Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Purification of the Ca2+-and Mg2+-requiring ATPase from rat brain synaptic plasma membrane.

A Ca2+-ATPase (Ca2+- and Mg2+-requiring ATPase) was purified from a synaptic plasma-membrane fraction of rat brain. This enzyme had properties similar to those of plasma-membrane Ca2+-ATPases from other organs: its splitting of ATP was dependent on both Ca2+ and Mg2+, it bound in a Ca2+-dependent fashion to calmodulin-Sepharose and it cross-reacted with specific antibodies raised against human erythrocyte-membrane Ca2+-ATPase. It had an apparent Mr of 138 000, similar to those of plasma-membrane ATPases from human erythrocyte and from dog heart sarcolemma. Previous high-Ca2+-affinity ATPases observed in brain had Mr 100 000; in at least one case, such an ATPase probably represented a different type of enzyme, derived from coated vesicles.     

Ordering of bulk membrane lipid or protein promotes activity of plasma membrane Mg2+ATPase

Treatment of liver plasma membranes with phospholipase A2 or high doses of concanavalin A enhances the activity of Mg2+ATPase assayed at temperatures greater than 30 degrees C. The effects of the two treatments are not additive. Both the removal of phospholipids and binding of the lectin increase the degree of polarization of fluorescence of the lipid-soluble fluorophores, diphenylhexatriene and beta-parinaric acid, suggesting that decreased lipid fluidity may activate Mg2+-ATPase. In fact modification of lipid fluidity by reconstitution of phospholipase-treated membranes with phosphatidylcholines of defined fatty acid composition or by addition of cis-vaccenic acid showed a strong inverse correlation between Mg2+ATPase activity and lipid fluidity as monitored by fluorescence polarization. However, despite the ability of concanavalin A to nonspecifically order membrane lipid, its effect on Mg2+ATPase is apparently not mediated in this manner because other enzyme-activating lectins such as Ricinus communis agglutinin and wheat germ agglutinin are without effect on lipid fluidity. The facts that lectins of lower valency than tetravalent native concanavalin A such as divalent succinyl concanavalin A are far less effective in activating the enzyme and that paraformaldehyde treatment also activates suggests that cross-linking of membrane proteins is responsible. Hence, the diminution in activity of this membrane enzyme due to the disordering effect of heat in the physiological temperature range can be counteracted by isothermally increasing the order of either membrane lipid or protein.     

Localization of a Mg2+-activated ATPase in the plasma membrane of Trypanosoma cruzi

The Wachstein and Meisel incubation medium was used to detect ATPase activity in epimastigote, spheromastigote (amastigote), and bloodstream trypomastigote forms of Trypanosoma cruzi. Reaction product, indicative of enzyme activity, was associated with the plasma membrane covering the cell body and the flagellum of the parasite. No reaction product was found in the portion of the plasma membrane lining the flagellar pocket. The plasma membrane-associated ATPase activity was not inhibited by ouabain or oligomycin, was detected in incubation medium without K+, was inhibited by prolonged glutaraldehyde fixation, and its activity was diminished when Mg2+ was omitted from the incubation medium. The Ernst medium was used to detect Na+-K+-ATPase activity in T. cruzi. No reaction product indicative of the presence of this enzyme was detected. Reaction product indicative of 5'-nucleotidase was not detected in T. cruzi. Acid phosphatase activity was detected in lysosomes. Those results indicate that a Mg2+-activated ATPase is present in the plasma membrane of T. cruzi and that it can be used as an enzyme marker, provided that the mitochondrial and flagellar ATPases are inhibited, to assess the purity of plasma membrane fractions isolated from this parasite.     

