Rabbit sarcoplasmic reticulum Ca(2+)-ATPase replaces yeast PMC1 and PMR1 Ca(2+)-ATPases for cell viability and calcineurin-dependent regulation of calcium tolerance

SERCA1a, the fast-twitch skeletal muscle isoform of sarco(endo)plasmic reticulum Ca(2+)-ATPase, was expressed in yeast using the promoter of the plasma membrane H(+)-ATPase. In the yeast Saccharomyces cerevisiae, the Golgi PMR1 Ca(2+)-ATPase and the vacuole PMC1 Ca(2+)-ATPase function together in Ca2+ sequestration and Ca2+ tolerance. SERCA1a expression restored growth of pmc1 mutants in media containing high Ca2+ concentrations, consistent with increased Ca2+ uptake in an internal compartment. SERCA1a expression also prevented synthetic lethality of pmr1 pmc1 double mutants on standard media. Electron microscopy and subcellular fractionation analysis showed that SERCA1a was localized in intracellular membranes derived from the endoplasmic reticulum. Finally, we found that SERCA1a ATPase activity expressed in yeast was regulated by calcineurin, a Ca2+/calmodulin-dependent phosphoprotein phosphatase. This result indicates that calcineurin contributes to calcium homeostasis by modulating the ATPase activity of Ca2+ pumps localized in intra-cellular compartments.     

An endoplasmic reticulum-bound Ca(2+)/Mn(2+) pump,ECA1, supports plant growth and confers tolerance to Mn(2+) stress

Plants can grow in soils containing highly variable amounts of mineral nutrients, like Ca(2+) and Mn(2+), though the mechanisms of adaptation are poorly understood. Here, we report the first genetic study to determine in vivo functions of a Ca(2+) pump in plants. Homozygous mutants of Arabidopsis harboring a T-DNA disruption in ECA1 showed a 4-fold reduction in endoplasmic reticulum-type calcium pump activity. Surprisingly, the phenotype of mutant plants was indistinguishable from wild type when grown on standard nutrient medium containing 1.5 mM Ca(2+) and 50 microM Mn(2+). However, mutants grew poorly on medium with low Ca(2+) (0.2 mM) or high Mn(2+) (0.5 mM). On high Mn(2+), the mutants failed to elongate their root hairs, suggesting impairment in tip growth processes. Expression of the wild-type gene (CAMV35S::ECA1) reversed these conditional phenotypes. The activity of ECA1 was examined by expression in a yeast (Saccharomyces cerevisiae) mutant, K616, which harbors a deletion of its endogenous calcium pumps. In vitro assays demonstrated that Ca(2+), Mn(2+), and Zn(2+) stimulated formation of a phosphoenzyme intermediate, consistent with the translocation of these ions by the pump. ECA1 provided increased tolerance of yeast mutant to toxic levels of Mn(2+) (1 mM) and Zn(2+)(3 mM), consistent with removal of these ions from the cytoplasm. These results show that despite the potential redundancy of multiple Ca(2+) pumps and Ca(2+)/H(+) antiporters in Arabidopsis, pumping of Ca(2+) and Mn(2+) by ECA1 into the endoplasmic reticulum is required to support plant growth under conditions of Ca(2+) deficiency or Mn(2+) toxicity.     

Sarcoplasmic-endoplasmic reticulum Ca2+-ATPase inhibition prevents endothelin A receptor antagonism in rat aorta

This study tested whether sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase regulates the ability of endothelin receptor antagonist to inhibit the endothelin-1 constriction. The endothelin A receptor antagonist BQ-123 (1 microM) completely relaxed constriction to 10 nM endothelin-1 in endothelium-denuded rat aorta. Challenge with cyclopiazonic acid (10 microM), a sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase inhibitor, during the plateau of endothelin-1 constriction enhanced the constriction by approximately 30%. BQ-123 relaxed the endothelin-1 plus cyclopiazonic acid constriction by only approximately 10%. In contrast, prazosin (1 microM), an alpha-adrenergic receptor antagonist, still completely relaxed the 0.3 muM phenylephrine constriction in the presence of cyclopiazonic acid. Verapamil relaxed the endothelin-1 plus cyclopiazonic acid constriction by approximately 30%, whereas Ni(2+) and 2-aminoethoxydiphenyl borate, nonselective cation channel and store-operated channel blockers, respectively, completely relaxed the constriction. These results suggest that lowered sarcoplasmic-endoplasmic reticulum Ca(2+)-ATPase activity selectively decreases the ability of endothelin receptor antagonist to inhibit the endothelin A receptor. The decreased antagonism may be related to the opening of store-operated channels and subsequent greater internalization of endothelin A receptor.     

