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PubMed1986

Chemical modification of sarcoplasmic reticulum. Stimulation of Ca2+ release.

Pretreatment of sarcoplasmic membranes with acetic or maleic anhydrides, which interact principally with amino groups, resulted in an inhibition of Ca2+ accumulation and ATPase activity. The presence of ATP, ADP or adenosine 5'-[beta, gamma-imido]triphosphate in the modification medium selectively protected against the inactivation of ATPase activity by the anhydride but did not protect against the inhibition of Ca2+ accumulation. Acetic anhydride modification in the presence of ATP appeared to increase specifically the permeability of the sarcoplasmic reticulum membrane to Ca2+ but not to sucrose, Tris, Na+ or Pi. The chemical modification stimulated a rapid release of Ca2+ from sarcoplasmic reticulum vesicles passively or actively loaded with calcium, from liposomes reconstituted with the partially purified ATPase fraction but not from those reconstituted with the purified ATPase. The inactivation of Ca2+ accumulation by acetic anhydride (in the presence of ATP) was rapid and strongly pH-dependent with an estimated pK value above 8.3 for the reactive group(s). The negatively charged reagents pyridoxal 5-phosphate and trinitrobenzene-sulphonate, which also interact with amino groups, did not stimulate Ca2+ release. Since these reagents do not penetrate the sarcoplasmic reticulum membranes, it is proposed that Ca2+ release is promoted by modification of internally located, positively charged amino group(s).

Acetic Anhydrides↗
PubMed1992

Sarcoplasmic-reticulum biogenesis in contraction-inhibited skeletal-muscle cultures.

We have previously shown that inhibition of the spontaneous contractile activity of cultured embryonic-chick skeletal-muscle fibres with tetrodotoxin (TTX) leads to decreased sarcoplasmic-reticulum Ca(2+)-transport rates and steady-state concentrations of the high-energy Ca(2+)-ATPase phosphoenzyme intermediate [Charuk & Holland (1983) Exp. Cell Res. 144, 143-157]. In the present study we used a monoclonal antibody to the Ca(2+)-ATPase to show that there is a decreased amount of enzyme accumulated by contraction-inhibited myotubes. Indirect immunofluorescence microscopy using the monoclonal antibody to the Ca(2+)-ATPase also revealed a disordered subcellular organization of the sarcotubular system in contraction-inhibited myotubes. The biogenesis of sarcoplasmic-reticulum proteins in TTX-paralysed myofibres was studied by labelling cells with [35S]methionine before isolation of the active Ca(2+)-pump membrane fraction. Protein turnover was selectively increased in that fraction from TTX-treated muscle cultures. Electrophoretic analysis and quantitative fluorography confirmed that decreased accumulation of the Ca(2+)-ATPase enzyme in contraction-inhibited myotubes was associated with increased turnover of this protein. The present results demonstrate that biogenesis of the sarcoplasmic-reticulum Ca(2+)-ATPase is regulated by the contractile activity of skeletal-muscle fibres.

Animals↗
PubMed1992

Characterization of ruthenium red-binding sites of the Ca(2+)-ATPase from sarcoplasmic reticulum and their interaction with Ca(2+)-binding sites.

Sarcoplasmic reticulum Ca(2+)-ATPase has previously been shown to bind and dissociate two Ca2+ ions in a sequential mode. This behaviour is confirmed here by inducing sequential Ca2+ dissociation with Ruthenium Red. Ruthenium Red binds to sarcoplasmic reticulum vesicles (6 nmol/mg) with a Kd = 2 microM, producing biphasic kinetics of Ca2+ dissociation from the Ca(2+)-ATPase, decreasing the affinity for Ca2+ binding. Studies on the effect of Ca2+ on Ruthenium Red binding indicate that Ruthenium Red does not bind to the high-affinity Ca(2+)-binding sites, as suggested by the following observations: (i) micromolar concentrations of Ca2+ do not significantly alter Ruthenium Red binding to the sarcoplasmic reticulum; (ii) quenching of the fluorescence of fluorescein 5'-isothiocyanate (FITC) bound to Ca(2+)-ATPase by Ruthenium Red (resembling Ruthenium Red binding) is not prevented by micromolar concentrations of Ca2+; (iii) quenching of FITC fluorescence by Ca2+ binding to the high-affinity sites is achieved even though Ruthenium Red is bound to the Ca(2+)-ATPase; and (iv) micromolar Ca2+ concentrations prevent inhibition of the ATP-hydrolytic capability by dicyclohexylcarbodi-imide modification, but Ruthenium Red does not. However, micromolar concentrations of lanthanides (La3+ and Tb3+) and millimolar concentrations of bivalent cations (Ca2+ and Mg2+) inhibit Ruthenium Red binding as well as quenching of FITC-labelled Ca(2+)-ATPase fluorescence by Ruthenium Red. Studies of Ruthenium Red binding to tryptic fragments of Ca(2+)-ATPase, as demonstrated by ligand blotting, indicate that Ruthenium Red does not bind to the A1 subfragment. Our observations suggest that Ruthenium Red might bind to a cation-binding site in Ca(2+)-ATPase inducing fast release of the last bound Ca2+ by interactions between the sites.

