The formation of phosphoenzyme of sarcoplasmic reticulum. Requirement for membrane-bound Ca2+.

Membrane-bound Ca or Mg of sarcoplasmic reticulum fragments were removed by treating the membrane with EDTA or an acidic solution, and the changes in the enzymatic activities of sarcoplasmic reticulum fragments induced by these treatments were examined. With the decrease in the amount of membrane-bound Ca below 1-3-10(-8) mol/mg protein, it was demonstrated that the activity of (Ca2+ + Mg2+)-ATPase transiently increased and then diminished, that the Ca-uptake and phosphoenzyme formation declined gradually, and that the activity of Mg2+-ATPase was affected to a less extent. Sodium dodecyl sulfate-gel electrophoretic patterns of peptides from the metal-deficient membranes were the same as those of the untreated material. The level of the phosphoenzyme formation of the metal-deficient membrane was restored by increasing the amount of membrane-bound Ca, but not by increasing the amount of membrane-bound Mg.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum.

The Ca2+ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca2+ and surface membrane-bound Ca2+ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca2+ in the absence of ATP, 10-12 h are necessary for measurable amount of Ca2+ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca2+ from 'loaded' vesicles only after this period of incubation. A fraction of Ca2+ of 50-60 nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg2+ and K+ in the medium and is readily released by ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca2+ (30-40 nmol/mg protein) is rapidly released X-537A. The results indicate that most of the Ca2+ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca2+ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.     

Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase.Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9–10S and 4.5–4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G₁ form.     

Analysis of the arrangement of protein components in the sarcomplasmic reticulum of rat skeletal muscle.

The nature of the protein components and their location in the sarcoplasmic reticulum membrane were studied using sarcoplasmic reticulum vesicles isolated from rat skeletal muscle and purified by a density gradient centrifugation system. On the basis of analysis by means of sodium dodecyl sulfate gel electrophoresis, the protein components appear to be similar if not identical with those reported by others for rabbit sarcoplasmic reticulum, and the relative amount of each component is also similar to that found with rabbit sarcoplasmic reticulum. Evidence is presented that radioiodine-labeled diazotized diiodosulfanilic acid is a nonpermeant labeling agent of the protein components of sarcoplasmic reticulum vesicles; this agent minimally disturbs the functional activities of these membranes. By means of this labeling agent and perturbing agents, it is concluded that the protein components with molecular weights greater than 120,000 and the (Ca2+ + Mg2+)-adenosine triphosphatase partially or totally reside on or at the external surface of the sarcoplasmic reticulum vesicles. In the case of the adenosine triphosphatase, highly controlled trypsin treatment cleaves the molecule into two products, a 65,000 molecular weight fragment and a 56,000 molecular weight fragment. The evidence indicates that the 65,000 molecular weight component of the (Ca2+ + Mg2+)-adenosine triphosphatase is located in a more exposed fashion on the external surface of the vesicles than the 56,000 molecular weight compoenet and that some adenosine triphosphatase molecules have a more exposed position on the external surface of the vesicle than others. The protein components designated by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261) as "calsequestrin" and "high affinity Ca2+ binding protein" are shown not to be on the external surface of the rat sarcoplasmic reticulum vesicle but rather to reside either within the core of the membrane or on the inside surface of the vesicle. The results of this study are in agreement with the model for the organization of the protein components of the sarcoplasmic reticulum membrene recently proposed by MacLennan (MacLennan, D. H. (1975) Can. J. Biochem. 53, 251-261).     