Baseline cytosolic Ca2+ oscillations derived from a non-endoplasmic reticulum Ca2+ store

Cytosolic Ca(2+) oscillations can be due to cycles of release and re-uptake of internally stored Ca(2+). To investigate the nature of these Ca(2+) stores, we expressed the Pmr1 Ca(2+) pump of Caenorhabditis elegans in COS-1 cells and pretreated the cells with thapsigargin to prevent Ca(2+) uptake by the sarco(endo)plasmic reticulum Ca(2+)-ATPase. Pmr1 co-localized with the Golgi-specific 58K protein and was targeted to a Ca(2+) store that was less leaky for Ca(2+) than the endoplasmic reticulum and whose inositol trisphosphate receptors were less sensitive to inositol trisphosphate and ATP than those in the endoplasmic reticulum. ATP-stimulated Pmr1-overexpressing cells responded after a latency to extracellular Ca(2+) with a regenerative Ca(2+) signal, which could be prevented by caffeine. They also produced very stable ilimaquinone-sensitive baseline Ca(2+) spikes, even in the presence of thapsigargin. Such responses never occurred in non-transfected cells or in cells that overexpressed the type-1 sarco(endo)plasmic reticulum Ca(2+)-ATPase. Abortive Ca(2+) spikes also occurred in histamine-stimulated untransfected HeLa cells pretreated with thapsigargin, and they too were inhibited by ilimaquinone. We conclude that the Pmr1-induced Ca(2+) store, which probably corresponds to the Golgi compartment, can play a crucial role in setting up baseline Ca(2+) spiking.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Phenotypic screening of mutations in Pmr1, the yeast secretory pathway Ca2+/Mn2+-ATPase, reveals residues critical for ion selectivity and transport

Thirty-five mutations were generated in the yeast secretory pathway/Golgi ion pump, Pmr1, targeting oxygen-containing side chains within the predicted transmembrane segments M4, M5, M6, M7, and M8, likely to be involved in coordination of Ca(2+) and Mn(2+) ions. Mutants were expressed in low copy number in a yeast strain devoid of endogenous Ca(2+) pumps and screened for loss of Ca(2+) and Mn(2+) transport on the basis of hypersensitivity to 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and Mn(2+) toxicity, respectively. Three classes of mutants were found: mutants indistinguishable from wild type (Class 1), mutants indistinguishable from the pmr1 null strain (Class 2), and mutants with differential sensitivity to BAPTA and Mn(2+) toxicity (Class 3). We show that Class 1 mutants retain normal/near normal properties, including (45)Ca transport, Golgi localization, and polypeptide conformation. In contrast, Class 2 mutants lacked any detectable (45)Ca transport; of these, a subset also showed defects in trafficking and protein folding, indicative of structural problems. Two residues identified as Class 2 mutants in this screen, Asn(774) and Asp(778) in M6, also play critical roles in related ion pumps and are therefore likely to be common architectural components of the cation-binding site. Class 3 mutants appear to have altered selectivity for Ca(2+) and Mn(2+) ions, as exemplified by mutant Q783A in M6. These results demonstrate the utility of phenotypic screening in the identification of residues critical for ion transport and selectivity in cation pumps.     

The creatine kinase system is essential for optimal refill of the sarcoplasmic reticulum Ca2+ store in skeletal muscle.

Muscle function depends on an adequate ATP supply to sustain the energy consumption associated with Ca(2+) cycling and actomyosin sliding during contraction. In this regulation of energy homeostasis, the creatine kinase (CK) circuit for high energy phosphoryl transfer between ATP and phosphocreatine plays an important role. We earlier established a functional connection between the activity of the CK system and Ca(2+) homeostasis during depolarization and contractile activity of muscle. Here, we show how CK activity is coupled to the kinetics of spontaneous and electrically induced Ca(2+) transients in the sarcoplasm of myotubes. Using the UV ratiometric Ca(2+) probe Indo-1 and video-rate confocal microscopy in CK-proficient and -deficient cultured cells, we found that spontaneous and electrically induced transients were dependent on ryanodine-sensitive Ca(2+) release channels, sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps, extracellular calcium, and functional mitochondria in both cell types. However, at increasing sarcoplasmic Ca(2+) load (induced by electrical stimulation at 0.1, 1, and 10 Hz), the Ca(2+) removal rate and the amount of Ca(2+) released per transient were gradually reduced in CK-deficient (but not wild-type) myotubes. We conclude that the CK/phosphocreatine circuit is essential for efficient delivery of ATP to the sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase pumps and thereby directly influences sarcoplasmic reticulum refilling and the kinetics of the sarcoplasmic Ca(2+) signals.     

