The localization of (Ca2+ or Mg2+)-ATPase in plasma membranes of renal proximal tubular cells

Basolateral and brush-border vesicles from pig kidney cortex were prepared by differential centrifugation followed by free-flow electrophoresis. A low-affinity (Ca2+ or Mg2+)-ATPase which co-migrated with alkaline phosphatase was demonstrated. A considerable enrichment (by a factor of 10) of this ATPase activity was only observed in the brush-border and not in the basolateral membrane fractions. Maximal stimulation of this brush-border enzyme by Ca2+ was achieved when the ratio of Ca2+ to ATP reached a value between 1 and 2. The enzyme was not inhibited by excess Ca2+ or Mg2+. A kinetic analysis of the azide-insensitive (Ca2+ or Mg2+)-ATPase gave a Km of 0.43 mM for Ca-ATP and of 0.14 mM for Mg-ATP.     

Effect of chloride and bicarbonate ions on Mg2+ -ATPase of plasma membranes of bream (Abramis brama L.) brain sensitive to GABAA-ergic compounds

Effect of Cl and HCO3- ions on the Mg2+ -ATPase activity of the plasma membrane of bream brain was investigated. Cl- (5 or 10 mM) and HCO3- (1-5 mM) individually have low effect on the "basal" Mg2+ -ATPase. Simultaneous presence of Cl- and HCO3- in the incubation medium significantly increased the enzyme activity. Maximum effect of anions on the enzyme is observed in the presence of Cl- (approximately 7 mM) and HCO3- (approximately 3 mM). Br- can replace Cl- under joint effect with HCO3-, while I- has half maximum activity compared with Cl-. Bicuculline (7 microM) inhibits completely the joint effect of Cl- and HCO3- on the enzyme, while it has no effect on the "basal" Mg2+ -ATPase activity. SH-reagents (5, 5-dithiobis-2-nitrobenzoic acid, N-ethylmaleimide), oligomycine and orthovanadate inhibited the Cl-, HCO3- -activated Mg2+ -ATPase. The obtained results demonstrated that Mg2+ -ATPase of the bream brain sensitive to GABAergic ligands at a fixed concentrations of Cl- and HCO3- ions in the incubation medium is Cl-, HCO3- -activated by Mg2+ -ATPase, whose activity meets a number of requirements to the system which may be involved by GABAA receptors in the Cl-/HCO3- -exchange processes.     

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.     

Membrane adenosine triphosphatase activities in rat pancreas

The membrane ATPase activities present in rat pancreas were studied to investigate the possible role of ATPase enzymes in HCO3(-) secretion in the pancreas. It was found that all the HCO3(-)-sensitive (anion-sensitive) ATPase activity was accountable as pancreatic mitochondrial ATPase, thus supporting the view that a distinct plasma membrane 'bicarbonate-ATPase' is not involved in HCO3(-) secretion in pancreas. A remarkably high Mg+- and CA2+-requiring ATPase activity (30 mumol ATP hydrolysed/min per mg) was found in the plasma membrane fraction (rho = 1.10-1.13). This activity has been characterized in some detail. It is inhibited by p-fluorosulfonylbenzoyladenosine, an affinity label analogue of ATP and the analogue appears to label covalently a protein of Mr approximately 35 000. The (Ca2+ + Mg2+)-ATPase activity did not form a 'phosphorylated-intermediate' and was vanadate-insensitive. These and other tests have served to demonstrate that the (Ca2+ + Mg2+)-ATPase activity is different in properties from (Na+ + K+)-ATPase, Ca2+-ATPase, (H+ + K+)-ATPase or mitochondrial H+-ATPase. Apart from the (Ca2+ + Mg2+)-ATPase of plasma membrane and mitochondrial ATPase, the only other membrane ATPase activities noted were (Na+ + K+)-ATPase, which occurred in the same fractions as the (Ca2+ + Mg2+)-AtPase at rho = 1.10-1.13 and was of surprisingly low activity, and an ATPase activity in light membrane fractions (rho - 1.08-1.09) derived from zymogen granule membranes. At this time, therefore, there is no obvious candidate for an ATPase activity at the luminal surface of pancreatic cells which is directly involved in ion transport, but the results presented here direct attention to the high activity (Ca2+ + Mg2+)-ATPase in the plasma membrane fraction.     

