) bound at equilibrium is much greater with decavanadate than with monovanadate. The binding data can be rationalized in terms of one binding site per ATPase molecule for monovanadate and two sites per ATPase for decavanadate. The Ca-ATPase crystals formed with monovanadate and with decavanadate are similar in appearance, but decavanadate is particularly effective in promoting the crystallization of Ca2+-ATPase at low V concentration (10-100 microM) in a Ca2+-free medium.     

Uncoupling of Ca2+ transport in sarcoplasmic reticulum as a result of labeling lipid amino groups and inhibition of Ca2+-ATPase activity by modification of lysine residues of the Ca2+-ATPase polypeptide

Limited labeling of amino groups with fluorescamine in fragmented sarcoplasmic reticulum vesicles inhibits Ca2+-ATPase activity and Ca2+ transport. Under the labeling conditions used, 80% of the label reacts with phosphatidylethanolamine and 20% with the Ca2+-ATPase polypeptide. This degree of labeling does not result in vesicular disruption or in loss of vesicular proteins and does not increase the membrane permeability to Ca2+. Fluorescamine labeling of a purified Ca2+-ATPase devoid of aminophospholipids also inhibits Ca2+-ATPase activity, suggesting that labeling of lysine residues of the enzyme polypeptide is responsible for the inhibition of Ca2+-ATPase activity in sarcoplasmic reticulum. Fluorescamine labeling interferes with phosphoenzyme formation and decomposition in both the native vesicles and the purified enzyme; addition of ATP during labeling, and with less effectiveness ADP or AMP, protects both partial reaction steps. Addition of a nonhydrolyzable ATP analog protects phosphoenzyme formation but not decomposition. The inhibition of Ca2+ transport but not of Ca2+-ATPase occurs in sarcoplasmic reticulum vesicles labeled in the presence of ATP, indicating that the transport reaction is uncoupled from the Ca2+-ATPase reaction. The inhibition of Ca2+ transport but not of Ca2+-ATPase activity is also found in sarcoplasmic reticulum vesicles in which only phosphatidylethanolamine has reacted with fluorescamine. Furthermore, the extent of labeling of phosphatidylethanolamine is correlated with the inhibition of Ca2+ transport rates. The inhibition of Ca2+ transport is a reflection of the inhibition of Ca2+ translocation and is not due to an increase in Ca2+ efflux. We propose that labeling of phosphatidylethanolamine perturbs the lipid environment around the enzyme, producing a specific defect in the Ca2+ translocation reaction.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

The effect of hypothyroidism on sarcoplasmic reticulum in fast-twitch muscle of the rat

The effects of hypothyroidism on the Ca2+-transport capabilities of fast-twitch muscle (m. gastrocnemius) of the rat were studied in whole-muscle homogenate and isolated sarcoplasmic reticulum. Hypothyroidism did not affect the percentage recovery and the vesicle composition of the sarcoplasmic reticulum fraction, the total lipid and phospholipid-to-protein ratios and the protein composition (both qualitative and quantitative). Also the Ca2+-loading capacity of purified sarcoplasmic reticulum, in the presence of oxalate, and the Ca2+ and pH dependence of both the uptake reaction and the coupled ATPase activity were unchanged. However, the homogenate Ca2+-loading capacity and the Ca2+-uptake activity were depressed, as was the yield of purified sarcoplasmic reticulum. The results indicate a 31% reduction of the entire sarcoplasmic reticulum membrane system per volume of muscle. Ca2+/ATP coupling ratios, determined in purified sarcoplasmic reticulum vesicles by measurement of initial rates of net Ca2+ uptake and Ca2+-Mg2+-dependent hydrolysis of ATP, were found to be 1.48 +/- 0.06 and 2.08 +/- 0.05 in the euthyroid and hypothyroid groups, respectively. Identical values were obtained with a recently described Ca2+-pulse method (Meltzer, S. and Berman, M.C. (1984) Anal. Biochem. 138, 458-464), i.e., 1.53 +/- 0.06 and 2.01 +/- 0.03 in the euthyroid and hypothyroid groups, respectively. Passive Ca2+ efflux from sarcoplasmic reticulum was the same in both groups (30 nmol/mg per min), as was the fraction of vesicles that did not show net uptake of Ca2+ (less than 10%), which makes it unlikely that these parameters provide an explanation for the differences in the coupling ratio. The energy of activation of the (Ca2+ + Mg2+)-ATPase was increased in hypothyroidism, which may point to changes in the phospholipid environment of the enzyme. Physiological concentrations of T3 and T4 had no effect on the (Ca2+ + Mg2+)-ATPase in vitro, but all observed changes in the hypothyroid state could be reversed within 14 days by administration of T3 to hypothyroid animals. Approximate calculations indicate that the observed changes in the sarcoplasmic reticulum as a result of thyroid-hormone depletion may contribute significantly to the decrease in relaxation rate and the decrease in energy consumption during contraction.     

