Purification and characterization of a Ca2+- or Mg2+-stimulated ATPase from plasma membrane enriched fractions of Dictyostelium discoideum

Evidence is presented for the presence of both diethylstilbestrol (DES)-sensitive and DES-insensitive Mg2+-ATPase activities in plasma membrane enriched fractions of Dictyostelium discoideum. When removed from the membrane, the DES-sensitive activity is markedly less stable than the DES-insensitive activity, and the two activities display a number of quite distinct properties. The DES-sensitive enzyme has a decided preference for Mg2+ over Ca2+, displays saturation kinetics in response to ATP as substrate (Km = 0.2 mM) and has a narrow pH optimum range. In contrast, the DES-insensitive activity is stimulated equally by Mg2+ or Ca2+, is not saturable by ATP within the mM concentration range and has a much broader pH optimum. The DES-insensitive activity has been purified extensively. The purified enzyme is inhibited by vanadate and fluoride, but is insensitive to N,N'-dicyclohexylcarbodiimide (DCCD), N-ethylmaleimide and thimerosal. In the absence of divalent cations, the enzyme displays a sigmoidal activity curve in response to substrate concentration, which is abolished by addition of either Mg2+ or Ca2+, suggesting a binding site for a divalent cation and a positive cooperative interaction. The enzyme is capable of hydrolyzing other nucleotide triphosphates and ADP, but is without activity on AMP, p-nitrophenyl phosphate and pyrophosphate. The enzyme has an apparent molecular weight of approximately 64,000.     

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.     

Stimulation of synaptosomal ATP-dependent Ca2+-uptake by N-ethylmaleimide

Treatment of rat brain synaptosomal lysate with N-ethylmaleimide (NEM) was found to stimulate ATP-dependent Ca2+-uptake. This Ca2+-uptake stimulation was blocked by dithioerythritol (DTE), mitochondrial inhibitors oligomycin and sodium azide, but not by vanadate, an inhibitor of plasma membrane Ca2+ pump. Maximal stimulation of Ca2+-uptake was observed at a NEM/protein ratio of 0.1 mumole/0.5-1.0 mg. On fractionation, it was found that NEM did not affect synaptic plasma membrane Ca2+-uptake, but almost doubled it in synaptic mitochondria.     

A plasma-membrane associated ATPase from the acidophilic bacterium Acidiphilium cryptum

A membrane-bound ATPase of Acidiphilium cryptum, an acidophilic bacterium of mine origin, has been studied. The enzyme has a pH optimum of 8.4 Mg2+ is required for its activity and could be replaced by Mn2+, but not by Ca2+. The enzyme shows a strong preference for ATP as substrate, with the apparent Km of about 0.2 mM. Sulphite ion significantly stimulated the enzyme activity. N,N'-Dicyclohexylcarbodiimide, oligomycin, and azide strongly inhibited the enzyme, whereas vanadate was without effect, suggesting that the A. cryptum ATPase might be of F0F1 type.     

Isolation of rat liver endocytic vesicles using the proton pump as a marker

ATP-driven acidification visualized by the delta pH indicator acridine orange was used as marker for isolation of endocytic vesicles from rat liver. By differential and Percoll density gradient centrifugation, a vesicle fraction was obtained with an approx. 80-fold enriched H+-pump activity. The preparation contained vesicles that had taken up fluorescein isothiocyanate-labeled dextran or horseradish peroxidase injected into rats in vivo, proving the presence of endosomes. The H+-pump in these vesicles showed: (a) strict preference for ATP; (b) stimulation by Mg2+ and Mn2+, but not by monovalent cations; (c) stimulation by Cl-, I- and Br-; (d) electrogenicity; (e) insensitivity to vanadate, slight inhibition by oligomycin and strong inhibition by N-ethylmaleimide (NEM) and N,N'-dicyclohexylcarbodimide (DCCD). The vesicles exhibited an ouabain-, oligomycin- and levamisole-resistant ATPase activity, which was slightly stimulated by Cl-, unaffected by vanadate and inhibited by NEM and DCCD. Thus, a simple and efficient high-speed centrifugation method is available for isolation of endocytic vesicles from mammalian liver.     

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.     

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

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

Inhibition of red cell Ca2+-ATPase by vanadate

1. The Mg2+- plus Ca2+-dependent ATPase (Ca2+-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate. 2. Several natural activators of Ca2+-ATPase (Mg2+, K+, Na+ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate. 3. Among the ligands tests, K+, in combination with Mg2+, had the most pronounced effect on inhibition by vanadate. 4. Under conditions optimal for inhibition of Ca2+-ATPase, the K 1/2 for vanadate was 1.5 microM and inhibition was nearly complete at saturating vanadate concentrations. 5. There are similarities between the kinetics of inhibition of red cell Ca2+-ATPase and (Na+ + K+)-ATPase prepared from a variety of sources; however, (Na+ + K+)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.     