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Evidence that the platelet plasma membrane does not contain a (Ca2+ + Mg2+)-dependent ATPase

The present study was designed to determine the subcellular distribution of the platelet (Ca2+ + Mg2+)-ATPase. Human platelets were surface labeled by the periodate-boro[3H]hydride method. Plasma membrane vesicles were then isolated to a purity of approx. 90% by a procedure utilizing wheat germ agglutinin affinity chromatography. These membranes were found to be 2.6-fold enriched in surface glycoproteins compared to an unfractionated vesicle fraction and almost 7-fold enriched compared to intact platelets. In contrast, the isolated plasma membranes showed a decreased specific activity of the (Ca2+ + Mg2+)-ATPase compared to the unfractionated vesicle fraction. This decrease in specific activity was found to be similar to that of an endoplasmic reticulum marker, glucose-6-phosphatase, and to that of a platelet inner membrane marker, phospholipase A2. We conclude, therefore, that the (Ca2+ + Mg2+)-ATPase is not located in the platelet plasma membrane but is restricted to membranes of intracellular origin.     

Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.     

Characterisation of a high affinity Ca2+-stimulated, Mg2+-dependent ATPase in the rat parotid plasma membrane

Two Ca2+-stimulated ATPase activities have been identified in the plasma membrane of rat parotid: (a) a (Ca2+ + Mg2+)-ATPase with high affinity for free Ca2+ (apparent Km = 208 nM, Vmax = 188 nmol/min per mg) and requiring micromolar concentration of Mg2+ and (b) a (Ca2+ or Mg2+)-ATPase with relatively low affinity for free Ca2+ (K0.5 = 23 microM) or free Mg2+ (K0.5 = 26 microM). The low-affinity (Ca2+ or Mg2+)-ATPase can be maximally stimulated by Ca2+ alone or Mg2+ alone. The high-affinity (Ca2+ + Mg2+)-ATPase exhibits sigmoidal kinetics with respect to ATP concentration with K0.5 = 0.4 mM and a Hill coefficient of 1.91. It displays low substrate specificity with respect to nucleotide triphosphates. Although trifluoperazine inhibits the activity of the high affinity (Ca2+ + Mg2+)-ATPase only slightly, it inhibits the activity of the low-affinity (Ca2+ or Mg2+)-ATPase quite potently with 22 microM trifluoperazine inhibiting the enzymic activity by 50%. Vanadate, inositol 1,4,5-trisphosphate, phosphatidylinositol 4,5-bisphosphate, Na+,K+ and ouabain had no effect on the activities of both ATPases. Calmodulin added to the plasma membranes does not stimulate the activities of both ATPases. The properties of the high-affinity (Ca2+ + Mg2+)-ATPase are distinctly different from those of the previously reported Ca2+-pump activity of the rat parotid plasma membrane.     

Rat liver plasma membrane Ca2+- or Mg2+-activated ATPase. Evidence for proton movement in reconstituted vesicles

The Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane was partly purified by treatments with sodium cholate and lysophosphatidylcholine, and by isopycnic centrifugation on sucrose gradients. The ATPase activity had high sensitivity to detergents, poor nucleotide specificity and broad tolerance for divalent cations. It was insensitive to mitochondrial ATPase inhibitors such as oligomycin and to transport ATPase inhibitors such as vanadate and ouabain. Using the cholate dialysis procedure, the partly purified enzyme was incorporated into asolectin vesicles. Upon addition of Mg2+-ATP, fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine (ACMA) was observed. The quenching was abolished by a protonophore, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Asolectin vesicles or purified ATPase alone failed to promote quenching. These data suggest that the Ca2+- or Mg2+-activated ATPase from rat liver plasma membrane is able of H+-translocation coupled to ATP hydrolysis.     

A high affinity Ca2+-stimulated and Mg2+-dependent ATPase in rat corpus luteum plasma membrane fractions