Further evidence for the existence of an intrinsic bicarbonate-stimulated Mg2+-ATPase in brush border membranes isolated from rat kidney cortex

Summary The aim of this study was to provide further evidence for the existence of a nonmitochondrial bicarbonate-stimulated Mg²⁺-ATPase in brush border membranes derived from rat kidney cortex. A plasma membrane fraction rich in brush border microvilli and a mitochondrial fraction were isolated by differential centrifugation. Both fractions contain a Mg²⁺-ATPase activity which can be stimulated by bicarbonate. The two Mg²⁺-ATPases are stimulated likewise by chloride, bicarbonate, and sulfite or inhibited by oligomycin and aurovertin, though to different degrees. In contrast to these similarities, only the Mg²⁺-ATPase activity of the mitochondrial fraction is inhibited by atractyloside, a substance which blocks an adenine nucleotide translocator in the inner mitochondrial membrane. On the other hand, filipin, an antibiotic that complexes with cholesterol in the membranes inhibits exclusively the Mg²⁺-ATPase of the cholesterol-rich brush border membranes. Furthermore it could be demonstrated by the use of bromotetramisole, an inhibitor of alkaline phosphatase activity, that the Mg²⁺-ATPase activity in the membrane fraction is not due to the presence of the highly active alkaline phosphatase in these membranes. These results support the assumption that an intrinsic bicarbonate-stimulated Mg²⁺-ATPase is present in rat kidney brush border membranes.     

Purification and properties of H+-translocating, Mg2+-adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae

H+-translocating, Mg2+-ATPase was solubilized from vacuolar membranes of Saccharomyces cerevisiae with the zwitterionic detergent N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate and purified by glycerol density gradient centrifugation. Partially purified vacuolar membrane H+-ATPase, which had a specific activity of 18 units/mg of protein, was separated almost completely from acid phosphatase and alkaline phosphatase. The purified enzyme required phospholipids for maximal activity and hydrolyzed ATP, GTP, UTP, and CTP, with this order of preference. Its Km value for Mg2+-ATP was determined to be 0.21 mM and its optimal pH was 6.9. ADP inhibited the enzyme activity competitively, with a Ki value of 0.31 mM. The activity of purified ATPase was strongly inhibited by N,N'-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, tributyltin, 7-chloro-4-nitrobenzoxazole, diethylstilbestrol, and quercetin, but was not affected by oligomycin, sodium azide, sodium vanadate, or miconazole. It was not inhibited at all by antiserum against mitochondrial F1-ATPase or mitochondrial F1-ATPase inhibitor protein. These results indicated that vacuolar membrane H+-ATPase is different from either yeast plasma membrane H+-ATPase or mitochondrial F1-ATPase. The vacuolar membrane H+-ATPase was found to be composed of two major polypeptides a and b of Mr = 89,000 and 64,000, respectively, and a N,N'-dicyclohexylcarbodiimide binding polypeptide c of Mr = 19,500, whose polypeptide composition was also different from those of either plasma membrane H+-ATPase or mitochondrial F1-ATPase of S. cerevisiae.     

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.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Comparative properties of sensitive to GABAA-ergic ligands, Cl(-), HCO3(-)-activated Mg2+-ATPase from brain plasma membranes of fish and rats

