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.     

Is there a plasma membrane-located anion-sensitive ATPase? III. Identity of the erythrocyte enzyme with (Ca2+ + Mg2+)-ATPase.

The characteristics of the anion-sensitive Mg2+-ATPase activity of the rabbit erythrocyte have been studied in a lyophilized ghost preparation. The enzyme appears to be different from the anion-sensitive Mg2+-ATPase activity of other tissues in many parameters, such as optimal pH, effects of various anions, oligomycin sensitivity and effects of Triton X-100. The enzyme is insensitive towards inhibition by irreversibly bound 4,4'-diisothiocyano-dihydrostilbene-2,2'-disulfonic acid (H2DIDS). This excludes a relationship between the enzyme and the "band 3" protein, which is thought to be involved in the anion exchange over the erythrocyte membrane. From the effects of ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), CaCl2, chlorpromazine and ruthenium red it is concluded that the enzyme activity does not represent a separate entity but is part of the (Ca2+ + Mg2+)-ATPase system of the erythrocyte membrane. A reported stimulatory effect of carbonic anhydrase is attributed to a contamination of the carbonic anhydrase preparation by calcium and/or (Ca2+ + Mg2+)-ATPase activator protein.     

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.     

Lipid composition of different preparations of brain Na+, K+-ATPase

The content and composition of phospholipids is determined in beef microsomal and synaptosomal fractions and also in these fractions preparations solubilized with triton X-100 (0.1%) and digitonin (0.2%). It is shown that the microsomal fraction is richer in phospholipids. The solubilized fragments of microsomes have less or the same amount of phospholipids per protein unit than the initial fraction of microsomes, and the solubilized fragments of synaptosomes contain a higher quantity of phospholipids than the initial fraction. The content of phospholipids in "the riton" fragments of synaptosomes is higher than in "those" of microsomes. Contrary to digitonin which solubilizes the active Na+, K+-ATPase complex of microsomes and synaptosomes, triton X-100 solubilizes the active enzyme of microsomes only. A higher total content of phospholipids in "the triton" extracts of synaptosomes does not probably correlate with the presence of Na+, K+-ATPase activity in them. But these extracts are found to contain less phosphatidylserine whose addition recovers Mg2+, Na+, K+-ATPase activity in them. The effect of phosphatidylserine is not strictly specific for "the triton" extracts of synaptosomes, this lipid activates to a definite extent the extracts of microsomes as well. It is shown that at the first stages of bull brain Na+, K+-ATPase purification the total content of phospholipids and cholesterol in the preparations increases but the composition of phospholipids remains unchanged.     

Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Hydroperoxides selectively inhibit human erythrocyte membrane enzymes

Treatment of washed erythrocytes with tert-butyl hydroperoxide (0.5 mM, 10 min) inhibited basal Ca2+ + Mg2+-ATPase activity by 40% and calmodulin-stimulated activity by 54%. The inhibition was accompanied by the formation of methemoglobin and the aggregation of some membrane proteins into a high-molecular-weight polymer. Membranes, isolated from washed erythrocytes, showed a similar pattern of inhibition. Basal Ca2+ + Mg2+-ATPase activity was inhibited 50% at 10 min and 70% at 30 min while calmodulin-stimulated activity was inhibited 70% at 10 min and 84% at 30 min. Thiobarbituric acid-reactive products formed slowly during the first 10 min and then increased sharply between 10 and 30 min. The polymerization of membrane proteins was also observed during the tert-butyl hydroperoxide exposure. Inhibition of erythrocyte membrane enzymes was selective. The Na+ + K+-stimulated Mg2+ ATPase, like the Ca2+ + Mg2+-ATPase, was sensitive to membrane oxidation but the activities of Mg2+-ATPase and acetylcholinesterase were less inhibited by tert-butyl hydroperoxide. Acetylcholinterase was found to be very resistant to hydroperoxide treatment with less than 10% loss of activity. The effects of two other hyproperoxides on enzyme inhibition were studied also. Cumene hydroperoxide (0.5 mM) was found to be as potent as tert-butyl hydroperoxide but hydrogen peroxide at 10 mM did not produce thiobarbituric acid-reactive products or inhibit Ca2+ + Mg2+-ATPase activity until after 20 min. The selective effects of peroxides on these enzyme activities are discussed.     

