Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.     

Characterization of the Ca2+- and Mg2+-Dependent ATPases in Electrophorus Electroplax Microsomes

Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.     

The rat liver plasma membrane high affinity (Ca2+-Mg2+)-ATPase is not a calcium pump. Comparison with ATP-dependent calcium transporter

The high affinity (Ca2+-Mg2+)-ATPase purified from rat liver plasma membrane (Lin, S.-H., and Fain, J. N. (1984) J. Biol. Chem. 259, 3016-3020) has been further characterized. This enzyme also possesses Mg2+-stimulated ATPase activity with K0.5 of 0.16 microM free Mg2+. However, the Vm of the Mg2+-stimulated activity is only half that of the Ca2+-stimulated ATPase activity. The effects of Ca2+ and Mg2+ on this enzyme are not additive. Both the Ca2+-stimulated ATPase and Mg2+-stimulated ATPase activities have similar affinities for ATP (0.21 mM and 0.13 mM, respectively) and similar substrate specificities (they are able to utilize ATP, GTP, UTP, CTP, ADP, and GDP as substrates); both activities are not inhibited by vanadate, p-chloromercuribenzoate, ouabain, dicyclohexylcarbodiimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, oligomycin, F-, N-ethylmaleimide, La3+, and oxidized glutathione. These properties of the Mg2+- and Ca2+-ATPases indicate that both activities reside on the same protein. A comparison of the properties of this high affinity (Ca2+-Mg2+)-ATPase with those of the liver plasma membrane ATP-dependent Ca2+ transport activity reconstituted into artificial liposomes (Lin, S.-H. (1985) J. Biol. Chem. 260, 7850-7856) suggests that this high affinity (Ca2+-Mg2+)-ATPase is not the biochemical expression of the liver plasma membrane Ca2+ pump. The function of this high affinity (Ca2+-Mg2+)-ATPase remains unknown.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase. I. characterization and general properties

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Purification and Characterization of a Cation-stimulated Adenosine Triphosphatase from Corn Roots

A membrane-bound, monovalent cation-stimulated ATPase from Zea mays roots has been purified to a single band on sodium dodecyl sulfate gel electrophoresis. Microsomal preparations with K⁺ -stimulated ATPase activity were extracted with 1 m NaClO₄, and the solubilized enzyme was purified by chromatography on columns of n-hexyl-Sepharose, DEAE-cellulose, and Sephadex G-100 Superfine. A 500-fold purification over the activity present in the microsomes was obtained. The K⁺ -stimulated activity shows positive cooperativity with increasing KCl concentrations. The purified enzyme shows K⁺ -stimulated activity with ATP, GTP, UTP, CTP, ADP, α + β-glycerophosphate, p-nitrophenyl phosphate, and pyrophosphate as substrates. Under most conditions ATP is the best substrate. Although dicyclohexyl carbodiimide and Ca²⁺ inhibit and alkylguanidines stimulate the K⁺ -ATPase while bound to microsomes, they have no effect on the purified enzyme.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Measurement of microsomal ATPase activities: a comparison between the inorganic phosphate-release assay and the NADH-coupled enzyme assay

The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted.     

Bilayer orientation of membrane-bound NH2 groups across microsomal vesicles and their role in the function of gastric K+-stimulated adenosine triphosphatase

The transversal distribution of the free NH₂ groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K⁺-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH₂ groups was studied after modification with the probes under various conditions and relating the inhibition of the K⁺-stimulated ATPase to the ATPase-dependent H⁺ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K⁺-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K⁺. Both the K⁺-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH₂ groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH₂ groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH₂ appeared to be associated with the K⁺-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH₂ groups of the K⁺-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K⁺-stimulated ATPase and dye uptake.     

Chemical modification of gastric microsomal potassium-stimulated ATPase

Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K⁺-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K⁺-stimulated ATPase and H⁺-transporting activities of the gastric microsomes, while the Mg²⁺-ATPase was not affected. Half-maximal inhibition occurred at about 3 μg 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent K_m for ATP protection was about 50 μM. Nucleotide selectivity for protection was ATP > ADP > ITP > GTP > CTP > AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the M_r 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the M_r 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg²⁺-ATPase and K⁺-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K⁺-ATPase and may be actually at the ATP binding site.     