Characterisation of Ca2+ or Mg(2+)-dependent nucleoside triphosphatase from rat mesenteric small arteries.

When isolated rat mesenteric small arteries were submitted to 2 s of sonication, a nucleoside triphosphatase activity was released to the medium, mainly from the plasma membrane of the vascular smooth muscle cells. The activity was kinetically characterized: It hydrolysed ATP, UTP and GTP with the same substrate affinity and the same specific activity. CaATP, as well as MgATP were substrates for the enzyme with an apparent Km in the micromolar range. ATPase inhibitors: ouabain, vanadate, AlF4-, oligomycin and N-ethylmaleimide were without effect on the hydrolytic activity. Among other modifiers tested only N,N'-dicyclohexylcarbodiimide caused significant (greater than 30%) inhibition. In the presence of micromolecular concentrations of Ca2+ and Mg2+, small (less than 20 mM) concentrations of Na+, K+, Rb+, Cs+ and choline+, irrespective of the nature of the anion, activated the hydrolysis with an equilibrium ordered pattern, but concentrations of monovalent cation salts above 20 mM decreased the hydrolysis rate. No activation by monovalent cation salts was seen at millimolar concentrations of divalent cations and substrate. On the basis of the results a standard mixture is proposed, which allows a sensitive assay of the specific enzyme activity.     

Rapid calcium release from cardiac sarcoplasmic reticulum vesicles is dependent on Ca2+ and is modulated by Mg2+, adenine nucleotide, and calmodulin

A subpopulation of canine cardiac sarcoplasmic reticulum vesicles has been found to contain a "Ca2+ release channel" which mediates the release of intravesicular Ca2+ stores with rates sufficiently rapid to contribute to excitation-contraction coupling in cardiac muscle. 45Ca2+ release behavior of passively and actively loaded vesicles was determined by Millipore filtration and with the use of a rapid quench apparatus using the two Ca2+ channel inhibitors, Mg2+ and ruthenium red. At pH 7.0 and 5-20 microM external Ca2+, cardiac vesicles released half of their 45Ca2+ stores within 20 ms. Ca2+-induced Ca2+ release was inhibited by raising and lowering external Ca2+ concentration, by the addition of Mg2+, and by decreasing the pH. Calmodulin reduced the Ca2+-induced Ca2+ release rate 3-6-fold in a reaction that did not appear to involve a calmodulin-dependent protein kinase. Under various experimental conditions, ATP or the nonhydrolyzable ATP analog, adenosine 5'-(beta, gamma-methylene)triphosphate (AMP-PCP), and caffeine stimulated 45Ca2+ release 2-500-fold. Maximal release rates (t1/2 = 10 ms) were observed in media containing 10 microM Ca2+ and 5 mM AMP-PCP or 10 mM caffeine. An increased external Ca2+ concentration (greater than or equal to 1 mM) was required to optimize the 45Ca2+ efflux rate in the presence of 8 mM Mg2+ and 5 mM AMP-PCP. These results suggest that cardiac sarcoplasmic reticulum contains a ligand-gated Ca2+ channel which is activated by Ca2+, adenine nucleotide, and caffeine, and inhibited by Mg2+, H+, and calmodulin.     

Identification of a vanadate-sensitive, membrane-bound ATPase in the archaebacterium Methanococcus voltae.

Membrane-bound ATPase activity was detected in the methanogen Methanococcus voltae. The ATPase was inhibited by vanadate, a characteristic inhibitor of E1E2 ATPases. The enzyme activity was also inhibited by diethylstilbestrol. However, it was insensitive to N,N'-dicyclohexylcarbodiimide, ouabain, and oligomycin. The enzyme displayed a high preference for ATP as substrate, was dependent on Mg2+, and had a pH optimum of approximately 7.5. The enzyme was completely solubilized with 2% Triton X-100. The enzyme was insensitive to oxygen and was stabilized by ATP. There was no homology with the Escherichia coli F0F1 ATPase at the level of DNA and protein. The membrane-bound M. voltae ATPase showed properties similar to those of E1E2 ATPases.     