Characterization of the plasma membrane Mg2+-ATPase from the yeast, Saccharomyces cerevisiae.

The plasma membrane of Saccharomyces cerevisiae has a Mg2+-dependent ATPase which is distinct from the mitochondrial Mg2+-ATPase and at the pH optimum of 5.5 has a Km for ATP of 1.7 mM and a Vmax of 0.42 mumol of ATP hydrolyzed/mg/min. At least three protein components of the crude membrane (Mr = 210,000, 160,000 and 115,000) are labeled with [gamma"32P]ATP at pH 5.5. These phosphoproteins form rapidly in the presence of Mg2+, rapidly turn over the bound phosphate when unlabeled ATP is added, and dephosphorylate after incubation in the presence of hydroxylamine. Vanadate, an inhibitor of the Mg2+-ATPase activity, blocks the phosphorylation of the 210,000- and 115,000-dalton proteins. At pH 7.0, only the 210,000- and 160,000-dalton proteins are phosphorylated. While these three phosphorylated intermediates have not been unambiguously identified as components of the Mg2+-ATPase, the finding of such phosphorylated components in association with that activity implies that this enzyme differs in mechanism from the mitochondrial proton pump and that it is similar in mechanism to the metal ion pumps ((Na+-K+)-ATPase and Ca2+-ATPase) of the mammalian plasma membrane.     

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.     

Myometrial (Na+ + K+)-activated ATPase and its Ca2+ sensitivity

Ouabain-sensitive (Na+ + K+)-ATPase activity in the rat myometrial microsome fraction could only be determined following detergent treatment. The (Na+ + K+)-ATPase activity manifested by detergent treatment proved very stable even to high concentrations of NaN3, in contrast Mg+-ATPase activity was reduced to about 30 percent of the control. The major part of the Mg2+-ATPase in the myometrial membrane preparation was found to be identical with the NaN3-sensitive ATP diphosphohydrolase capable of ATP and ADP hydrolysis. This monovalent-cation-insensitive ATP hydrolysis could be extensively reduced by DMSO. Furthermore DMSO prevented the inactivation of the (Na+ + K+)-ATPase activity. 10-100 microM Ca2+ inhibited the (Na+ + K+)-ATPase activity obtained in the presence of SDS by 15-50 percent. The Ca2+ sensitivity of the enzyme was considerably decreased if the proteins solubilized by the detergent had been separated from the membrane fragments by ultracentrifugation. The inhibitory effect could be regained by combining the supernatant with the pellet. Ca2+ sensitivity of the (Na+ + K+)-ATPase activity was preserved even after removal of the solubilized proteins provided that DMSO had been applied. It appears that a factor in the plasma membrane solubilized by SDS may be responsible for the loss of Ca2+ sensitivity of the (Na+ + K+)-ATPase activity, the solubilization of which can be prevented by DMSO.     

Calcium transport in human erythrocytes. Separation and reconstitution of high and low Ca affinity (Mg mca)-AT Pase activities in membranes prepared at low ionic strength.