The effect of temperature on the acetylcholinesterase activity of muscle microsomes

Membrane vesicles which constitute the sarcotubular system were separated and the fraction enriched in T-tubules purified by a calcium loading procedure. The preparations of unfractioned microsomes and T-tubules have been analyzed for their relative content of enzyme markers and acetylcholinesterase. The amount of this enzyme in the T-tubule fraction was higher than in mixed microsomes but less than two-fold the value of vesicles derived from sarcoplasmic reticulum. Arrhenius plots of membrane-bound and soluble acetylcholinesterase from either mixed microsomes or fractions enriched in T-tubules show an anomalous behaviour as two break points were obtained. The first discontinuity was found at about 17 degrees C for membrane-bound, and 12-14 degrees C for soluble acetylcholinesterase. The second one being at about 25 degrees C for both particulate and detergent-solubilized enzyme. The changes in activity with temperature suggest that lipid-protein, detergent-protein and protein-protein interactions might be involved in the stabilization of the enzyme both in the natural membrane and in the soluble state.     

Isolated bovine myocardial sarcolemma and sarcoplasmic reticulum vesicles contain tightly bound calcium-dependent protease inhibitor

Bovine myocardial sarcolemma and sarcoplasmic reticulum vesicle preparations contained calcium-dependent protease inhibitor protein. No inhibitor was detected in mitochondrial membranes. The membrane-bound inhibitor co-purified with the marker enzymes for sarcolemma and sarcoplasmic reticulum, Na+,K+-ATPase and Ca2+,K+-ATPase respectively, on isopycnic ultracentrifugation through linear sucrose density gradients. Sarcolemma and sarcoplasmic reticulum vesicles contained about 1 mg of inhibitor per g of membrane protein. However, about one-half of the inhibitor in sarcoplasmic reticulum vesicles was not tightly associated with the membrane. The membrane-bound inhibitor may function to modulate calcium-dependent proteolytic cleavage of sarcolemmal or sarcoplasmic reticulum-associated proteins.     

A role of H+ flux in active Ca2+ transport into sarcoplasmic reticulum vesicles. II. H+ ejection during Ca2+ uptake

Proton efflux during Ca2+ transport into sarcoplasmic reticulum vesicles was examined. Although a rapid H+ ejection was observed during the initial phase of Ca2+ uptake and the amount of the liberated H+ was more than that due to hydrolysis of ATP, generation of a pH difference as a result of the H+ efflux could not be detected by direct pH measurement with a pH meter. Alkalinization of the inside of the vesicles during Ca2+ uptake was more precisely examined by flow dialysis assay and a significant uptake of acetate or salicylate into the vesicles was found, suggesting the generation of a small pH difference across the SR membrane. From these results, it was concluded that counter-transport of H+ was operative in Ca2+ uptake but that only a relatively small pH difference was generated as a result of the H+ efflux. The intrinsic buffering capacity of sarcoplasmic reticulum vesicles was measured and a relatively large value (130 nmol H+/pH unit/mg at pH 6.2) was obtained.     

Synthesis of adenosine triphosphate during release of intravesicular and membrane-bound calcium ions from passively loaded sarcoplasmic reticulum.

Sarcoplasmic reticulum isolated from rabbit skeletal muscle and incubated in a medium containing Ca2+ in the absence of ATP retains intravesicular and/or membrane-bound Ca2+. The synthesis of ATP coupled with the release of intravesicular Ca2+ is totally inhibited by the ionophore X-537A. Release of the membrane-bound Ca2+, retained after short periods of incubation (10min) or after release of the intravesicular Ca2+ by ionophore X-537A, still supports some synthesis of ATP. The ratios of Ca2+ released to ATP synthesized are 2.5-3.2, when bound and intravesicular Ca2+ are released simultaneously, and 3.1-4.0, when only bound Ca2+ is released. The results show that the synthesis of ATP by sarcoplasmic reticulum during release of passively accumulated Ca2+ by EGTA [ethanedioxybis(ethylamine)tetra-acetic acid] is accompanied by a loss of membrane-bound Ca2+.     