Comparison of the catalytic and structural properties of Ca2+-ATPase in longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum

The catalytic behavior and structural features of Ca2+-ATPase in the vesicles of longitudinal tubules and terminal cisternae of the sarcoplasmic reticulum isolated from rabbit skeletal muscles was analysed. pH measurements have shown under optimal conditions Ca2+-ATPase has similar catalytic behavior both in the fractions of longitudinal tubules and terminal cisternae. Under non-optimal conditions, the behavior similarity was not observed. The specific activity of the ATPase enzyme under optimal conditions was shown to be much higher in the fraction of longitudinal tubules than in the fraction of terminal cisternae. Caffeine added to both fractions had no effect on the catalytic behavior of Ca2+-ATPase. As judged from fluorescence analysis, the structure of Ca2+-ATPase of longitudinal tubules differs from that structure of terminal cisternae. In sarcoplasmic reticulum membrane, at least half of the tryptophan residues of Ca2+-ATPase was shown to be buried in the lipid bilayer. Our findings suggest that in terminal cisternae some of the Ca2+-ATPase molecules exist as an oligomeric protein and do not participate in ATP hydrolysis (named "silent" Ca2+-ATPase).     

Purification, calcium binding properties, and ultrastructural localization of the 53,000- and 160,000 (sarcalumenin)-dalton glycoproteins of the sarcoplasmic reticulum

The 53-kDa glycoprotein and sarcalumenin (160-kDa glycoprotein) were extracted from rabbit skeletal muscle sarcoplasmic reticulum with EGTA and purified by fractionation on DEAE-Sephadex A-25 and lentil lectin-Sepharose 4B. Sarcalumenin was shown to bind up to 400 nmol of Ca2+/mg of protein at pH 7.5, which is equivalent to binding of approximately 35 mol of Ca2+/mol of protein. The apparent dissociation constant was 300 microM in the presence of 20 mM KCl and 600 microM in 150 mM KCl. The 53-kDa glycoprotein did not bind any Ca2+ under the conditions examined. Immunoblot analysis of isolated sarcoplasmic reticulum subfractions demonstrated the presence of the two glycoproteins in both the longitudinal sarcoplasmic reticulum and the terminal cisternae. Their concentrations were higher, however, in the longitudinal sarcoplasmic reticulum vesicles. Comparative immunoelectron microscopic studies using monoclonal antibodies revealed a codistribution of the 53-kDa glycoprotein with the Ca2(+)-ATPase in all regions of the free sarcoplasmic reticulum. A similar distribution was found for sarcalumenin, although immunolabeling was much weaker. The colocalization of the 53-kDa glycoprotein and sarcalumenin with the Ca2(+)-ATPase and the Ca2+ binding properties of sarcalumenin suggest that the glycoproteins may be involved in the sequestration of Ca2+ in the nonjunctional regions of the sarcoplasmic reticulum.     

Ca2+-ATPase membrane crystals in sarcoplasmic reticulum. The effect of trypsin digestion

Vanadate induces the formation of two-dimensional crystalline arrays of Ca2+-ATPase molecules in sarcoplasmic reticulum. The Ca2+-ATPase membrane crystals are evenly distributed among the terminal cisternae and longitudinal tubules of sarcoplasmic reticulum, but very few crystals were observed in the T tubules. Tryptic cleavage of the Ca2+ transport ATPase into two major fragments (A and B) did not interfere with the vanadate-induced formation of membrane crystals. The ability of Ca2+-ATPase to crystallize was lost after further cleavage of the A fragment into the A1 and A2 subfragments that is known to be accompanied by loss of Ca2+ uptake. Vanadate (0.1-5 mM) inhibited the secondary cleavage of Ca2+-ATPase by trypsin suggesting that the susceptibility of the tryptic cleavage sites is influenced either by the conformation of the enzyme or by the formation of ATPase crystals.     

Ca2+ uptake, Ca2+-ATPase activity, phosphoprotein formation and phosphate turnover in a microsomal fraction of smooth muscle

Vesicles capable of phosphate-stimulated calcium uptake were isolated from the microsomal fraction of the smooth muscle of the pig stomach according to a previously described procedure which consists in increasing the density of the vesicles by loading them with calcium phosphate and isolating them by centrifugation [Raeymaekers, L., Agostini, B., and Hasselbach, W. (1981) Histochemistry, 70, 139--150]. These vesicles, which contain calcium phosphate deposits, are able to accumulate an additional amount of calcium. This calcium uptake is accompanied by calcium-stimulated ATPase activity and by the formation of an acid-stable phosphoprotein. The acid-denatured phosphoprotein is dephosphorylated by hydroxylamine, which indicates that an acylphosphate is formed. This phosphoprotein probably represents a phosphorylated transport intermediate similar to that seen with the Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle. As with the Ca2+-ATPase of sarcoplasmic reticulum vesicles, this vesicular fraction catalyses an exchange between inorganic phosphate and the gamma-phosphate of ATP (ATP-Pi exchange) which is dependent on the presence of intravesicular calcium, and an exchange of phosphate between ATP and ADP (ATP-ADP exchange). The results further indicate that the turnover rate of the calcium pump, calculated from the ratio of calcium-stimulated ATPase activity to the steady-state level of phosphoprotein, is similar to that of Ca2+-ATPase of sarcoplasmic reticulum of skeletal muscle.