Functional expression in yeast of the human secretory pathway Ca(2+), Mn(2+)-ATPase defective in Hailey-Hailey disease.

The discovery and biochemical characterization of the secretory pathway Ca(2+)-ATPase, PMR1, in Saccharomyces cerevisiae, has paved the way for identification of PMR1 homologues in many species including rat, Caenorhabditis elegans, and Homo sapiens. In yeast, PMR1 has been shown to function as a high affinity Ca(2+)/Mn(2+) pump and has been localized to the Golgi compartment where it is important for protein sorting, processing, and glycosylation. However, little is known about PMR1 homologues in higher organisms. Loss of one functional allele of the human gene, hSPCA1, has been linked to Hailey-Hailey disease, characterized by skin ulceration and improper keratinocyte adhesion. We demonstrate that expression of hSPCA1 in yeast fully complements pmr1 phenotypes of hypersensitivity to Ca(2+) chelators and Mn(2+) toxicity. Similar to PMR1, epitope-tagged hSPCA1 also resides in the Golgi when expressed in yeast or in chinese hamster ovary cells. (45)Ca(2+) transport by hSPCA1 into isolated yeast Golgi vesicles shows an apparent Ca(2+) affinity of 0.26 microm, is inhibitable by Mn(2+), but is thapsigargin-insensitive. In contrast, heterologous expression of vertebrate sarcoplasmic reticulum and plasma membrane Ca(2+)-ATPases in yeast complement the Ca(2+)- but not Mn(2+)-related phenotypes of the pmr1-null strain, suggesting that high affinity Mn(2+) transport is a unique feature of the secretory pathway Ca(2+)-ATPases.     

A distinct endosomal Ca2+/Mn2+ pump affects root growth through the secretory process

Ca(2+) is required for protein processing, sorting, and secretion in eukaryotic cells, although the particular roles of the transporters involved in the secretory system of plants are obscure. One endomembrane-type Ca-ATPase from Arabidopsis (Arabidopsis thaliana), AtECA3, diverges from AtECA1, AtECA2, and AtECA4 in protein sequence; yet, AtECA3 appears similar in transport activity to the endoplasmic reticulum (ER)-bound AtECA1. Expression of AtECA3 in a yeast (Saccharomyces cerevisiae) mutant defective in its endogenous Ca(2+) pumps conferred the ability to grow on Ca(2+)-depleted medium and tolerance to toxic levels of Mn(2+). A green fluorescent protein-tagged AtECA3 was functionally competent and localized to intracellular membranes of yeast, suggesting that Ca(2+) and Mn(2+) loading into internal compartment(s) enhanced yeast proliferation. In mesophyll protoplasts, AtECA3-green fluorescent protein associated with a subpopulation of endosome/prevacuolar compartments based on partial colocalization with the Ara7 marker. Interestingly, three independent eca3 T-DNA disruption mutants showed severe reduction in root growth normally stimulated by 3 mm Ca(2+), indicating that AtECA3 function cannot be replaced by an ER-associated AtECA1. Furthermore, root growth of mutants is sensitive to 50 microm Mn(2+), indicating that AtECA3 is also important for the detoxification of excess Mn(2+). Curiously, Ateca3 mutant roots produced 65% more apoplastic protein than wild-type roots, as monitored by peroxidase activity, suggesting that the secretory process was altered. Together, these results demonstrate that the role of AtECA3 is distinct from that of the more abundant ER AtECA1. AtECA3 supports Ca(2+)-stimulated root growth and the detoxification of high Mn(2+), possibly through activities mediated by post-Golgi compartments that coordinate membrane traffic and sorting of materials to the vacuole and the cell wall.     