Evidence for the presence of calsequestrin in two structurally different regions of myocardial sarcoplasmic reticulum

Localization of calsequestrin in chicken ventricular muscle cells was determined by indirect immunofluorescence and immuno-Protein A-colloidal gold labeling of cryostat and ultracryotomy sections, respectively. Calsequestrin was localized in the lumen of peripheral junctional sarcoplasmic reticulum, as well as in the lumen of membrane-bound structures present in the central region of the I-band, while being absent from the lumen of the sarcoplasmic reticulum in the A-band region of the cardiac muscle cells. Since chicken ventricular muscle cells lack transverse tubules, the presence of calsequestrin in membrane bound structures in the central region of the I-band suggests that these cells contain nonjunctional regions of sarcoplasmic reticulum that are involved in Ca2+ storage and possibly Ca2+ release. It is likely that the calsequestrin containing structures present throughout the I-band region of the muscle cells correspond to specialized regions of the free sarcoplasmic reticulum in the I-band called corbular sarcoplasmic reticulum. It will be of interest to determine whether Ca2+ storage and possibly Ca2+ release from junctional and nonjunctional regions of the sarcoplasmic reticulum in chicken ventricular muscle cells are regulated by the same or different physiological signals.     

Calreticulin, and not calsequestrin, is the major calcium binding protein of smooth muscle sarcoplasmic reticulum and liver endoplasmic reticulum

The distribution of calsequestrin and calreticulin in smooth muscle and non-muscle tissues was investigated. Immunoblots of endoplasmic reticulum proteins probed with anti-calreticulin and anti-calsequestrin antibodies revealed that only calreticulin is present in the rat liver endoplasmic reticulum. Membrane fractions isolated from uterine smooth muscle, which are enriched in sarcoplasmic reticulum, contain a protein band which is immunoreactive with anti-calreticulin but not with anti-calsequestrin antibodies. The presence of calreticulin in these membrane fractions was further confirmed by 45Ca2+ overlay and "Stains-All" techniques. Calreticulin was also localized to smooth muscle sarcoplasmic reticulum by the indirect immunofluorescence staining of smooth muscle cells with anti-calreticulin antibodies. Furthermore, both liver and uterine smooth muscle were found to contain high levels of mRNA encoding calreticulin, whereas no mRNA encoding calsequestrin was detected. We have employed an ammonium sulfate precipitation followed by Mono Q fast protein liquid chromatography, as a method by which calsequestrin and calreticulin can be isolated from whole tissue homogenates, and by which they can be clearly resolved from one another, even where present in the same tissue. Calreticulin was isolated from rabbit and bovine liver, rabbit brain, rabbit and porcine uterus, and bovine pancreas and was identified by its amino-terminal amino acid sequence. Calsequestrin cannot be detected in preparations from whole liver tissue, and only very small amounts of calsequestrin are detectable in ammonium sulfate extracts of uterine smooth muscle. We conclude that calreticulin, and not calsequestrin, is a major Ca2+ binding protein in liver endoplasmic reticulum and in uterine smooth muscle sarcoplasmic reticulum. Calsequestrin and calreticulin may perform parallel functions in the lumen of the sarcoplasmic and endoplasmic reticulum.     

Stimulation of Ca2+ uptake by cyclic AMP and protein kinase in sarcoplasmic reticulum-rich and sarcolemma-rich microsomal fractions from rabbit heart.

The effect of cyclic AMP on Ca2+ uptake by rabbit heart microsomal vesicular fractions representing mainly fragments of either sarcoplasmic reticulum or sarcolemma was investigated in the presence and absence of soluble cardiac protein kinase and with microsomes prephosphorylated by cyclic AMP-dependent protein kinase. The acceleration of oxalate-promoted Ca2+ uptake by fragmented sarcoplasmic reticulum following cyclic AMP-dependent membrane protein phosphorylation, observed by other authors, was confirmed. In addition it was found that the acceleration was greatest at pH 7.2 and almost negligible at pH 6.0 and pH 7.8. A very marked increase in Ca2+ uptake by cyclic AMP-dependent membrane protein phosphorylation was observed in the presence of boric acid, a reversible inhibitor of Ca2+ uptake. In addition to the microsomal fraction thought to represent mainly fragments of the sarcoplasmic reticulum, the effect of protein kinase and cyclic AMP on Ca2+ uptake was investigated in a cardiac sarcolemma-enriched membrane fraction. Ca2+ uptake by sarcolemmal vesicles, unlike Ca2+ uptake by sarcoplasmic reticulum vesicles, was inhibited by low doses of digitoxin. The acceleration of oxalate-promoted Ca2+ uptake by cyclic AMP and soluble cardiac protein kinase, however, was quite similar to what was seen in preparations of fragmented sarcoplasmic reticulum, which suggests that it may reflect an acceleration of active Ca2+ transport across the myocardial cell surface membrane.     