Plasma membrane fractions from rat corpus luteum contain two kinds of Ca2+-stimulated ATPase, one having a high affinity for Ca2+, the other a low affinity for Ca2+. The high affinity ATPase had a specific Ca2+ requirement with a K 1/2 of 0.2 to 0.3 microM; it had a Vmax of 105 nmol min-1 mg-1 and distributed, upon subcellular fractionation, with recognized plasma membrane enzymes. The properties of this enzyme indicate that it is a CA2+ extrusion pump. The low affinity pump (K 1/2 for Ca2+, about 15 microM) was nonspecific, being stimulated equally well by Ca2+ of Mg2+; its function is unknown. Although the high affinity ATPase resembled the erythrocyte Ca2+-pumping ATPase in the properties mentioned above, it differed in that it failed to respond to Mg2+ or calmodulin. The lack of response to Mg2+ was due to the enzyme's retention of endogenous Mg2+; it did, after incubation with chelators, show a Mg2+ requirement. However, we were unable to show any effect of added calmodulin or trifluoperazine. This failure may be related to the high content of tightly bound calmodulin in these membranes. Much of this calmodulin could not be extracted even by washing with 1 mM EGTA and/or 0.1% (w/v) Triton X-100. This enzyme, the erythrocyte enzyme, and the adipocyte plasma membrane Ca2+ ATPase all belong to the class of Ca2+ ATPases with plasma membrane distribution and high affinity for Ca2+, indicating that they are Ca2+ extrusion pumps. However, the data indicate that tissue-specific differences exist within this class, with the enzyme from adipocytes and rat corpus luteum belonging to a subclass in which the requirement for Mg2+ and any response to calmodulin are difficult to demonstrate.     

Ca2+-dependent ATPase activity of alveolar macrophage plasma membrane.

A plasma membrane fraction was isolated from lysates of Bacillus Calmette-Guérin-induced alveolar macrophages of rabbit. On the basis of morphological and biochemical criteria this fraction appeared to be minimally contaminated by other subcellular organelles. Concentrations of Ca2+, but not of Mg2+, from 6.10(-8) to 1.10(-5) M markedly stimulated the basal ATPase (EC 3.6.1.3) activity of the plasma membrane, with an apparent Km (Ca2+) of 1.10(-6) M. The specific activity of the Ca2+-ATPase assayed at pCa = 5.5 was enriched about 8-fold in the plasma membrane fraction over the macrophage lysate. In contrast, the specific activity of the K+, EDTA-activated ATPase, associated to macrophage myosin, increased only 1.3-fold. Oligomycin and -SH group reagents exerted no influence on the Ca2+-ATPase activity, which was on the contrary inhibited by detergents such as Triton X-100 and deoxycholate. The activity of the Ca2+-ATPase was maximal at pH 7, and was decreased by 50 mM Na+ and 5 mM K+. On the contrary, the activity of Mg2+-ATPase, also present in the plasma membrane fraction, had a peak at about pH 7.8, and was stimulated by Na+ plus K+. On account of its properties, it is suggested that the Ca2+-ATPase is a component of the plasma membrane of the alveolar macrophage, and that its function may be that of participating in the maintenance of low free Ca2+ concentrations in the macrophage cytosol.     

Ca2+- or Mg2+-dependent ATPase in plasma membrane of cultured endothelial cells from bovine carotid artery

ATPase was found in plasma membrane of cultured endothelial cells from bovine carotid artery. The activity of the enzyme solubilized by octaethyleneglycol mono-n-dodecyl ether was enhanced by the addition of Ca2+ or Mg2+ and was not affected by F-actin and ouabain. Vmax was 2.8 and 10.0 mumol Pi/mg protein per h for Ca2+- and Mg2+-dependent activity, respectively, and the corresponding Km was 4.8 X 10(-4) M and 3.2 X 10(-4) M. Molecular weight of the protein was estimated to be approx. 250 000, as determined by activity-staining electrophoresis with polyacrylamide gels.     

Covalent phosphorylation of the Mg2+-dependent ATPase of yeast plasma membranes

Phosphorylation by [gamma-32P]ATP of proteins associated with the plasma membrane of Saccharomyces cerevisiae has been studied both in vivo and in vitro. Although at least nine proteins are labeled in vivo, there is only one major protein labeled in vitro. This species with an apparent molecular weight of 114,000 has been identified as the plasma membrane Mg2+-ATPase. Phosphorylation of this enzyme occurs exclusively on serine residues. This is the first report that the proton-translocating ATPase of fungal plasma membranes is subject to phosphorylation by a protein kinase.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.