Action of Cl(-) + HCO3(-) ions on Mg2+-ATPase from brain plasma membranes of fish and rats has been studied. Maximal effect of the anions on the Mg2+-ATPase activity is revealed in the presence of 10 mM Cl(-) and 3 mM HCO3(-) at physiological values of pH of incubation medium. The studied Cl(-), HCO3(-)-activated Mg2+-ATPases of both animal species, by their sensitivity to SH-reagents (5,5-dithiobis-nitrobenzoic acid, N-ethylmaleimide), oligomycin, and orthovanadate, are similar to transport ATPase of the P-type, but differ from them by molecular properties and by sensitivity to ligands of GABAA-receptors. It has been established that the sensitive to GABAA-ergic ligands, Cl(-), HCO3(-)-activated Mg2+-ATPase from brain of the both animal species is protein of molecular mass around 300 kDa and of Stocks' radius 5.4 nm. In fish the enzyme is composed of one major unit of molecular mass approximately 56 kDa, while in rats--of three subunits of molecular masses about 57, 53, and 45 kDa. A functional and structural coupling of the ATP-hydrolyzing areas of the studied enzyme to sites of binding of GABAA-receptor ligands is suggested.     

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.     

Observations on the (Ca2+ plus Mg2+)-ATPase activator found in various mammalian erythrocytes.

1. A soluble activator of membrane (Ca2+ plus Mg2)-ATPase is present in hemolysates of the newborn calf and cow, the new born and adult pig as well as human erythrocytes. 2. The activator is also found in reticulocytes of the adult pig. 3. The activator obtained from any of the above species is capable of stimulating the membrane (Ca2+ plus Mg2+)-ATPases of the other species, regardless of the age of the animals. 4. The results obtained from density fractionation of human erythrocytes revealed that the soluble factor has little simulatory effect on membranes of young erythrocytes from which it is derived but caused a marked stimulation on (Ca2+ plus Mg2+)-ATPase activity of the intermediate aged and old erythrocyte membranes. 5. The above observations support the following conclusions: (a) the extremely low levels of (Ca2+ plus Mg2+)-ATPase in cow erythrocytes is not due to the lack of a (Ca2+ plus Mg2+)-ATPase activator; (b) the distribution of (Ca2+ plus Mg2+)-atpase activator is not species specific and the differences in the level of membrane (Ca2+ plus Mg2+)-ATPase activity in various species of cells is an inherent property of that particular membrane (c) the (Ca2+ plus Mg2+)-ATPase activator is present at least from the time of reticulocyte formation and remain during tthe life span of the erythrocyte.     

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.     

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.     

Characterization of the Ca2+- or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle

Transverse tubule membranes isolated from rabbit skeletal muscle have high levels of a Ca2+- or Mg2+-ATPase with Km values for Ca-ATP or Mg-ATP in the 0.2 mM range, but do not display detectable levels of ATPase activity activated by micromolar [Ca2+]. The transverse tubule enzyme is less temperature or pH dependent than the Ca2+-ATPase of sarcoplasmic reticulum and hydrolyzes equally well ATP, ITP, UTP, CTP, and GTP. Of several ionic, non-ionic, and zwitterionic detergents tested, only lysolecithin solubilizes the transverse tubule membrane while preserving ATPase activity. After extraction of about 50% of the transverse tubule proteins by solubilization with lysolecithin most of the ATPase activity remains membrane bound, indicating that the Ca2+- or Mg2+-ATPase is an intrinsic membrane enzyme. A second extraction of the remaining transverse tubule proteins with lysolecithin results in solubilization and partial purification of the enzyme. Sedimentation of the Ca2+- or Mg2+-ATPase, partially purified by lysolecithin solubilization, through a continuous sucrose gradient devoid of detergent leads to additional purification, with an overall 3- to 5-fold purification factor. The purified enzyme preparation contains two main protein components of molecular weights 107,000 and 30,000. Cholesterol, which is highly enriched in the transverse tubule membrane, copurifies with the enzyme. Transverse tubule membrane vesicles also display ATP-dependent calcium transport which is not affected by phosphate or oxalate. The possibility that the Ca2+- or Mg2+-ATPase is the enzyme responsible for the Ca2+ transport displayed by isolated transverse tubules is discussed.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

Increase of (Ca2+ +Mg2+)-ATPase activity of renal basolateral membranes by platelet-derived growth factor through a specific receptor

