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.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

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 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+-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.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Characterization of Ca2+ uptake in plasma membrane vesicles isolated from guinea pig ileum smooth muscle

We have characterized ATP-dependent Ca2+ transport into highly purified plasma membrane fraction isolated from guinea pig ileum smooth muscle. The membrane fraction contained inside-out sealed vesicles and was enriched 30-40-fold in 5'-nucleotidase and phosphodiesterase I activity as compared to post nuclear supernatant. Plasma membrane vesicles showed high rate (76 nmol/mg/min) and high capacity for ATP dependent Ca2+ transport which was inhibited by addition of Ca2+ ionophore A23187. The inhibitors of mitochondrial Ca2+ transport, i.e., sodium azide, oligomycin and ruthenium red did not inhibit ATP-dependent Ca2+ uptake into plasma membrane vesicles. The energy dependent Ca2+ uptake into plasma membranes showed very high specificity for ATP as energy source and other nucleotide triphosphates were ineffective in supporting Ca2+ transport. Phosphate was significantly better as Ca2+ trapping anion to potentiate ATP-dependent Ca2+ uptake into plasma membrane fraction as compared to oxalate. Orthovanadate, an inhibitor of cell membrane (Ca2+-Mg2+)-ATPase activity, completely inhibited ATP-dependent Ca2+ transport and the Ki was approximately 0.6 microM. ATP-dependent Ca2+ transport and formation of alkali labile phosphorylated intermediate of (Ca2+-Mg2+)-ATPase increased with increasing concentrations of free Ca2+ in the incubation mixture and the Km value for Ca2+ was approximately 0.6-0.7 microM for both the reactions.     

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.     

Partial purification and characterization of the (Ca2+ + Mg2+)-ATPase from squid optic nerve plasma membrane

A membrane fraction enriched in axolemma was obtained from optic nerves of the squid (Sepiotheutis sepioidea) by differential centrifugation and density gradient fractionation. The preparation showed an oligomycin- and NaN3-insensitive (Ca2+ + Mg2+)-ATPase activity. The dependence of the ATPase activity on calcium concentration revealed the presence of two saturable components. One had a high affinity for calcium (K1 1/2 = 0.12 microM) and the second had a comparatively low affinity (K2 1/2 = 49.5 microM). Only the high-affinity component was specifically inhibited by vanadate (K1 = 35 microM). Calmodulin (12.5 micrograms/ml) stimulated the (Ca2+ + Mg2+)-ATPase by approx. 50%, and this stimulation was abolished by trifluoperazine (10 microM). Further treatment of the membrane fraction with 1% Nonidet P-40 resulted in a partial purification of the ATPase about 15-fold compared to the initial homogenate. This (Ca2+ + Mg2+)-ATPase from squid optic nerve displays some properties similar to those of the uncoupled Ca2+-pump described in internally dialyzed squid axons, suggesting that it could be its enzymatic basis.     

Calmodulin-stimulated Ca(2+)-ATPases in the vacuolar and plasma membranes in cauliflower.

The subcellular locations of Ca(2+)-ATPases in the membranes of cauliflower (Brassica oleracea L.) inflorescences were investigated. After continuous sucrose gradient centrifugation a 111-kD calmodulin (CaM)-stimulated and caM-binding Ca(2+)-ATPase (BCA1; P. Askerlund [1996] Plant Physiol 110: 913-922; S. Malmström, P. Askerlund, M.G. Plamgren [1997] FEBS Lett 400: 324-328) comigrated with vacuolar membrane markers, whereas a 116-kD caM-binding Ca(2+)-ATPase co-migrated with a marker for the plasma membrane. The 116 kD Ca(2+)-ATPase was enriched in plasma membranes obtained by aqueous two-phase partitioning, which is in agreement with a plasma membrane location of this Ca(2+)-ATPase. Countercurrent distribution of a low-density intracellular membrane fraction in an aqueous two-phase system resulted in the separation of the endoplasmic reticulum and vacuolar membranes. The 111-kD Ca(2+)-ATPase co-migrated with a vacuolar membrane marker after countercurrent distribution but not with markers for the endoplasmic reticulum. A vacuolar membrane location of the 111-kD Ca(2+)-AtPase was further supported by experiments with isolated vacuoles from cauliflower: (a) Immunoblotting with an antibody against the 111-kD Ca(2+)-ATPase showed that it was associated with the vacuoles, and (b) ATP-dependent Ca2+ uptake by the intact vacuoles was found to be CaM stimulated and partly protonophore insensitive.     

