Transport ATPase Of Sarcoplasmic Reticulum Research Articles

Ca2+-ATPase Molecules as a Calcium-Sensitive Membrane-Endoskeleton of Sarcoplasmic Reticulum.

The Ca2+-transport ATPase of sarcoplasmic reticulum (SR) is an integral, transmembrane protein. It sequesters cytoplasmic calcium ions released from SR during muscle contraction, and causes muscle relaxation. Based on negative staining and transmission electron microscopy of SR vesicles isolated from rabbit skeletal muscle, we propose that the ATPase molecules might also be a calcium-sensitive membrane-endoskeleton. Under conditions when the ATPase molecules scarcely transport Ca2+, i.e., in the presence of ATP and ≤ 0.9 nM Ca2+, some of the ATPase particles on the SR vesicle surface gathered to form tetramers. The tetramers crystallized into a cylindrical helical array in some vesicles and probably resulted in the elongated protrusion that extended from some round SRs. As the Ca2+ concentration increased to 0.2 µM, i.e., under conditions when the transporter molecules fully carry out their activities, the ATPase crystal arrays disappeared, but the SR protrusions remained. In the absence of ATP, almost all of the SR vesicles were round and no crystal arrays were evident, independent of the calcium concentration. This suggests that ATP induced crystallization at low Ca2+ concentrations. From the observed morphological changes, the role of the proposed ATPase membrane-endoskeleton is discussed in the context of calcium regulation during muscle contraction.

Rearrangement of boundary, stand-by, and fluid lipids during the formation of two-dimensional crystals of Ca2+-ATPase

Ca2+-transport ATPase of sarcoplasmic reticulum of rabbit skeletal and carp abdominal muscle is essential for the removal of large amounts of Ca2+ ions during the relaxation of the muscle. As in the case of other lipid–protein systems, fluid lipid and motionally restricted boundary lipid sites lead to multicomponent spin label EPR spectra. Having subtracted the dominating fluid component a two-component lineshape is recovered as a subtraction endpoint, suggesting the presence of a third, motionally less restricted stand-by lipid shell as fraction of boundary lipids. On adding 5mM decavanadate the phosphate binding site is blocked and the protein dimers form a two dimensional array, at least in the case of the sarcoplasmic reticulum of rabbit. During two-dimensional crystal formation of rabbit Ca2+-ATPase an increase of lipids was found in boundary and stand-by shells. No such change was observed in the case of the sarcoplasmic reticulum of carp abdominal muscle.

Functional evidence of a transmembrane channel within the Ca 2+ transport ATPase of sarcoplasmic reticulum

Ca 2+ efflux can be studied conveniently following dilution of sarcoplasmic reticulum (SR) vesicles preloaded with 45Ca 2+ by active transport. The rates of efflux are highly dependent on ATPase substrates and cofactors (P i, Mg 2+, Ca 2+ and ADP) in the efflux medium. On the other hand, pbenothiazines stimulate efflux through a passive permeability channel with no coupled catalytic events. Efflux activation by manipulation of catalytically active ATPase ligands, as well as by the catalytically inactive phenothiazines, can be prevented by thapsigargin, which is a highly specific inhibitor of the Ca 2+-ATPase. This demonstrates that the passive channel activated by phenothiazines is an integral part of the ATPase, and can operate either uncoupled or coupled to catalytic events.

The Modulation of the Calcium Transport by Skeletal Muscle Sarcoplasmic Reticulum in the Hibernating European Hamster

Calcium transport of skeletal muscle sarcoplasmic reticulum was comparatively studied in hibernating and summer active European hamsters (Cricetus cricetus L.). Crude homogenates from psoas, soleus and mixed skeletal muscles were used. Protein yield was strongly reduced in the muscle homogenates of hibernating hamsters. The calcium concentration in the muscle of hibernating hamsters was increased to a much higher content than in the serum. In the same animals the maximal rate of calcium uptake and the calcium storing capacity of sarcoplasmic reticulum were augmented by 43% and respectively 17%. Kinetic experiments with various concentrations of free calcium revealed in the hibernating animals higher uptake rates and a lower apparent calcium affinity than in the summer active hamsters. Some shift of calcium uptake rate and calcium affinity similar to that of a fast-twitch muscle was also observed in winter active animals kept at 22 degrees C under natural photoperiod. By contrast, the activity of the calcium dependent ATPase was not increased, suggesting a tighter coupling during hibernation between calcium dependent ATP-hydrolysis and calcium transport. No seasonal difference was observed in the calcium release by KCl-caffeine from calcium loaded vesicles of sarcoplasmic reticulum. Proportion and size of fibre types were studied with cold cross sections from psoas and soleus muscles. An average atrophy of about 25% was found during hibernation in both muscles. Cytochemistry revealed, however, a different reduction of cross area between type-I- and type-II-fibres, which reaches values up to 46% in the type-II-fast-fibres of the slow soleus muscle. Electron microscopy did not show any definite change in the distribution and amount of sarcoplasmic reticulum. The results suggest that during hibernation a modulation in the properties of calcium transport ATPase of sarcoplasmic reticulum occurs to better support the calcium transport function at low temperatures, which in turn warrants the restoration of ion homeostasis in the course of the arousal.