Studies on the microsomal sodium-plus-potassium ion-stimulated adenosine triphosphatase system in rat ventral prostate

A Mg(2+)+Na(+)+K(+)-stimulated adenosine triphosphatase (ATPase) preparation was isolated from rat ventral prostate by flotation of microsomal membranes in high-density sucrose solutions. The reaction medium for optimum Na(+)+K(+)-stimulated ATPase activity was found to be: Na(+), 115mm; K(+), 7-10mm; Mg(2+), 3mm; ATP, 3mm; tris buffer, pH7.4 at 38 degrees , 20mm. The average DeltaP(i) (Mg(2+)+Na(+)+K(+) minus Mg(2+)+Na(+)) was 9mumoles/mg. of protein/hr., representing a 30% increase over the Mg(2+)+Na(+)-stimulated ATPase activity. At high concentrations, K(+) was inhibitory to the enzyme activity. Half-maximal inhibition of Na(+)+K(+)-stimulated ATPase activity was elicited by ouabain at 0.1mm. The preparation exhibited phosphatase activity towards ribonucleoside triphosphates other than ATP. However, stimulation of P(i) release by Na(+)+K(+) was observed only with ATP as substrate. The apparent K(m) for ATP for Na(+)+K(+)-stimulated activity was about 0.3x10(-3)m. Ca(2+) inhibited only the Na(+)+K(+)-stimulated ATPase activity. Mg(2+) could be replaced by Ca(2+) but then no Na(+)+K(+) stimulation of ATPase activity was noticed. The addition of testosterone or dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one) in vitro at 0.1-10mum under a variety of experimental conditions did not significantly increase the Na(+)+K(+)-stimulated ATPase activity. The enzyme preparations from prostates of orchidectomized rats, however, exhibited a drastic decrease in the specific activity of Na(+)+K(+)-stimulated ATPase; these changes were prevented in the orchidectomized rats by injection of testosterone propionate.     

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.     

High-affinity calcium-stimulated, magnesium-dependent adenosine triphosphatase in Trypanosoma cruzi.

1. A high-affinity (Ca2+ + Mg2+)-ATPase and a low-affinity Mg(2+)-ATPase were identified in the 105,000 g fraction from epimastigote forms of Trypanosoma cruzi, the agent of Chagas' disease (Tulahuen strain). 2. Activities were conserved after enzyme solubilization with deoxycholate. 3. The Ca(2+)-stimulated ATPase activity was (a) lower than that of the Mg(2+)-ATPase; (b) inhibited by p-chloromercurobenzoate and orthovanadate and (c) insensitive to oligomycin. 4. Optimal stimulation by Ca2+ was observed at pH 6.5-6.8 in the presence of 1 mM MgCl2 and 0.1 M KCl. 5. The Mg(2+)-ATPase was insensitive to p-chloromercurobenzoate and orthovanadate and did not require KCl for activity. 6. Kinetic analysis of the (Ca2+ + Mg2+)-ATPase yielded a half-maximal stimulating concentration of 1.1 microM for Ca2+ and a Km of 66 microM for ATP. 7. The (Ca2+ + Mg2+)-ATPase clearly differed from the Ca(2+)- or Mg(2+)-ATPases previously characterized in the same strain of T. cruzi (Frasch et al., 1978; Comp. Biochem. Physiol. 60B, 271-275).     

Vanadate inhibition of brain (Ca+Mg)-ATPase

Vanadate was a potent inhibitor of the membrane-bound (Ca+Mg)-ATPase from rat brain, the concentration required for 50% inhibition under conditions optimal for enzymatic activity being 3 M. Vanadate inhibition increased with the MgCl₂ concentration, half-maximal inhibition occurring at 2 mM MgCl₂, near the MgCl₂ concentration required for half-maximal activation of the ATPase activity. MnCl₂ could substitute for MgCl₂, and at concentrations of 1 mM (Ca+Mn)-ATPase activity was greater than (Ca+Mg)-ATPase activity, although sensitivity to vanadate was less. Vanadate inhibition increased also with the KCl concentration, half-maximal inhibition occurring at 8 mM, again near the concentration required for half-maximal activation of ATPase activity. By contrast, NaCl stimulated (Ca+Mg)-ATPase activity without potentiating vanadate inhibition. These effects of cations on ATPase activity and vanadate inhibition resemble properties of certain transport ATPases and thus suggest mechanistic and functional similarities.     