A comparison of brush-border membranes prepared from rabbit small intestine by procedures involving Ca2+ and Mg2+ precipitation

Brush-border membranes were isolated from rabbit small intestine by procedures involving precipitation of undesired membranes with either 10 mM MgCl2 or 10 mM CaCl2. The membranes were compared on the basis of marker enzyme content and lipid composition. Ca2+-prepared membranes displayed a greater enrichment of alkaline phosphatase and sucrase activity compared to homogenate than did the Mg2+-prepared membranes. The former also displayed an impoverishment of (Na+ + K+)-ATPase activity, the specific activity of which increased several-fold in Mg2+-prepared membranes. Membranes prepared with Ca2+ were characterized by a lower phosphoacylglycerol-protein ratio and a higher phosphatidylethanolamine-phosphatidylcholine ratio. Although lysophosphoacylglycerols accounted for about 6% of the total phospholipids in these membranes compared to 2% in Mg2+-prepared membranes, the free fatty acid content was similar in both types of membranes. It was concluded that Ca2+ prepared membranes were less contaminated by basolateral membranes than were Mg2+-prepared membranes and the use of Ca2+ did not notably enhance degradation of endogenous lipids by brush-border membrane phospholipase A.     

An endogenous inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase

An inhibitor protein of synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was purified to apparent homogeneity from rat cerebrum by a molecular weight cut followed by chromatography of cytosol proteins with molecular weights between 10 000 and 3500 on DEAE-Sephadex at pH 5.2. The inhibitor could be partially inactivated by proteinases and dithiothreitol, but was heat-stable. Gel filtration gave a molecular weight of about 6000. Like the (Ca2+ + Mg2+)-ATPase inhibitor protein isolated from erythrocytes, the inhibitor from brain contains a characteristic high proportion of glutamic acid (36%) and glycine (37%) residues. Synaptic plasma membrane Mg2+-ATPase and microsomal membrane (Ca2+ + Mg2+)-ATPase did not respond to the inhibitor. Synaptic plasma membrane and erythrocyte membrane (Ca2+ + Mg2+)-ATPases, however, were affected. Inhibitory influence on synaptic membrane (Ca2+ + Mg2+)-ATPase was reversible, since inhibition could be relieved upon removal of inhibitor from saturable sites on the membrane. The inhibitor is not a calmodulin-binding protein, since the concentration of calmodulin for half-maximal activation of the ATPase was unaffected by its presence. Mode of inhibition of the (Ca2+ + Mg2+)-ATPase by the inhibitor was non-competitive.     

The inhibitor of liver plasma membrane (Ca2+-Mg2+)-ATPase. Purification and identification as a mediator of glucagon action

Rat liver plasma membranes contain (Ca2+-Mg2+)-ATPase sensitive to inhibition by both glucagon and Mg2+. We have previously shown that Mg2+ inhibition is mediated by a 30,000-dalton inhibitor, originally identified as a membrane-bound protein. In fact, this inhibitor is also present in the 100,000 X g supernatant of the total liver homogenate. Its purification was achieved from this fraction by a combination of ammonium sulfate washing, gel filtration, and cationic exchange chromatography. N-Ethylmaleimide (NEM) treatment caused the inactivation of the purified inhibitor, which suggested that this protein possesses at least one NEM-sensitive sulfhydryl group essential for its activity. Treatment of the liver plasma membranes with NEM resulted in a 2- and 5-fold decrease in the affinity of the (Ca2+-Mg2+)-ATPase for glucagon and Mg2+, respectively, while the basal enzyme activity remained unchanged. This effect of NEM was concentration-, pH-, and time-dependent, optimal conditions being obtained by a 60-min treatment of plasma membranes with 50 mM NEM, at pH 7 and at 4 degrees C. The presence of 0.5 mM Mg2+ during NEM treatment of the plasma membranes prevented NEM inactivation. Reconstitution experiments showed that addition of the purified inhibitor to NEM-treated plasma membranes restored the inhibitions of the (Ca2+-Mg2+)-ATPase by both magnesium and glucagon. It is proposed that the (Ca2+-Mg2+)-ATPase inhibitor not only confers its sensitivity of the liver (Ca2+-Mg2+)-ATPase to Mg2+, but also mediates the inhibition of this system by glucagon.     