21] Analysis of two-dimensional crystals of Ca2+-ATPase in sarcoplasmic reticulum

Publisher Summary This chapter focuses on the analysis of two-dimensional crystals of Ca 2+ -ATPase in sarcoplasmic reticulum. The Ca 2+ -transport ATPase of sarcoplasmic reticulum (SR) is an intrinsic membrane protein with a molecular weight of 109,000. It is asymmetrically distributed in the SR membrane with much of its mass exposed on the cytoplasmic surface, where it can be visualized by negative staining in the form of 40-A-diameter surface particles. The intramembranous portion of the Ca 2+ -ATPase is revealed by freeze-etching as particles of about 75 A diameter that are more numerous in the cytoplasmic than in the luminal fracture face of the membrane. During ATP-dependent Ca 2+ transport, the Ca 2+ -ATPase alternates between two distinct conformations, E l and E 2 . The Ca 2+ transport is initiated by interaction of the Ca 2+ -ATPase with Ca 2+ and ATP in the E l conformation. Phosphorylation of the enzyme by ATP is followed by conversion from the E l into the E 2 enzyme form. The Ca 2+ transport is completed by the release of Ca 2+ from the enzyme in the E2 conformation, and the subsequent hydrolysis of the phosphoenzyme intermediate. The E l conformation is stabilized by saturation of the high-affinity Ca 2+ binding site of the enzyme with Ca 2+ or lanthanides.

Competition between decavanadate and fluorescein isothiocyanate on the Ca2+-ATPase of sarcoplasmic reticulum.

The binding of vanadate and fluorescein isothiocyanate to the Ca2+-transport ATPase of sarcoplasmic reticulum (EC 3.6.1.3) was analyzed. Monovanadate binds to the Ca2+-transport ATPase at a single high affinity site (site 1), that is presumably related to the binding site for inorganic orthophosphate, and to one of the two sites for decavanadate. Binding of vanadate to this site stabilizes the enzyme in the E2 conformation, with inhibition of ATPase activity and the formation of crystalline arrays of Ca2+-ATPase. Decavanadate also binds with high affinity to a second site on the Ca2+-ATPase (site 2), that is blocked by fluorescein isothiocyanate and may be part of the binding site for ATP. Crystallization of Ca2+-ATPase in sarcoplasmic reticulum, labeled with fluorescein isothiocyanate, by either monovanadate or decavanadate implies that occupation of site 1, but not of site 2, by vanadate is required for the conformational change of Ca2+-ATPase leading to the formation of crystalline arrays.

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase.

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.

Generation, characterization and ELISA of monospecific antibodies against the subunits of a Ca 2+-dependent protein kinase and a Ca 2+-transport ATPase from rabbit skeletal muscle

Monospecific precipitating sheep antibodies were generated for the first time against the purified, homogeneous α-, β- and γ-subunits of the Ca 2+-dependent protein kinase, phosphorylase kinase, from rabbit muscle. As reference, antibodies against the holoenzyme and the Ca 2+-transport ATPase of sarcoplasmic reticulum were induced. In all cases antibody titers could be quantitated (standard error 5–10%) by enzyme-linked immunosorbent assay. Differentiation of antibody binding was achieved by quantitative precipitation and complement fixation assays. In general maximal antibody titers were reached 56 days after primary immunization and high titers (5000) were maintained for several weeks. Anti-α, anti-β and anti-γ avidly precipitate the denatured subunits employed as immunogens as well as the native enzyme. No cross-reactivity between antibodies against a specific subunit and any of the other heterologous subunits was demonstrable in double immunodiffusion assays providing no evidence for immunologically identical sites on the α-, β- and γ-subunits. Since anti-α, anti-β and anti-γ strongly inhibit enzyme activity, it is likely that they do so primarily by sterically interfering with the binding of the large substrate phosphorylase b (M r 2.0 × 10 5) to phosphorylase kinase (M r 1.3 × 10 6). It cannot be excluded, however, that anti-β and anti-γ bind to the active sites on these 2 subunits.