Characteristics of Mg2+-dependent, ATP-activated Ca2+ transport in synaptic and microsomal membranes and in permeabilized synaptosomes

Synaptic plasma membranes isolated from rat brain exhibited a Ca2+ transport process that was strictly dependent on the presence of Mg2+ and activated by ATP hydrolysis. The characteristics of this ATP-activated transport process included a high affinity for Ca2+ and ATP with the Kact for these two substrates being 0.7 and 5 microM, respectively, and a lower affinity for Mg2+, Kact = 54 microM. The estimated constants for ATP-activated Ca2+ transport into synaptic membrane vesicles and the dependence of such transport on Mg2+ were indicative that such transport was related to the previously described high affinity (Ca2+ + Mg2+)-ATPase in synaptic membranes. An ATP- and Mg2+-dependent Ca2+ transport process with very similar kinetic characteristics was present also in a general microsomal membrane fraction obtained from brain tissue. The synaptic and microsomal membrane ATP-activated transport processes exhibited differences in their sensitivity to vanadate inhibition. Interaction with vanadate was fairly complex and best analyzed by a two-component model. Thus, the estimated Ki values for vanadate were 0.2 and 6.6 microM for the synaptic membranes and 0.7 and 13.8 microM for the microsomes. Since the microsomal membranes contain a substantial population of intraneuronal endoplasmic reticulum vesicles, the effects of vanadate on Ca2+ transport into intraneuronal membrane organelles, other than mitochondria, was determined in saponin-permeabilized synaptosomes. The estimated Ki values for vanadate inhibition of Ca2+ transport activity were 0.7 and 13 microM. The accumulation of Ca2+ into synaptic plasma membrane vesicles was readily reversed by activation of the Na+-Ca2+ exchange carrier, whereas the Ca2+ associated with intrasynaptosomal organelles was not affected by changes in [Na+]. Thus, there are at least two ATP-dependent Ca2+ transporting processes localized on two distinct neuronal membranes, one on the plasma membrane and the second on intraneuronal membranes.     

Dolichol kinase in rat sarcoplasmic reticulum membrane preparations

A dolichol kinase (EC 2.7.1.108) was found in sarcoplasmic reticulum membrane fractions from rat leg muscle. This enzyme specifically required CTP as a phosphoryl donor and relatively little activity was found in the absence of exogenous detergent-suspended dolichol. Unlike other reported dolichol kinases, the kinase from skeletal muscle was activated almost equally well by Ca2+, Zn2+, or Mg2+, but not Mn2+. No effect of calmodulin was seen. The kinase exhibited a single pH optimum at pH 7-8 in contrast to kinases from certain other tissues. Despite the low level of dolichol present in skeletal muscle, the kinase in the sarcoplasmic reticulum fraction had an activity comparable to that of microsomal preparations from tissues such as brain and liver, which may indicate that skeletal muscle has a high capacity for dolichol phosphorylation and protein glycosylation.     

Ca2+- or Mg2+-Dependent Enzymic ATP Hydrolysis Associated with the Microsomal Fraction of Frog Sciatic Nerves

The microsomal fraction of frog sciatic nerves was found to contain Ca2+- or Mg2+-dependent hydrolytic activity toward different nucleoside di- and triphosphates. In the presence of Ca2+ substrate specificity was in the order CTP > UTP > GTP > ATP. When Mg2+ was used, the triphosphates were approximately equally good substrates. ATP hydrolytic activity was very similar with Ca2+ or Mg2+ as the cofactor, whereas Ca2+ was the more potent activator of hydrolysis of the other triphosphates tested. The preparation showed some activity toward the nucleoside diphosphates but none toward the monophosphates or p-nitrophenylphosphate. The enzymic properties of ATP hydrolysis were more closely studied. The hydrolysis was optimal at 18--24 degrees C in the presence of 1 mM-Ca2+ or 1 mM-Mg2+. Ca2+- and Mg2+-ATP hydrolysis displayed pH maxima around 8.0--8.5 and 7.4--8.0, respectively. Vmax values for Ca2+- and Mg2+-ATP hydrolysis similar: approx. 12 mumol Pi per h per mg protein with a Km value of approx. 0.05 mM. The ATP hydrolysis activity was inhibited by NaF but unaffected by ouabain, vanadate, cytochalasin B, and various drugs known to influence ATPase activity of mitochondria. Zn2+ stimulated the ATP hydrolysis activity at low concentrations (10(-6)-10(-5) M) and inhibited it at higher concentrations. The possibility that these observations account for stimulation and inhibition of axonal transport in frog sciatic nerves exposed to similar concentrations of Zn2+ is discussed.     

Inhibition of an ecto-ATP-diphosphohydrolase by azide.