Human erythrocyte membranes obtained by freeze-thawing of ghosts prepared in the absence or presence of EDTA, by washing with a 12 mosm medium at pH 7.7 or a 2 mosm medium at pH 6.5 contain both high and low Ca affinity (Mg + Ca)-ATPase activities. Incubation of ghosts in a less than 2 mosm medium at pH 7.5 or in 0.1 mm EDTA + 1 Him Tris-maleate (pH 8.0) results in removal of the high affinity (Mg + Ca)-ATPase activity from the membrane in a time dependent manner. Under similar conditions up to 25% of membrane proteins are removed. The soluble protein fraction extracted, although devoid of ATPase activity, reconstitutes with the remaining membrane residue with restoration of original (Mg + Ca)-ATPase activity. Addition of the soluble protein fraction to heat-treated membranes devoid of low affinity (Mg + Ca)-ATPase activity allows reconstitution of more than 33% of the original high affinity (Mg + Ca)-ATPase activity which has a Ca dissociation constant of approximately 1.6μm. Temperature and phospholipase A₂ studies indicate that low affinity (Mg + Ca)-ATPase activity is phospholipid dependent in contrast to high affinity (Mg + Ca)-ATPase activity. Ruthenium red and LaCl₃ inhibit both high and low affinity (Mg + Ca)-ATPase activities with similar potencies. The ease of removal of high affinity (Mg + Ca)-ATPase activity from the membrane by relatively mild conditions suggests that an activator protein or the high affinity (Mg + Ca)-ATPase itself is only loosely attached to the membrane. These studies show that low affinity (Mg + Ca)-ATPase activity is not an artifact and is distinct from high affinity (Mg + Ca)-ATPase activity. The low affinity (Mg + Ca)-ATPase activity is sensitive to Ca²⁺ in the concentration range from below 0.3 μm to 300 μm compatible with an association of this enzyme with Ca transport.     

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.     

Detergent activation of the ATPase activity of chloroplast coupling factor 1

The activation of the ATPase activity of coupling factor 1 (CF1) from chloroplasts by several detergents was studied. Further evidence that detergent micelles are important in the activation of Ca2+-ATPase was obtained. Maximal activation of CA2+-ATPase was achieved with short-chain alkyl-beta-D-glucopyranoside (alkylglucosides) detergents. Treatment of CF1 with hexylglucoside or heptylglucoside followed by hydroxylapatite chromatography caused nearly total removal of the epsilon subunit of the enzyme, whereas treatment with decylglucoside caused less ATPase activation and less loss of the epsilon subunit. The ATPase activity of detergent-activated CF1 was inhibited by purified epsilon subunit. Detergents that form small micelles appear to be most effective in removing the epsilon subunit and in activating the Ca2+-ATPase of CF1. When present during assay, the alkylglucosides also induce a Mg2+-ATPase activity in CF1. Octyl- and nonylglucoside are most effective in promoting this reaction. If, however, CF1 deficient in the epsilon subunit was used, even decylglucoside elicited rapid Mg2+-ATPase hydrolysis. It is concluded that removal of the epsilon subunit, although necessary for the expression of Mg2+-ATPase, is not sufficient. The detergents that cause maximal displacement of the epsilon subunit are less effective in inducing Mg2+-ATPase activity. The selective removal of subunits from CF1 by specific detergents points to potential problems with the use of these detergents in the solubilization of oligomeric membrane proteins.     

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.     

Effect of calixarene-phosphonic acid on Na+, K+-ATPase activity in plasma membranes of the smooth-muscle cells

Effect of calix[4]arenes C-97, C-99, C-107, functionalized by fragments of alpha-hydroxy-phosphonic, alpha-aminophosphonic- and methylene-bisphosphonic acid on enzymatic activity of oubaine-sensitive Na+, K+-ATPase and oubaine-resistant basal Mg2+- ATPase (specific activity - 10.6 +/- 0.9 and 18.1 +/- 1.2 micromol Pi/h per 1 mg of protein, respectively; n = 7) was studied in experiments made on the suspension of myometrium cell plasma membranes treated by 0.1% solution of digitonin. It was found that calixarene-phosphonic acids in concentration of 100 microM inhibited enzymatic activity of Na+, K+-ATPase by 86-98% and did not practically affect activity of Mg2+-ATPase. These calixarenes were more efficient than oubaine in suppressing enzymatic activity of the sodium pump: in case of the effect of calixerenes the value of the appearence constant of inhibition I0.5 was < 0.1 microM. Calixarene-methylene-bisphosphonic acid (calixarene C-97; I0.5 =33 +/- 4 microM (n = 6) takes the most efficient inhibitory effect on Na+,K+-ATPase activity among the studied calixarenes. A phenomenon of negative cooperation: the Hill coefficient value etaH =0.1-0.5<1 is characteristic of both the inhibiting effect of calixarenes and oubaine. Reguliarities of calixarenes C-97 effect on enzymatic activity of Na+,K+-ATPase were studied. As it appeared its inhibiting effect cannot be caused by trivial factors - potentially possible binding of Mg ions by it and (or) this substance effect on Mg2+ interaction with ATP4- in the incubation medium. Calixerene C-97 does not also decrease the enzyme affinity for Mg ions or ATP. However this calixerenes decreases the affinity of Na+,K+-ATPase for Na ions (the value of activation constant K(Na+)) from 50 +/- 4 (control) to 76 +/- 6 microM in the control and under the effect of calixerene, respectively). A conclusion is made that calixerene C-97 is highly-efficient (with respect to oubaine) and selective (with respect to lack of its effect on basal Mg2+-ATPase) inhibitor of Na+,K+-ATPase of plasma membrane. In the practical aspect it may be used in concentration of 1-10 microM in biochemical membranology when testing and studying kinetic and catalytic properties of the sodium pump in case of such experimental model, as the plasma membrane fraction.     