Calmodulin-dependent elevation of calcium transport associated with calmodulin-dependent phosphorylation in cardiac sarcoplasmic reticulum

The rate of calcium transport by sarcoplasmic reticulum vesicles from dog heart assayed at 25°C, pH 7.0, in the presence of oxalate and a low free Ca 2+ concentration (approx. 0.5 μM) was increased from 0.091 to 0.162 μmol·mg −1·min −1 with 100 nM calmodulin, when the calcium-, calmodulin-dependent phosphorylation was carried out prior to the determination of calcium uptake in the presence of a higher concentration of free Ca 2+ (preincubation with magnesium, ATP and 100 μM CaCl 2; approx. 75 μM free Ca 2+). Half-maximal activation of calcium uptake occurs under these conditions at 10–20 nM calmodulin. The rate of calcium-activated ATP hydrolysis by the Ca 2+-, Mg 2+-dependent transport ATPase of sarcoplasmic reticulum was increased by 100 nM calmodulin in parallel with the increase in calcium transport; calcium-independent ATP splitting was unaffected. The calcium-, calmodulin-dependent phosphorylation of sarcoplasmic reticulum, preincubated with approx. 75 μM Ca 2+ and assayed at approx. 10 μM Ca 2+ approaches maximally 3 nmol/mg protein, with a half-maximal activation at about 8 nM calmodulin; it is abolished by 0.5 mM trifluperazine ∗∗ ∗∗ 10-[3-(4-Methylpiperazin-1-yl)propyl]-2-trifluoromethyl-phenothiazine; trifluoperazine. . More than 90% of the incorporated [ 32P]phosphate is confined to a 9–11 kDa protein, which is also phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and most probably represents a subunit of phospholamban. The stimulatory effect of 100 nM calmodulin on the rate of calcium uptake assayed at 0.5 μM Ca 2+ was smaller following preincubation of sarcoplasmic reticulum vesicles with calmodulin in the presence of approx. 75 μM Ca 2+, but in the absence of ATP, and was associated with a significant degree of calmodulin-dependent phosphorylation. However, the stimulatory effect on calcium uptake and that on calmodulin-dependent phosphorylation were both absent after preincubation with calmodulin, without calcium and ATP, suggestive of a causal relationship between these processes.

Synthesis of the calcium transport ATPase of sarcoplasmic reticulum and other muscle proteins during development of muscle cells in vivo and in vitro

The effect of medium Ca 2+ concentration upon the concentration and the rate of synthesis of muscle proteins was investigated in chicken pectoralis muscle cultures. There is an easily identifiable class of muscle protein which includes the Ca 2+-ATPase of sarcoplasmic reticulum, myosin, troponin C, ATP : creatine phosphotransferase, muscle specific actin, tropomyosin 1 and 2, and muscle hemagglutinin, which show a large increase in concentration during normal development. The increased synthesis of these proteins was inhibited, without inhibition of cell proliferation, in culture media of relatively low Ca 2+ concentration, 0.05–0.3 mM, where fusion was prevented. Similar medium Ca 2+ concentration was required for the expression of all these proteins, suggesting their coordinate regulation. The proteins are denoted as ‘calcium-modulated proteins’. The increased Ca 2+ transport activity of sarcoplasmic reticulum in cultured chicken pectoralis muscle cells during development at 1.8 mM medium calcium concentration represents de novo synthesis of the Ca 2+ transport ATPase, as shown by immunoprecipitation, active site labeling and direct identification of the Ca 2+ transport ATPase on two-dimensional gel electropherograms of whole muscle homogenates. The concentration and the turnover rate of the majority of the muscle proteins is not affected significantly by medium Ca 2+ concentration between 0.06 and 1.8 mM. It is proposed that increase in cytoplasmic free Ca 2+ concentration during fusion plays a central role in the regulation of the synthesis of calcium-modulated proteins.

Effect of temperature on the Ca2+ transport ATPase of sarcoplasmic reticulum

Effect of temperature on the Ca2+ transport ATPase of sarcoplasmic reticulum

The effect of anti-ATPase antibodies upon the Ca ++ transport of sarcoplasmic reticulum

Sheep or guinea pig antisera against the purified Ca ++ transport ATPase of sarcoplasmic reticulum inhibit Ca ++ transport due to a complement-dependent damage of the membrane, which causes massive leakage of Ca ++. The Ca ++-activated ATPase activity is only slightly affected even at ten times higher antibody concentration than that required for inhibition of Ca ++ transport. Antibodies prepared against the Ca ++ binding protein (C 1 protein) have no influence upon either ATPase activity or Ca ++ transport and ferritin-labeled anti-C 1 antibodies do not bind to microsomes.