Studies were made on the direct effect of platelet-derived growth factor (PDGF) on the high-affinity (Ca2+ +Mg2+)-ATPase, a membrane bound Ca2+-extrusion pump enzyme of the basolateral membranes (BLM) of canine kidney (Km for free Ca2+ = 1.0 x 10(-7) M, Vmax = 180 nmol Pi/mg/min). At 1 x 10(-7) M free Ca2+, PDGF (10(-10)-10(-8) M) stimulated the enzyme activity significantly. Addition of 5 - 200 microM suramin, a compound that blocks binding of PDGF to its receptors on cell membranes, inhibited the stimulatory effect of PDGF dose-dependently (IC50 = 40 microM). A high affinity specific receptor for PDGF (Kd = 4.4 x 10(-10) M, Bmax = 460 fmol/mg protein) was detected on BLM preparations by radioreceptor assay with 125I-PDGF and unlabelled PDGF. Suramin (10-1000 microM) also inhibited the binding of PDGF to BLM preparations dose-dependently. From these results, it is proposed that PDGF stimulates (Ca2+ +Mg2+)-ATPase activity of kidney BLM preparations by enhancing its affinity for free Ca2+ through a specific receptor.     

The calmodulin-stimulated (Ca2+ + Mg2+)-ATPase in hemoglobin S erythrocyte membranes: effects of sickling and oxidative agents

A decrease in the reactivity of erythrocyte membrane (Ca2+ + Mg2+)-ATPase to calmodulin stimulation has been observed in aging red cells and in various types of hemolytic anemias, particularly in sickle red cell membranes. Unlike the aging process, the defect in the (Ca2+ + Mg2+)-ATPase from SS red blood cells is not secondary to a decrease in calmodulin activity and is already present in the least dense SS red blood cells separated on a discontinuous density gradient. Deoxygenated AS red cells were forced to sickle by lowering the pH, raising the osmolarity of the buffer (sickling pulse). Under these conditions an inhibition of the calmodulin-stimulated enzyme was observed only if several cycles of oxygenation/deoxygenation were applied. No alteration of the enzyme could be detected after submitting AS red blood cells to other conditions or in AA red blood cells submitted to the same treatments. This suggests that oxidative processes are involved in the alterations of the (Ca2+ + Mg2+)-ATPase activity. Treatment of membranes from AA erythrocytes by thiol group reagents and malondialdehyde, a by-product of auto-oxidation of membrane unsaturated lipids and a cross-linking agent of cytoskeletal proteins, led to a partial inhibition of the calmodulin-stimulated (Ca2+ + Mg2+)-ATPase. We postulate that the hyperproduction of free radicals described in the SS red blood cells and involved in the destabilization of the membrane may be also responsible for the (Ca2+ + Mg2+)-ATPase failure.     

The role of a (Ca2+ + Mg2+)-ATPase of the rough endoplasmic reticulum in regulating intracellular Ca2+ during cholinergic stimulation of rat pancreatic acini

Rough endoplasmic reticulum membranes, purified from isolated rat pancreatic acini stimulated by carbachol, had a decreased Ca2+ content and increased (Ca2+ + Mg2+)-ATPase activity. Ca2+ was regained and ATPase activity reduced to control levels only after blockade by atropine. The (Ca2+ + Mg2+)-ATPase was activated by free Ca2+ (half-maximal at 0.17 microM; maximal at 0.7 microM) over the concentration range which occurs in the cell cytoplasm. Pretreatment with EGTA, at a high concentration (5 mM), inhibited ATPase activity which, our results suggest, was due to removal of a bound activator such as calmodulin. The rate of (Ca2+ + Mg2+)-ATPase actively declined during the 10-min period over which maximal active accumulation of Ca2+ by membrane vesicles occurs. In the presence of ionophore A23187, which released actively accumulated Ca2+ and stimulated the (Ca2+ + Mg2+)-ATPase, this time-dependent decline in activity was not observed. Our data provide evidence that the activity of the Ca2+-transporting ATPase of the rough endoplasmic reticulum is regulated by both extra and intravesicular Ca2+ and is consistent with a direct role of this enzyme in the release and uptake of Ca2+ during cholinergic stimulation of pancreatic acinar cells.