Functional and immunocytochemical evidence for the expression and localization of the secretory pathway Ca2+-ATPase isoform 1 (SPCA1) in cerebellum relative to other Ca2+ pumps

Membrane fractions of pig cerebellum show Ca2+-ATPase activity and Ca2+ transport due to the presence of the secretory pathway Ca2+-ATPase (SPCA). The SPCA1 isoform shows a wide distribution in the neurons of pig cerebellum, where it is found in the Golgi complex of the soma of Purkinje, stellate, basket and granule cells, and also in more distal components of the secretory pathway associated with a synaptic localization such as in cerebellar glomeruli. The SPCA1 may be involved in loading the Golgi complex and the secretory vesicles of these specific neuronal cell types with Ca2+ and also Mn2+. This study of the cellular and subcellular localization of SPCA1 pumps relative to the sarco(endo) plasmic reticulum Ca2+-ATPase and plasma membrane Ca2+-ATPase pumps hints to a possible specific role of SPCA1 in controlling the luminal secretory pathway Ca2+ (or Mn2+) levels as well as the local cytosolic Ca2+ levels. In addition, it helps to specify the zones that are most vulnerable to Ca2+ and/or Mn2+ dyshomeostasis, a condition that is held responsible of an increasing number of neurological disorders.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Ca2+-dependent protein kinase--a modulation of the plasma membrane Ca2+-ATPase in parotid acinar cells

Cross-talk between cAMP and [Ca(2+)](i) signaling pathways represents a general feature that defines the specificity of stimulus-response coupling in a variety of cell types including parotid acinar cells. We have reported recently that cAMP potentiates Ca(2+) release from intracellular stores, primarily because of a protein kinase A-mediated phosphorylation of type II inositol 1,4,5-trisphosphate receptors (Bruce, J. I. E., Shuttleworth, T. J. S., Giovannucci, D. R., and Yule, D. I. (2002) J. Biol. Chem. 277, 1340-1348). The aim of the present study was to evaluate the functional and molecular mechanism whereby cAMP regulates Ca(2+) clearance pathways in parotid acinar cells. Following an agonist-induced increase in [Ca(2+)](i) the rate of Ca(2+) clearance, after the removal of the stimulus, was potentiated substantially ( approximately 2-fold) by treatment with forskolin. This effect was prevented completely by inhibition of the plasma membrane Ca(2+)-ATPase (PMCA) with La(3+). PMCA activity, when isolated pharmacologically, was also potentiated ( approximately 2-fold) by forskolin. Ca(2+) uptake into the endoplasmic reticulum of streptolysin-O-permeabilized cells by sarco/endoplasmic reticulum Ca(2+)-ATPase was largely unaffected by treatment with dibutyryl cAMP. Finally, in situ phosphorylation assays demonstrated that PMCA was phosphorylated by treatment with forskolin but only in the presence of carbamylcholine (carbachol). This effect of forskolin was Ca(2+)-dependent, and protein kinase C-independent, as potentiation of PMCA activity and phosphorylation of PMCA by forskolin also occurred when [Ca(2+)](i) was elevated by the sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor cyclopiazonic acid and was attenuated by pre-incubation with the Ca(2+) chelator, 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA). The present study demonstrates that elevated cAMP enhances the rate of Ca(2+) clearance because of a complex modulation of PMCA activity that involves a Ca(2+)-dependent step. Tight regulation of both Ca(2+) release and Ca(2+) efflux may represent a general feature of the mechanism whereby cAMP improves the fidelity and specificity of Ca(2+) signaling.     

A Plasma Membrane-Type Ca2+-ATPase of 120 Kilodaltons on the Endoplasmic Reticulum from Carrot (Daucus carota) Cells (Properties of the Phosphorylated Intermediate)