Effects of hypochlorite-modified low-density and high-density lipoproteins on intracellular Ca2+ and plasma membrane Ca(2+)-ATPase activity of human platelets

The presence of hypochlorite-modified lipoproteins in atherosclerotic lesions suggests that HOCl, a naturally occurring oxidant formed by the myeloperoxidase-catalyzed reaction of H2O2 and Cl-, is a candidate for generation of modified lipoproteins in vivo. We have previously demonstrated that Cu(2+)-oxidized LDL inhibits platelet plasma membrane Ca(2+)-ATPase (PMCA) in isolated membranes and causes an increase in cytosolic Ca2+ in resting whole platelets. However, Cu(2+)-oxidized LDL may not be identical in structure and function to the physiologically modified lipoprotein. Since platelet function may be affected by native and modified lipoproteins, the effect of HOCl-modified LDL and HDL3 on platelet PMCA and on the free intracellular Ca2+ concentration ([Ca2+]i) of whole platelets has been investigated. We demonstrate that in contrast to Cu(2+)-oxidized LDL, HOCl-modified LDL and HDL3 stimulate platelet PMCA activity in isolated membranes and that this effect results in a decrease of [Ca2+]i in vivo. Thus, HOCl-oxidation produces modified lipoproteins with the potential for altering platelet function and with properties different from those of the Cu(2+)-oxidized counterparts.     

Biosynthesis of glycerolipids by hepatoma and liver microsomes. I. Fatty acyl-CoA ligase and acyl-CoA:sn-glycerol-3-phosphate acyltransferase

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on ouabain-Sepharose. By the method used two subfractions were obtained, one eluting freely from the affinity gel (MF1oua) and a second specifically retained by matrix-bound ouabain (MF2oua), with a total recovery of 95 per cent. Fractionation required the binding of matrix-bound ouabain to its plasma membrane receptor, i.e. (Na+ + K+)-ATPase. Increasing the temperature and binding time did not significantly alter the fractionation of plasma membranes into the two subfractions. Both plasma membrane subfractions separated by ouabain-Sepharose were of plasma membrane origin, as revealed by the identical specific activities of several membrane bound enzymes, gamma-glutamyl transpeptidase, alkaline phosphatase and Mg2+-ATPase in unseparated plasma membranes and in both subfractions, and by the identical amounts of the cytoskeletal protein actin in unseparated plasma membranes and subfractions. The plasma membrane subfractions MF1oua and MF2oua showed different structural and functional properties. In SDS-polyacrylamide gel electrophoresis polypeptides of 170, 150, 110, 94, 39, and 30 kDa were several-fold enriched in the adherent fraction, MF2oua. The phospholipid fatty acid composition of the plasma membrane subfractions proved to be different, as well. MF2oua contained significantly higher amounts of saturated fatty acids as compared to MF1oua. The specific activities of (Na+ + K+)-ATPase, Ca2+-ATPase and lysolecithin acyltransferase were highly enriched in the adherent fraction MF2oua, as compared to MF1oua. The data suggest that by the means of affinity chromatography on ouabain-Sepharose plasma membrane domains of the lymphocyte plasma membrane can be isolated, most probably implicated in the initiation of lymphocyte activation.     

Effects of calcium and soluble cytoplasmic activator protein (calmodulin) on various states of (Ca2+ + Mg2+)-ATPase activity in isolated membranes of human red cells