Comparative study of properties of Na+, K+-ATPase and Mg2+-ATPase of the myometrium plasma membrane

The comparative research of catalytic properties of two ATP-hydrolases of the sarcolemma of the smooth muscle of the uterus--ouabaine-sensitive Na+,K+-ATPase and ouabaine-resistent Mg2+-ATPase is carried out. The specific enzymatic activity of Na+,K+-ATPase and Mg2+-ATPase makes 10.2 +/- 0.7 and 18.1 +/- 1.2 mmol P/mg of protein for 1 hour, accordingly. The action of ouabaine on Na+,K+-ATPase is characterized by magnitude of quotient of inhibition I0.5=21.3 +/- 1.5 mkM. Processing of the sarcolemma fraction by digitonin in concentrations 0.001 +/- 0.1% promotes an activation of Na+,K+ATPase and Mg2+- ATPase, and in the first case much more efficiently than in the second. The kinetics of accumulation of the product of ATP-hydrolase reactions of phosphate satisfies the laws of the zero order reaction (incubation time--about 10 min). Na+,K+-ATPase is highly specific concerning the univalent cations--Na+, K+, however Li+ can partially substitute K+. Activity of Mg2+-ATPase is not specific concerning univalent cations. The dependence of Na+,K+-ATPase activity on pH in the range of 6.0-8.0 is characterized by the bell-shaped curve, at the same time the linear dependence on pH is peculiar to Mg2+-ATPase. The functioning of Na+,K+-ATPase is provided only by ATP, in the case of Mg2+-ATPase ATP can be successfully replaced with other nucleotidetriphosphates. It is supposed that the obtained experimental data can be beneficial in further research of membranous mechanisms underlying the cation exchange in the smooth muscles, in particular when studying the role of the plasma membrane in the maintenance of electromechanical coupling in them, and also in the regulation of ionic homeostasis in myocytes.     

Purification of a low affinity Mg2+ (Ca2+)-ATPase from the plasma membranes of a human oat cell carcinoma

Plasma membranes of many mammalian cells contain a Mg2+-dependent ATPase activity which is easily inactivated by detergents. This activity is the combined expression of at least two ATP-hydrolyzing enzymes (Knowles, A.F., Isler, R.E., and Reece, J.F. (1983) Biochim. Biophys. Acta 731, 88-96). We have purified one of these enzymes from the plasma membranes of a human oat cell carcinoma xenograft. The enzyme was extracted from the membranes by 0.5% digitonin and purified on a DE52 column. The purified enzyme contained a major protein band of Mr = 30,000 when dissociated by sodium dodecyl sulfate. It hydrolyzed all nucleoside triphosphates in the presence of Mg2+ or Ca2+, but showed little activity toward nucleoside diphosphates. The enzyme was inhibited by p-chloromercuriphenyl sulfonate, slowly inactivated by p-fluorosulfonylbenzoyl-5'-adenosine and dithiothreitol at room temperature, and lost activity readily in solutions containing low concentrations of several detergents. This knowledge of the macromolecular structure of the Mg2+(Ca2+)-ATPase and its catalytic properties is important in determining the orientation of the enzyme in the membrane and its physiological function.     