Cytosolic Ca2+ levels are regulated in part by Ca2+-pumping ATPases that export Ca2+ from the cytoplasm; however, the types and properties of Ca2+ pumps in plants are not well understood. We have characterized the kinetic properties of a 120-kD phosphoenzyme (PE) intermediate formed during the reaction cycle of a Ca2+-ATPase from suspension-cultured carrot (Daucus carota) cells. Only one Ca2+-dependent phosphoprotein was formed when carrot membrane vesicles were incubated with [[gamma]-32P]ATP (W.L. Hsieh, W.S. Pierce, and H. Sze [1991] Plant Physiol 97: 1535-1544). Formation of this 120-kD phosphoprotein was inhibited by vanadate, enhanced by La3+, and decreased by hydroxylamine, confirming its identification as an intermediate of a phosphorylated-type Ca2+-translocating ATPase. The 120-kD Ca2+-ATPase was most abundant in endoplasmic reticulum-enriched fractions, in which the Ca2+-ATPase was estimated to be 0.1% of membrane protein. Direct quantitation of Ca2+-dependent phosphoprotein was used to examine the kinetics of PE formation. PE formation exhibited a Km for Ca2+ of 1 to 2 [mu]M and a Km for ATP of 67 nM. Relative affinities of substrates, determined by competition experiments, were 0.075 [mu]M for ATP, 1 [mu]M for ADP, 100 [mu]M for ITP, and 250 [mu]M for GTP. Thapsigargin and cyclopiazonic acid, specific inhibitors of animal sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, had no effect on PE formation; erythrosin B inhibited with 50% inhibition at <0.1 [mu]M. Calmodulin (1 [mu]M) stimulated PE formation by 25%. The results indicate that the carrot 120-kD Ca2+-ATPase is similar but not identical to animal plasma membrane-type Ca2+- ATPase and yet is located on endomembranes, such as the endoplasmic reticulum. This type of Ca2+ pump may reside on the cortical endoplasmic reticulum, which is thought to play a major role in anchoring the cytoskeleton and in facilitating secretion.     

Myeloperoxidase-derived oxidants inhibit sarco/endoplasmic reticulum Ca2+-ATPase activity and perturb Ca2+ homeostasis in human coronary artery endothelial cells

The sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) plays a critical role in Ca(2+) homeostasis via sequestration of this ion in the sarco/endoplasmic reticulum. The activity of this pump is inhibited by oxidants and impaired in aging tissues and cardiovascular disease. We have shown previously that the myeloperoxidase (MPO)-derived oxidants HOCl and HOSCN target thiols and mediate cellular dysfunction. As SERCA contains Cys residues critical to ATPase activity, we hypothesized that HOCl and HOSCN might inhibit SERCA activity, via thiol oxidation, and increase cytosolic Ca(2+) levels in human coronary artery endothelial cells (HCAEC). Exposure of sarcoplasmic reticulum vesicles to preformed or enzymatically generated HOCl and HOSCN resulted in a concentration-dependent decrease in ATPase activity; this was also inhibited by the SERCA inhibitor thapsigargin. Decomposed HOSCN and incomplete MPO enzyme systems did not decrease activity. Loss of ATPase activity occurred concurrent with oxidation of SERCA Cys residues and protein modification. Exposure of HCAEC, with or without external Ca(2+), to HOSCN or HOCl resulted in a time- and concentration-dependent increase in intracellular Ca(2+) under conditions that did not result in immediate loss of cell viability. Thapsigargin, but not inhibitors of plasma membrane or mitochondrial Ca(2+) pumps/channels, completely attenuated the increase in intracellular Ca(2+) consistent with a critical role for SERCA in maintaining endothelial cell Ca(2+) homeostasis. Angiotensin II pretreatment potentiated the effect of HOSCN at low concentrations. MPO-mediated modulation of intracellular Ca(2+) levels may exacerbate endothelial dysfunction, a key early event in atherosclerosis, and be more marked in smokers because of their higher SCN(-) levels.     

Molecular cloning and chromosomal localization of a sarco/endoplasmic reticulum-type Ca2(+)-ATPase of Drosophila melanogaster

We report here the characterization of a Drosophila melanogaster cDNA that encodes a sarco/endoplasmic reticulum-type Ca2(+)-ATPase. Previously we amplified the phosphorylation site - FITC-binding site fragment of this cDNA by Polymerase Chain Reaction utilizing homology primers (Váradi, A., Gilmore-Hebert, M. and Benz, E. J. Jr. (1989) FEBS Letters 258 203-207) and in the present work we used this fragment as probe for isolation of a cDNA clone with the intire coding region. The isolated 3.3 kb clone has been sequenced and the primary structure of the encoded protein has been deduced. It consists of 1002 amino acids with all the characteristic features of the P-type ATPases. It shows 67-71% amino acid identity and an apparently identical hydropathy profile with the mammalian sarco/endoplasmic reticulum-type Ca2(+)-ATPases. On basis of sequence similarity we suggest that the first transmembrane segment of sarco/endoplasmic reticulum type Ca2(+)-ATPases may be responsible for targeting of these transport proteins into the organellar membrane. The gene of this sarco/endoplasmic reticulum-type Ca2(+)-ATPase has been mapped on the right arm of chromosome 2, in section 60A.     

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.