Animals↗
PubMed1993

Intraluminal calcium of the liver endoplasmic reticulum stimulates the glucuronidation of p-nitrophenol.

The relationship between the intraluminal Ca2+ content of endoplasmic reticulum and the rate of the glucuronidation of p-nitrophenol was investigated in isolated rat hepatocytes. Different agents which decrease the Ca2+ level in the endoplasmic reticulum [calcium ionophores (A23187, ionomycin) or Ca(2+)-ATPase inhibitors(thapsigargin,2,5-di-(t-butyl)-1,4-benzohydroquinone+ ++)] inhibited the conjugation of p-nitrophenol. Depletion of intracellular Ca2+ stores by preincubation of hepatocytes in the absence of free Ca2+ (in the presence of excess EGTA) also decreased the rate of glucuronidation; Ca2+ re-admission to EGTA-treated hepatocytes restored glucuronidation. In intact liver microsomes the p-nitrophenol UDP-glucuronosyl-transferase activity was not modified by varying the external free Ca2+ concentrations within a cytosol-like range. Emptying of the Ca2+ from the lumen of microsomal vesicles by A23187, after MgATP-stimulated Ca2+ sequestration, decreased the glucuronidation of p-nitrophenol. A similar effect was observed in filipin-permeabilized hepatocytes. In native and in detergent-treated microsomes, Ca2+ (1-10 mM) increased the p-nitrophenol UDP-glucuronosyltransferase activity. It is suggested that the physiological concentration of Ca2+ in the lumen of the endoplasmic reticulum is necessary for the optimal activity of p-nitrophenol UDP-glucuronosyltransferase; the depletion of Ca2+ decreases the activity of the enzyme.

Animals↗
PubMed1994

Binding of Ca2+ to the (Ca(2+)-Mg2+)-ATPase of sarcoplasmic reticulum: equilibrium studies.

Equilibrium fluorescence methods have been used to establish a model for Ca2+ binding to the (Ca(2+)-Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum and to define the effects of H+ and Mg2+ on Ca2+ binding. The basic scheme proposed is: E2 <--> E1 <--> E1Ca <--> El'Ca <--> E1'Ca2. The E1 conformation of the ATPase initially has one high-affinity binding site for Ca2+ exposed to the cytoplasmic side of the sarcoplasmic reticulum, but in the E2 conformation this site is unable to bind Ca2+; Ca2+ does not bind to luminal sites on E2. The second, outer, Ca(2+)-binding site on the ATPase is formed after binding of Ca2+ to the first, inner, site on E1 and the E1Ca <--> E1'Ca conformation change. The pH- and Mg(2+)-dependence of the E2 <--> E1 equilibrium has been established after changes in the fluorescence of the ATPase labelled with 4-nitrobenzo-2-oxa-1,3-diazole. It is proposed that Mg2+ from the cytoplasmic side of the sarcoplasmic reticulum can bind to the first Ca(2+)-binding site on both E1 and E2. It is proposed that the change in tryptophan fluorescence intensity after binding of Ca2+ follows from the E1Ca <--> E1'Ca change. The pH- and Mg(2+)-dependence of this change defines H(+)- and Mg(2+)-binding constants at the two Ca(2+)-binding sites. It is proposed that the change in tryptophan fluorescence observed on binding Mg2+ follows from binding at the second Ca(2+)-binding site. Effects of pH and Mg2+ on the fluorescence of the ATPase labelled with 4-(bromomethyl)-6,7-dimethoxycoumarin are proposed to follow from binding to a site on the ATPase, the 'gating' site, which affects the affinity of the first Ca(2+)-binding site for Ca2+ and affects the rate of dissociation of Ca2+ from the ATPase.