Differentiation between Ca2+ transport and ATP-induced Ca2+ binding by sarcoplasmic reticulum

The Ca²⁺ actively accumulated by sarcoplasmic reticulum isolated from skeletal muscle is composed of two fractions; one represented by intravesicular free Ca²⁺ and another represented by Ca²⁺ selectively bound to the membranes. Both of these Ca²⁺ fractions depend on ATP, although it is not clear whether ATP hydrolysis is essential for accumulation of the second Ca²⁺ fraction. The existence of the membrane-bound Ca²⁺ induced by ATP is clearly shown in experiments in which the Ca²⁺ retention by sarcoplasmic reticulum is measured in the presence and in the absence of X-537A, a Ca²⁺ ionophore, which makes the membrane permeable to Ca²⁺. Thus, in the presence of X-537A all Ca²⁺ accumulated due to ATP is bound to the membranes. This membrane-bound Ca²⁺ represents about 30 nmol/mg protein in the range of external $\text{p}\text{Ca}$ values of 7 to 3.5. The magnitude of this Ca²⁺ fraction is slightly higher whether or not the experiments are performed in the presence of oxalate, which greatly increased the intravesicular Ca²⁺ accumulation. Furthermore, taking advantage of the impermeability of sarcoplasmic reticulum to EGTA, it is possible to show the existence of the membrane-bound Ca²⁺ as a distinct fraction from that which exists intravesicularly.     

Changes in the fatty acid composition of sarcoplasmic reticulum lipids and calcium uptake activity

Supplementing the diet of rats with saflower oil or hydrogenated coconut oil resulted in marked changes in the fatty acid composition of the skeletal muscle sarcoplasmic reticulum hut did not affect such properties of the sarcoplasmic reticulum as the rate of Ca²⁺ uptake, the total amount of Ca²⁺ taken up, the rate and extent of Ca²⁺ release in the cold, and the basal and extra ATPase activities. Both of the oil supplements resulted in large increases in the proportion of linoleic acid in the sarcoplasmic reticulum hut neither of them significantly affected the proportion of polyenoic to saturated + monoenoic fatty acids. The relisons for the unexpectedly high linoleic acid content in the sarcoplasmic reticulum of the hydrogenated coconut oil supplemented rats are not known.     

Localization of the high affinity calcium binding protein and an intrinsic glycoprotein in sarcoplasmic reticulum membranes

Several proteins in sarcoplasmic reticulum preparations move in a band with a mobility, in sodium dodecyl sulfate-polyacrylamide gels (0.1 M phosphate buffer, pH 7.0), corresponding to a molecular mass of about 55,000 daltons. Only one of these proteins is the high affinity calcium binding protein. An intrinsic glycoprotein is also present in this band, and it is this glycoprotein which is found in vesicles reconstituted after dissolution of sarcoplasmic reticulum in deoxycholate. Both of these proteins are found in rather constant ratios with the ATPase in light, intermediate, and heavy sarcoplasmic reticulum vesicles. Transverse tubular vesicles can be isolated from the heavy sarcoplasmic reticulum vesicles after disruption of the membrane in a French pressure cell (Lau, Y.H., Caswell, A.H., and Brunschwig, J.P. (1977) J. Biol. Chem. 252, 5565-5574). These vesicles are enriched in their content of the high affinity calcium binding and depleted of the intrinsic glycoprotein. Cycloheptaamylose . fluorescamine complex (CFC) labels the intrinsic glycoprotein heavily indicating that it is at least partially exposed on the cytoplasmic surface of sarcoplasmic reticulum membranes. Since the carbohydrate component of the protein must lie in luminal spaces, it is inferred that the intrinsic glycoprotein is a transmembrane protein. The high affinity calcium binding protein is not labeled by CFC indicating that it is not exposed on the cytoplasmic surface of sarcotubular vesicles. The protein is also not affected by proteolytic digestion of sarcoplasmic reticulum vesicles and can be isolated intact from trypsin-digested vesicles. It is not removed from sarcoplasmic-reticulum vesicles by washing with buffers containing Chelex 100 or ethylene glycol bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). These data show that the high affinity calcium binding protein is localized in the interior of the sarcotubular system and suggest that it might be common to both sarcoplasmic reticulum and transverse tubular membranes.     

Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

Utilization of X-537A to distinguish between intravesicular and membrane-bound calcium ions in sarcoplasmic reticulum

The Ca²⁺ ionophore X-537A is employed as a tool to distinguish between intravesicular Ca²⁺ and surface membrane-bound Ca²⁺ in sarcoplasmic reticulum isolated from rabbit skeletal muscle. When sarcoplasmic reticulum is incubated in 20 mM Ca²⁺ in the absence of ATP, 10–12 h are necessary for measurable amounts of Ca²⁺ to penetrate into the vesicular space, as determined by the fact that X-537A releases Ca²⁺ from ‘loaded’ vesicles only after this period of incubation. A fraction of Ca²⁺ of 50–60nmol/mg protein, rapidly taken up by sarcoplasmic reticulum, exchanges with Mg²⁺ and K⁺ in the medium and is readily released by ethyleneglycol-bis-(β-aminoethyl ether)-N,N′-tetraacetic acid, but it is not released by X-537A. The slow-penetrating fraction of Ca²⁺ (30–40 nmol/mg protein) is rapidly released by X-537A. The results indicate that most of the Ca²⁺ retained by sarcoplasmic reticulum under conditions of passive uptake is bound to the external side of the membrane. The fraction of Ca²⁺ that slowly penetrates the vesicles remains essentially free inside the vesicles and only a small part is bound to the internal side of the membrane.     

Distinct immunopeptide maps of the sarcoplasmic reticulum Ca2+ release channel in malignant hyperthermia

Sarcoplasmic reticulum isolated from malignant hyperthermia-susceptible (MHS) muscle exhibits abnormalities in the regulation of calcium release. To identify the molecular basis of this abnormality, the Ca2+ release channel from both normal and MHS sarcoplasmic reticulum was examined using proteolytic digestion followed by immunoblot staining with a polyclonal antibody against the rabbit Ca2+ release channel protein. Under appropriate conditions, trypsin digestion of isolated sarcoplasmic reticulum vesicles from the two types of pigs revealed a distinct difference in the immunostaining pattern of the Ca2+ release channel-derived peptides. An approximate 86-kDa peptide was the predominant fragment in normal sarcoplasmic reticulum while an approximate 99-kDa peptide fragment was the major peptide detected in MHS sarcoplasmic reticulum. Digestion of sarcoplasmic reticulum vesicles isolated from four normal and four MHS pigs showed that the differences were highly reproducible. Trypsin digestion of sarcoplasmic reticulum isolated from heterozygous pigs, which contain one normal and one MHS allele, showed an antibody staining pattern that was intermediate between MHS and normal sarcoplasmic reticulum. These results can be explained by a primary amino acid sequence difference between the normal and MHS Ca2+ release channels and support the hypothesis that a mutation in the gene coding for the sarcoplasmic reticulum Ca2+ release channel is responsible for malignant hyperthermia.     