Interaction of chemical probes with sarcoplasmic reticulum membranes

The labelling of the sarcoplasmic reticulum membranes by the chemical probes, trinitrobenzenesulfonate (TNBS) and fluorodinitrobenzene (FDNB) has been investigated. The incorporation of TNBS, but not of FDNB, depends on the binding of Ca2+ or Mg2+ to the membranes. The labelling of lipids and of the various reticulum proteins by TNBS is increased by those agents, but the effect is not uniform for all membrane proteins. The Ca2+ -ATPase contributes only 2.2% for the total labelling of the sarcoplasmic reticulum proteins, whereas the proteins of molecular weight 90 000 and 30 000 contribute about 34 and 56%, respectively. However, the Ca2+-ATPase isolated from the membrane reacts with an amount of TNBS 5-fold higher than that which reacts with the enzyme in situ. Both probes, TNBS and FDNB, inhibit the Ca2+-ATPase activity and the Ca2+ uptake by sarcoplasmic reticulum, whereas the Mg2+-ATPase remains unaffected. The results indicate that FDNB is maximally incorporated into the sarcoplasmic reticulum membrane, whereas only some of the membrane amino groups are accessible to TNBS in the absence of Ca2+, Mg2+ or ATP which, when present, make additional amino groups available to TNBS. The highest degree of TNBS incorporation takes place into proteins, other than the ATPase, but sufficient reaction occurs with the enzyme to inhibit its activity.     

Effect of eosin Y on Ca2+ -independent, Mg2+ -dependent ATPase activity of smooth muscle cell plasma membranes

The effect of eosin Y (2',4',5',7'-tetrabromofluorescin) on basic kinetic parameters of the reaction of Mg2+ -dependent hydrolysis of ATP catalysed "basal" Mg2+ -ATPase myometrial cells plasma membrane has been studied. The eosin Y (10-100 microM) inhibited initial maximal velocity of the "basal" Mg2+ -ATPase of plasma membrane assayed for Mg2+ and ATP. At the same time the given inhibitor reduces the affinity of Mg2+ -ATPase for ATP. However, the difficult effect of the inhibitor action is observed for Mg ions: eosin Y in concentration of 10-50 microM increases the enzyme affinity for the ion-activator, while in concentration of 100 microM the affinity of Mg2+ -ATPase for Mg2+ is reduced. An analysis of eosin Y effect on catalytic efficiency of "basal" Mg2+ -ATPase of plasma membrane has shown, that at saturating concentrations of ATP (1 mM) the enzyme activity is less sensitive to the action of inhibitor. On this basis the conclusion is made that ATP in high concentrations can compete with eosin Y for active centre of Mg2+ -ATPase of smooth muscle cells plasma membrane.     

Seasonal variations in the renal cortical (Na+ + K+)-ATPase and Mg2+-ATPase of a hibernator, the ground squirrel (Spermophilus richardsonii).

1. The specific activity of renal cortical (Na+ + K+)-ATPase of the Richardson ground squirrel is markedly reduced during hibernation, in contrast to the specific activity of the accompanying Mg2+-ATPase which is markedly increased. 2. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is unchanged by hibernation. 3. Both the non-linear thermal dependence of (Na+ + K+)-ATPase and the linear thermal dependence of Mg2+-ATPase are also unchanged by hibernation. 4. The energy of activation of both enzymes is unchanged during hibernation, or by comparison with that determined in awake controls. 5. There is no evidence for inherent "cold resistance" in these enzyme preparations compared to similar preparations from the non-hibernating rabbit. This parameter does not change during hibernation. 6. Both the rate and amount of specific [3H]-ouabain binding to the renal cortical preparations of (Na+ + K+)-ATPase decrease during hibernation. This decrease matches the fall in enzyme activity so that the ratio of pumping sites/unit of enzyme activity shows no seasonal variations. 7. These findings suggest that the amount of renal cortical (Na+ + K+)-ATPase enzyme falls during hibernation, but that the enzyme which remains functions with the same thermodynamic efficiency and identical biochemical characteristics of that found in the awake summer controls.     

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.