Animals↗
PubMed1995

Characterization of N-ethylmaleimide-sensitive thiol groups required for the GTP-dependent fusion of endoplasmic reticulum membranes.

The GTP-dependent fusion activity of endoplasmic reticulum membranes is thought to be required for the structural maintenance and post-mitotic regeneration of the endoplasmic reticulum. This fusion is sensitive to the thiol-alkylating agent N-ethylmaleimide. In many intracellular fusion events N-ethylmaleimide-sensitivity is associated with a homotrimeric ATPase called N-ethylmaleimide-sensitive fusion protein or NSF. The addition of cytosol containing NSF is known to restore fusion activity to N-ethylmaleimide-treated membranes. We found that the inhibition of fusion of rat liver endoplasmic reticulum membranes (microsomes) by N-ethylmaleimide was not reversed by the addition of untreated cytosol. Fusion was also unaffected by treatment with a buffer known to remove NSF from membranes. Accordingly, no membrane-associated NSF was detected by immunoblot analysis. These data suggest that microsome fusion requires an N-ethylmaleimide-sensitive component distinct from NSF. This component was tightly associated with the membranes, so we used a number of chemical probes to characterize it in situ. Its thiol groups did not appear to be part of a GTP-binding site. They showed relatively low reactivity with sodium periodate, which induces the formation of disulphide bonds between proximate thiol groups. The thiols were not protected against N-ethylmaleimide by Zn2+, a potent inhibitor of fusion which is known to efficiently co-ordinate thiol groups. To characterize the topology of the fusion-related thiol groups we used bulky thiol-specific reagents prepared by conjugating BSA or 10 kDa aminodextran to the bifunctional reagent N-succinimidyl 3-(2-pyridyldithio)propionate. The inhibition of fusion by these reagents indicated that these thiols are highly exposed on the membranes. This exposure might be important for the function of these groups during GTP-triggered fusion.

Animals↗
PubMed1996

Isoforms of endoplasmic reticulum Ca(2+)-ATPase are differentially expressed in normal and diabetic islets of Langerhans.

Glucose-dependent sequestration of Ca2+ into endoplasmic reticulum and its subsequent release play an important role in the control of intracellular Ca2+ concentration, which regulates insulin secretion in pancreatic beta-cells. The active uptake of cytosolic Ca2+ into endoplasmic reticulum is mediated by sarco-(endo)plasmic reticulum Ca(2+)-ATPases (SERCAs). We found, using RT-PCR with isoform-specific primers, that SERCA 2 and SERCA 3 mRNAs are co-expressed in human and rat islets of Langerhans and in the RINm5F beta-cell line. Immunochemical analysis also revealed the existence of two SERCA proteins with molecular masses of 110 and 115 kDa in beta-cell membranes. The 115 kDa protein was identified as SERCA 2b by its reaction with an isoform-specific antibody and the 110 kDa protein most probably corresponds to SERCA 3. The presence of two functionally different SERCA isoforms raises the possibility that they are located in distinct Ca2+ stores. There is evidence that altered Ca2+ handling in the beta-cell may contribute to the decreased insulin secretion seen in non-insulin dependent diabetes mellitus (NIDDM). We therefore investigated SERCA 2 and SERCA 3 mRNA expression by quantitative RT-PCR in islets prepared from Goto-Kakizaki (GK) rats, a non-obese spontaneous model of NIDDM. We found a significant reduction (about 68%) in SERCA 3 isoform expression. Since SERCA 2 expression was not significantly reduced, these genes are independently regulated and probably play distinct roles in islets of Langerhans. The marked decrease of SERCA 3 expression may constitute a defect in Ca2+ signalling in GK rat islets which could be a component of NIDDM.

Animals↗
PubMed1999

Changes in voltage activation of contraction in frog skeletal muscle fibres as a result of sarcoplasmic reticulum Ca2+-ATPase activity.

The effects of cyclopiazonic acid, a specific sarcoplasmic reticulum Ca2+-ATPase inhibitor, on isometric tension were studied in response to prolonged steady-state depolarization induced by a rapid change in extracellular potassium concentration (potassium contractures) in frog semitendinosus muscle fibres. Cyclopiazonic acid (1-10 microM) enhanced the amplitude and time-course of relaxation of 146 mM potassium contracture. In the presence of cyclopiazonic acid 0.5 microM, the relationship between the amplitude of potassium contractures and the membrane potential shifted to more negative potentials, whereas the steady-state inactivation curve was unchanged. These observations suggest that cyclopiazonic acid has no effect on voltage sensors. The difference between potassium contractures in the absence and presence of cyclopiazonic acid in skeletal muscle fibres implies that the amplitude and slow relaxation of tension during prolonged steady-state depolarization may be expected to depend not only on inactivation of the process regulating calcium release from the sarcoplasmic reticulum but also on the ability of the sarcoplasmic reticulum to pump calcium.