(Ca2+ + Mg2+)-ATPase activity of red cells and their isolated membranes was investigated in the presence of various Ca2+ concentrations and cytoplasmic activator protein. Red cell ATPase activity was high at low Ca2+ concentrations, and low at moderate and high concentrations of Ca2+. In the case of isolated membranes, both low and moderate ca2+ concentrations produced higher (Ca2+ + Mg2+)-ATPase activity than high Ca2+ concentration. Membrane-free hemolysate containing soluble activator of (Ca2+ + Mg2+)-ATPase produced a significant increase in (Ca2+ + Mg2+)-ATPase activity only at low ca2+ concentration. Regardless of Ca2+ and activator concentrations, the enzyme activity in the membrane was lower than lysed red cells. The low level of (Ca2+ + Mg2+)-ATPase activity seen at high Ca2+ concentration can be augmented by lowering the Ca2+ concentration of EGTA in the assay medium. However, once the membrane was exposed to a high Ca2+ concentration, the activator could no longer exert it maximum stimulation at the low Ca2+ concentration brought about by addition of EGTA. This loss of activation was not attributable to the Ca2+-induced denaturation of activator protein but rather related to the alteration of (Ca2+ + Mg2+)-ATPase states in the membrane. On the basis of these data, it is suggested that only a small portion of (Ca2+ + Mg2+)-ATPase activity of isolated membranes can be stimulated by the soluble activator and that (ca2+ + Mg2+)ATPase most likely exists in various states depending upon ca2+ concentration and the presence of activator. The enzyme state exhibiting the high degree of stimulation by activator may undergo irreversible damage in the presence of high Ca2+ concentrations.     

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.     

Enhancement of (Ca2+ + Mg2+)-ATPase activity of human erythrocyte membranes by hemolysis in isosmotic imidazole buffer. II. Dependence on calcium and a cytoplasmic activator.

1. Activity of the (Ca2+ + Mg2+)-ATPase of erythrocyte membrane may be enhanced by a cytoplasmic protein activator. The presence of Ca2+ is necessary for the ionic strength-dependent interaction between the erythrocyte membrane and the activator. This is true no matter the purity of activator (unfractionated hemolysis supernatant or partially purified activator) or the major source of ionic strength (imidazole or NaCl). 2. When the endogenous activator enhances (Ca2+ + Mg2+)-ATPase activity of the erythrocyte membrane, there is a physical association between activator and membrane. This association is not disrupted by a decrease in ionic strength to 0.005 but is reversed by exposure to 5 mM ethyleneglycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. 3. Activator binding necessary for enhancement of (Ca2+ + Mg2+)-ATPase activity may occur during preparation of membranes or during incubation for assay of ATPase.     

A high-affinity, calmodulin-sensitive (Ca2+ + Mg2+)-ATPase and associated calcium-transport pump in the Ehrlich ascites tumor cell plasma membrane

A unique cytoplast preparation from Ehrlich ascites tumor cells (G. V. Henius, P. C. Laris, and J. D. Woodburn (1979) Exp. Cell. Res. 121, 337-345), highly enriched in plasma membranes, was employed to characterize the high-affinity plasma membrane calcium-extrusion pump and its associated adenosine triphosphatase (ATPase). An ATP-dependent calcium-transport system which had a high affinity for free calcium (K0.5 = 0.040 +/- 0.005 microM) was identified. Two different calcium-stimulated ATPase activities were detected. One had a low (K0.5 = 136 +/- 10 microM) and the other a high (K0.5 = 0.103 +/- 0.077 microM) affinity for free calcium. The high-affinity enzyme appeared to represent the ubiquitous high-affinity plasma membrane (Ca2+ + Mg2+)-ATPase (calcium-stimulated, magnesium-dependent ATPase) seen in normal cells. Both calcium transport and the (Ca2+ + Mg2+)-ATPase were significantly stimulated by the calcium-dependent regulatory protein calmodulin, especially when endogenous activator was removed by treatment with the calcium chelator ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid. Other similarities between calcium transport and the (Ca2+ + Mg2+)-ATPase included an insensitivity to ouabain (0.5 mM), lack of activation by potassium (20 mM), and a requirement for magnesium. These similar properties suggested that the (Ca2+ + Mg2+)-ATPase represents the enzymatic basis of the high-affinity calcium pump. The calcium pump/enzyme system was inhibited by orthovanadate at comparatively high concentrations (calcium transport: K0.5 congruent to 100 microM; (Ca2+ + Mg2+)-ATPase: K0.5 greater than 100 microM). Upon Hill analysis, the tumor cell (Ca2+ + Mg2+)-ATPase failed to exhibit cooperative activation by calcium which is characteristic of the analogous enzyme in the plasma membrane of normal cells.