Endogenous protease mediated manifestation of a Ca2+-stimulated ATPase in purified dog gastric microsomes

An endogenous soluble protease has been demonstrated to unmask a Ca²⁺-stimulated ATPase activity in purified dog gastric microsomes. The presence of ATP during protease treatment appears essential for the manifestation of the gastric Ca²⁺-stimulated ATPase activity. The endogenous protease appears to have trypsin-like activity, since soybean trypsin inhibitor completely blocks the protease effect. Manifestation of the Ca²⁺-stimulated ATPase occurs without affecting the microsomal (H⁺ +K⁺)-ATPase activity and associated H⁺ uptake ability. The unmasked Ca²⁺-stimulated ATPase appears insensitive to calmodulin. Possible roles of the enzyme in the regulation of gastric H⁺ transport have been discussed.     

2,5-Di(tert-butyl)-1,4-benzohydroquinone--a novel inhibitor of liver microsomal Ca2+ sequestration

Treatment of rat liver microsomes with 2,5-di(tert-butyl)-1,4-benzohydroquinone caused a dose-related inhibition (Ki congruent to 1 microM) of ATP-dependent Ca2+ sequestration. This was paralleled by a similar impairment of the microsomal Ca2+-stimulated ATPase activity. In contrast, the hydroquinose failed to induce Ca2+ release from Ca2+-loaded liver mitochondria (supplied with ATP), and inhibited neither the mitochondrial F1F0-ATPase nor the Ca2+-stimulated ATPase activity of the hepatic plasma membrane fraction. The inhibition of microsomal Ca2+ sequestration was not associated with any apparent alteration of membrane permeability or loss of other microsomal enzyme activities or modification of microsomal protein thiols. These findings suggest that 2,5-di(tert-butyl)-1,4-benzohydroquinone is a potent and selective inhibitor of liver microsomal Ca2+ sequestration which may be a useful tool in studies of Ca2+ fluxes in intact cells and tissues.     

Localization of endogenous ATPases at the nerve terminal

We have investigated the localization of a set of intrinsic ATPase activities associated with purified synaptic plasma membranes and consisting of (a) a Mg²⁺-ATPase; (b) an ATPase active at high concentrations of Ca²⁺ in the absence of Mg²⁺ (Ca_H-ATPase); (c) a Ca²⁺ requiring Mg²⁺-dependent ATPase (Ca + Mg)-ATPase, stimulated by calmodulin (Ca-CaM-ATPase); (d) a Ca²⁺-dependent ATPase stimulated by dopamine (DA-ATPase); and (e) the ouabain-sensitive (Na + K)-ATPase. The following results were obtained: (1) All ATPases are largely confined to the presynaptic membrane; (2) the DA-, (Ca + Mg)-, (Ca-CaM)-, and (Na + K)-ATPases are oriented with their ATP hydrolysis sites facing the synaptoplasm; (3) the Mg- and Ca_H-ATPases are oriented with their ATP hydrolysis sites on the junctional side of the presynaptic membrane and are therefore classified as ecto-ATPases of as yet unknown function.     

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.     

The effect of berbamine derivatives on activated Ca2+-stimulated Mg2+-dependent ATPase in erythrocyte membranes.

Ca2+-stimulated Mg2+-dependent ATPase (Ca2+ + Mg2+-ATPase) stimulated by calmodulin, by partial proteolysis or by oleic acid in erythrocyte membranes was inhibited by various derivatives of the naturally occurring alkaloid berbamine. The ability of these derivatives to inhibit trypsin-activated Ca2+ + Mg2+-ATPase correlated well with their ability to inhibit the calmodulin-stimulated enzyme. Inhibition of the trypsin-activated Ca2+ + Mg2+-ATPase by O-4-(ethoxybutyl)berbamine (EBB) was competitive with respect to ATP. The Ki for inhibition was about 8 microM. These results suggest that the binding site of EBB on the activated Ca2+ + Mg2+-ATPase may bear structural similarity to that on calmodulin, and may be closely related to the ATP-binding site on the enzyme.