Animals↗
PubMed1997

Age-dependent accumulation of hybrid vasopressin-oxytocin gene products but not hybrid oxytocin-vasopressin products in the endoplasmic reticulum of Brattleboro rats.

The age-dependence of the incidence of magnocellular neurosecretory neurons containing abnormal accumulations of peptide in the rough endoplasmic reticulum was examined in homozygous Brattleboro rats and in their wild-type Long Evans counterparts. Neurons in which the immunophenotype of the peptide aggregates indicate that somatic cross-over mutations involving the 5' end of the vasopressin gene and the 3' end of the oxytocin gene have occurred, increased with age in homozygous Brattleboro rats, reaching a maximum of 24 cells per hypothalamus (approximately 0.6% of the vasopressin neurons). The increase occurred in both male and female animals but was significantly greater in females. The average incidence of such cells was 6 times greater in the supraoptic than in the paraventricular nucleus. No such cells could be detected in either nucleus of Long Evans rats despite the evidence for hybrid mRNA in these animals. Moreover, no accumulation of peptide translated from the hybrid mRNAs derived from the 5' end of the oxytocin gene and the 3' end of the vasopressin gene could be detected in either Brattleboro or Long Evans animals. These results strongly suggest that the accumulation of peptide in the rough endoplasmic reticulum of vasopressin neurons in homozygous Brattleboro rats is due to an abnormality other than the somatic crossing-over mutation. A second type of abnormal magnocellular neuron with accumulations of peptide in the rough endoplasmic reticulum, in which the immunophenotype of the peptide reveals products derived only from the oxytocin precursor, was present in both Long Evans and Brattleboro rats, but did not increase with age in Brattleboro rats. The incidence of these cells was similar in the supraoptic and paraventricular nuclei.

Aging↗
PubMed2001

A subfraction of the yeast endoplasmic reticulum associates with the plasma membrane and has a high capacity to synthesize lipids.

Large parts of the endoplasmic reticulum of the yeast, Saccharomyces cerevisiae, are located close to intracellular organelles, i.e. mitochondria and the plasma membrane, as shown by fluorescence and electron microscopy. Here we report the isolation and characterization of the subfraction of the endoplasmic reticulum that is closely associated with the plasma membrane. This plasma membrane associated membrane (PAM) is characterized by its high capacity to synthesize phosphatidylserine and phosphatidylinositol. As such, PAM is reminiscent of MAM, a mitochondria associated membrane fraction of the yeast [Gaigg, B., Simbeni, R., Hrastnik, C., Paltauf, F. & Daum, G. (1995) Biochim. Biophys. Acta 1234, 214-220], although the specific activity of phosphatidylserine synthase and phosphatidylinositol synthase in PAM exceeds several-fold the activity in MAM and also in the bulk endoplasmic reticulum. In addition, several enzymes involved in ergosterol biosynthesis, namely squalene synthase (Erg9p), squalene epoxidase (Erg1p) and steroldelta24-methyltransferase (Erg6p), are highly enriched in PAM. A possible role of PAM in the supply of lipids to the plasma membrane is discussed.

Blotting, Western↗
PubMed1999

Sarcoplasmic reticulum in vascular cells in hypertension and during proliferation.

1. Multiple sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and two types of sarcoplasmic reticulum Ca2+ channels, the ryanodine receptor and the inositol 1,4,5 triphosphate (IP3) receptor are expressed. The heterogeneity of the Ca2+ pumps and Ca2+ channels in vascular cells will be discussed. 2. An age-related change in expression of the SERCA isoforms is observed in smooth muscle cells. 3. The sarcoplasmic reticulum Ca(2+)-uptake rate and the level of SERCA 2 mRNA are different in thoracic than in abdominal aortas and in aortas from spontaneously hypertensive rats than from normotensive rats. 4. Proliferation of vascular smooth muscle cells is associated with major changes in intracellular Ca(2+)-handling mechanisms.

Animals↗
PubMed2001

Immunolocalization of a novel cholesteryl ester hydrolase in the endoplasmic reticulum of murine and human hepatocytes.