Immunoelectron microscopical localization of phospholamban in adult canine ventricular muscle

The subcellular distribution of phospholamban in adult canine ventricular myocardial cells was determined by the indirect immunogold-labeling technique. The results presented suggest that phospholamban, like the Ca2+-ATPase, is uniformly distributed in the network sarcoplasmic reticulum but absent from the junctional portion of the junctional sarcoplasmic reticulum. Unlike the Ca2+-ATPase, but like cardiac calsequestrin, phospholamban also appears to be present in the corbular sarcoplasmic reticulum. Comparison of the relative distribution of phospholamban immunolabeling in the sarcoplasmic reticulum with that of the sarcolemma showed that the density of phospholamban in the network sarcoplasmic reticulum was approximately 35-fold higher than that of the cytoplasmic side of the sarcolemma, which in turn was found to be three- to fourfold higher than the density of the background labeling. However, a majority of the specific phospholamban labeling within 30 nm of the cytoplasmic side of the sarcolemma was clustered and present over the sarcoplasmic reticulum in the subsarcolemmal region of the myocardial cells, suggesting that phospholamban is confined to the junctional regions between the sarcolemma and the sarcoplasmic reticulum, but absent from the nonjunctional portion of the sarcolemma. Although the resolution of the immunogold-labeling technique used (60 nm) does not permit one to determine whether the specific labeling within 30 nm of the cytoplasmic side of the sarcolemma is associated with the sarcolemma and/or the junctional sarcoplasmic reticulum, it is likely that the low amount of labeling in this region represents phospholamban associated with sarcoplasmic reticulum. These results suggest that phospholamban is absent from the sarcolemma and confined to the sarcoplasmic reticulum in cardiac muscle.     

Changes in intravesicular pH during Ca2+ transport in the sarcoplasmic reticulum

Studies with the use of [3H]acetate as an delta pH-indicator have established that pH in the native vesicles of sarcoplasmic reticulum is by 0.54 unit lower, than its extra-molecular value (6.5 units). The double [3H] and radioactive [3H] and [45Ca2+] labels were used to show that Ca2+ transport into the sarcoplasmic reticulum vesicles is accompanied by an increase in intravesicular pH. Carbonylcyanide-m-chlorophenylhydrazone, a protonophore, stimulates the equalization of the pH gradient (H+ removal) which is not accompanied by changes in the Ca2+ transport. In the presence of ionophore A23187 Ca2+ and [3H]acetate do not accumulate in vesicles in the ATP-dependent process. This indicates H+ removal from the vesicles only when there is the Ca2+ gradient creation and the absence of the close conjugation of Ca3+/2H+ realized by Ca2+-ATPase of sarcoplasmic reticulum.     

Changes in the structure, composition and function of sarcoplasmic-reticulum membrane during development.

The structure, chemical composition and function of the microsomal fraction, isolated by differential centrifugation and purified on sucrose gradients, from muscle of fetal, newborn and young rabbits were characterized and compared with those of sarcoplasmic reticulum vesicles from adult muscle. Negative staining shows that the microsomal vesicles isolated from muscles of embryos and newborn animals are smooth, in contrast to vesicles obtained from adult muscle which contain 4-nm particles on their surface. The particles appear first in the microsomal vesicles from muscles of 5--8-day-old rabbits. Their number increases with the age of the animals. Ca2+-pump protein, with molecular weight about 100000, accounts for 10% of the total protein content in sarcoplasmic reticulum membrane, isolated at the earliest stages of development analysed. Its amount increases continuously with the rabbit's age to the adult value of about 70% of total sarcoplasmic reticulum protein. The low amount of 100000-dalton protein and lack of 4-nm surface particles in sarcoplasmic reticulum vesicles obtained from fetal and newborn rabbits are strictly correlated with the low activity of Ca2+-dependent ATPase and the ability to take up Ca2+. These activities rise in parallel with the age of the rabbits. On the other hand, Mg2+-dependent ATPase activity is very high at the early stages of development and declines continuously to a low value in sarcoplasmic reticulum from adult muscle. The sarcoplasmic reticulum membrane from fetal and newborn rabbits contains a higher amount of lipids as compared with the membrane present in the muscle of adult animals. The ratio of both phospholipid to protein and neutral lipid to protein decreases with the age of the rabbits. The composition of sarcoplasmic reticulum phospholipids also changes during development.