We have recently purified a cholesteryl ester hydrolase (CEH) from rat liver microsomes. Antibodies raised against the purified protein specifically reacted with a 106-kd protein and neutralized 90% of the CEH activity of rat liver microsomes (J Lipid Res 1999;40:715-725). In this work we have used the anti-CEH antibody to study both the subcellular distribution of the protein in hepatocytes as well as its tissue-specific expression in rat. Western blotting of subcellular fractions obtained from isolated rat hepatocytes revealed that the immunoreactive 106-kd CEH was exclusively localized in microsomes. The antibody also recognized a 106-kd protein in microsomes from mouse and human liver but not from rat nonparenchymal liver cells. Confocal microscopy of HepG2 cells revealed that CEH immunoreactive material colocalized with calnexin, a marker of the endoplasmic reticulum. Furthermore, high-resolution immunoelectron microscopy of rat liver thin sections exclusively localized the CEH immunoreactivity to the endoplasmic reticulum of the hepatocyte. No CEH immunoreactivity was observed in microsomes derived from adrenal glands, ovaries, testis, pancreas, intestine, white adipose tissue, mammary gland, lung, spleen, brain, aorta, and macrophages. We report a CEH localized to the endoplasmic reticulum, erCEH, in the mammalian hepatocyte. The subcellular localization and tissue-restricted pattern of expression of erCEH suggests that it might have unique functions in liver cholesterol metabolism.

Animals↗
PubMed2006

Human cytomegalovirus pUL37x1 induces the release of endoplasmic reticulum calcium stores.

The human CMV UL37x1-encoded protein, also known as the viral mitochondria-localized inhibitor of apoptosis, traffics to the endoplasmic reticulum and mitochondria of infected cells. It induces the fragmentation of mitochondria and blocks apoptosis. We demonstrate that UL37x1 protein mobilizes Ca(2+) from the endoplasmic reticulum into the cytosol. This release is accompanied by cell rounding, cell swelling, and reorganization of the actin cytoskeleton, and these morphological changes can be substantially blocked by a Ca(2+) chelating agent. The UL37x1-mediated release of Ca(2+) from the endoplasmic reticulum likely has multiple consequences, including induction of the unfolded protein response, modulation of mitochondrial function, induction of mitochondrial fission, and protection against apoptotic stimuli.

Actins↗
PubMed1971

Isolation of a calcium-sequestering protein from sarcoplasmic reticulum.

An acidic protein has been extracted from sarcoplasmic reticulum with KCl and deoxycholate. The protein, which remains soluble after extraction, has been highly purified by fractionation on DEAE-cellulose, Sephadex, and hydroxylaptite. It has a molecular weight of 44,000 and contains 392 amino acid residues per molecule, of which 146 are either glutamic or aspartic acid. No phosphorus, sialic acid, or lipid has been detected in the preparation. The protein has been shown to bind up to 970 nmol of Ca(++) per mg (43 mol/mol) at pH 7.5, with an apparent dissociation constant of 4 x 10(-5) M. Preliminary data indicate that the protein is unique to sarcoplasmic reticulum and that it is hydrophobically bonded on the interior of these vesicles. The protein is believed to play a role in sequestering calcium within sarcoplasmic reticulum. The name Calsequestrin is suggested for the protein.

Adenosine Triphosphatases↗
PubMed1982

Thyrotropin-releasing hormone mobilizes Ca2+ from endoplasmic reticulum and mitochondria of GH3 pituitary cells: characterization of cellular Ca2+ pools by a method based on digitonin permeabilization.

Treatment of 45Ca2+-loaded GH3 pituitary cells with various concentrations of digitonin revealed discrete pools (I and II) of cellular 45Ca2+ defined by differing detergent sensitivities. Markers for cytosol and intracellular organelles indicated that the two 45Ca2+ pools were correlated with the two major cellular Ca2+-sequestering organelles, endoplasmic reticulum (I) and mitochondria (II). Studies with various inhibitors were consistent with these assignments. Mitochondrial uncouplers preferentially depleted 45Ca2+ pool II while trifluoperazine selectively depleted 45Ca2+ pool I. Control experiments indicated that translocation of in situ organellar 45Ca2+ during and after permeabilization was negligible. We used the digitonin-permeabilization method to examine the effect of thyrotropin-releasing hormone (TRH) treatment on intracellular Ca2+ pools of GH3 pituitary cells. TRH was found to rapidly deplete both endoplasmic reticulum and mitochondrial exchangeable Ca2+ by 25-30%. The 45Ca2+ loss from both pools was maximal by 1 min after TRH addition and was followed by a recovery phase; mitochondrial 45Ca2+ content returned to control levels by 30 min. Previous treatment of cells with the mitochondrial uncoupler carbonyl cyanide p-trifluoromethoxy-phenylhydrazone blocked TRH-induced 45Ca2+ efflux from mitochondria, while previous treatment with valinomycin, an agent that depleted both 45Ca2+ pools, blocked any additional effect of TRH on these pools. We conclude that TRH rapidly promotes a net loss of exchangeable Ca2+ from GH3 cells as a result of hormone-induced mobilization of Ca2+ from endoplasmic reticulum and mitochondria.

Calcium↗
PubMed1983

Heavy metals induce rapid calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle.

Micromolar concentrations of mercury, silver, and other reagents known to react with sulfhydryl groups are shown to stimulate ATPase activity and inhibit active calcium uptake in sarcoplasmic reticulum vesicles derived from rabbit fast skeletal muscle. These effects are caused by a dramatic increase in the calcium permeability of the sarcoplasmic reticulum. Measurements of Ca2+ permeability were made using both isotopes and by spectrophotometric techniques using the Ca2+ indicator arsenazo III. Air oxidation of a sulfhydryl group to a disulfide group also leads to a large increase in the calcium permeability of the sarcoplasmic reticulum.

Animals↗
PubMed1984

Ca2+-ATPase of the sarcoplasmic reticulum shares a common domain with a membrane glycoprotein associated with the cytoskeleton of microvilli.

On the basis of structural observations, it has been proposed that cytoskeletal organization of the intestinal microvilli could be related to striated muscle structure. We have prepared antibodies against an amphipathic membrane glycoprotein (140 kilodaltons) associated with microvillar cytoskeleton and investigated its occurrence in striated muscle. Frozen sections of striated muscle were prepared according to the technique of Tokuyasu and visualized by indirect immunofluorescence with antibodies to the 140-kilodalton protein. In longitudinal sections the labeling was concentrated mainly in the area of the I band. In cross sections a honeycomb pattern was observed, suggesting that the recognized antigen was probably associated with the periphery of the myofibrils. Ultrathin frozen sections prepared for electron microscopy revealed that this antigen is closely associated with the membrane of the sarcoplasmic reticulum. In muscle extracts, the antibodies to the intestinal microvillar 140-kilodalton protein recognized a protein of 100 kilodaltons that comigrates with the Ca2+-ATPase of the sarcoplasmic reticulum. They recognized a purified preparation of the Ca2+-ATPase and, more specifically, the trypsin-generated fragment A2, the NH2-terminal part of the molecule that is exposed on the cytoplasmic face of the sarcoplasmic reticulum. Although these two proteins, expressed in unrelated cells, have a different molecular size and are inserted in different types of membranes, they share a common structural domain responsible for their crossreactivity. We propose that this domain could also be responsible for a common function--namely, the bridging of actin filaments to membranes.

Animals↗
PubMed1985

Modulation of stoichiometry of the sarcoplasmic reticulum calcium pump may enhance thermodynamic efficiency.

The coupling of calcium transport to ATP hydrolysis in rabbit muscle sarcoplasmic reticulum vesicles was determined under steady-state conditions in the presence of 5 mM oxalate and using various concentrations of vesicles to modulate the concentration of free Ca2+ in the medium. This experimental approach takes advantage of the fact that at high concentrations of vesicles the slow rate of liberation of Ca2+ from its oxalate complex becomes rate limiting for pumping, therefore pushing the steady-state levels of this cation to very low values. A reduction in the number of calcium ions transported per ATP cleaved from a value near 2 at a low concentration of vesicles (high medium Ca2+ concentration) to a limiting value of about 1 at a very high concentration of vesicles (low medium Ca2+ concentration) was observed. A marked decrease in the specific ATPase activity was also found to take place as the concentration of the sarcoplasmic reticulum vesicles was increased to high levels and the concentration of medium Ca2+ declined. The data presented indicate that binding of 1 Ca2+ to the sarcoplasmic reticulum ATPase is sufficient to activate the pump. Furthermore, these findings support the existence of a control mechanism for the calcium pump that helps to avoid a futile cycle of ATP cleavage with no net transport of calcium and that increases the pumping capability at low concentrations of free Ca2+.

Adenosine Triphosphate↗