Cell surface ATPases (ecto-ATPases or E-ATPases) hydrolyze extracellular ATP and other nucleotides. Regulation of extracellular nucleotide concentration is one of their major proposed functions. Based on enzymatic characterization, the E-ATPases have been divided into two subfamilies, ecto-ATPases and ecto-ATP-diphosphohydrolases (ecto-ATPDases). In the presence of either Mg2+ or Ca2+, ecto-ATPDases, including proteins closely related to CD39, hydrolyze nucleoside diphosphates in addition to nucleoside triphosphates and are inhibited by millimolar concentrations of azide, whereas ecto-ATPases appear to lack these two properties. This report presents the first systematic kinetic study of a purified ecto-ATPDase, the chicken oviduct ecto-ATPDase (Strobel, R.S., Nagy, A.K., Knowles, A.F., Buegel, J. & Rosenberg, M.O. (1996) J. Biol. Chem. 271, 16323-16331), with respect to ATP and ADP, and azide inhibition. Km values for ATP obtained at pH 6.4 and 7.4 are 10-30 times lower than for ADP and the catalytic efficiency is greater with ATP as the substrate. The enzyme also exhibits complicated behavior toward azide. Variable inhibition by azide is observed depending on nucleotide substrate, divalent ion, and pH. Nearly complete inhibition by 5 mm azide is obtained when MgADP is the substrate and when assays are conducted at pH 6-6.4. Azide inhibition diminishes when ATP is the substrate, Ca2+ as the activating ion, and at higher pH. The greater efficacy of azide in inhibiting ADP hydrolysis compared to ATP hydrolysis may be related to the different modes of inhibition with the two nucleotide substrates. While azide decreases both Vmax and Km for ADP, it does not alter the Km for ATP. These results suggest that the apparent affinity of azide for the E.ADP complex is significantly greater than that for the free enzyme or E.ATP. The response of the enzyme to three other inhibitors, fluoride, vanadate, and pyrophosphate, is also dependent on substrate and pH. Taken together, these results are indicative of a discrimination between ADP and ATP by the enzyme. A mechanism of azide inhibition is proposed.     

Detection of an ATPase activity in rat liver peroxisomes

An ATPase co-sedimenting with rat liver peroxisomes has been detected after subcellular fractionation. The activity is Mg2+ dependent, with pH optimum of 7.5 and is inhibited by NEM and DCCD but not by oligomycin. Partial inhibition of the mitochondrial ATPase allows to detect the peroxisomal activity in the gradients. Protease inactivation and solubilization data suggests that the activity resides in a protein of the peroxisomal membrane, exposed to the cytosol.     

Biochemical Properties and Inhibition Kinetics of Phosphatase from Wheat Thylakoid Membranes

A phosphetase that hydrolyses phosphate monoesters has been Isolated from wheat thylakold membranes. Biochemical properties and inhibition kinetics of the phosphatase were Investigated using several Ions, organlc solvents, and Inhlbltors. Wheat （Trltlcum aestivum L. cv. PH82-2-2） thylakold membrane phosphatase activity was activated by Mg^2＋, Ca^2＋, and Fe^2＋ and was inhibited by Mn^2＋ and Cu^2＋. For example, enzyme activity was acUvated 34.81% by 2 mmol/l. Mg^2＋, but was Inhibited 22.3% and 8.5% by 2 and 1 mmol/L Cu^2＋, respectively. Methanol, ethanol and glycol were all able to activate enzyme activity. Enzyme activity was activated 58.5%, 48.2%, and 8.7% by 40% ethanol, methanol and glycol, respectively. From these results, It can be seen that the degree of actlvetlon of the phosphetase was greatest for ethanol and the type of acUvatlon was uncompetltlve. Moreover, the activity of the thylakold membrane phosphetase was Inhibited by molybdate, vanadete, phosphate, and fluoride and the type of Inhibition produced by these elements was uncompetltlve, non-competitive, competltlve and mixed, respectively.     

Purification and partial characterization of a calmodulin-stimulated nucleoside triphosphatase from pea nuclei

A nucleoside triphosphatase/deoxynucleoside triphosphatase associated with the chromatin fraction from a highly purified preparation of pea nuclei has been isolated and characterized. The purified enzyme has a molecular weight of 47,000 as checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it has an isoelectric point of 6.6. In the presence of divalent cations (Mg2+ = Mn2+ greater than Ca2+), this enzyme hydrolyzes nucleoside triphosphates or deoxynucleoside triphosphates. Hydrolysis is optimal at pH 7.5 and is significantly inhibited by relatively low concentrations of quercetin, but is not sensitive to vanadate, nitrate, or oligomycin. The enzyme has a rather broad nucleotide substrate specificity and has a Km for MgATP2- of 0.6 mM. The enzyme activity is stimulated over 3-fold by Ca2+ and calmodulin, and the stimulation is blocked by the Ca2+ chelator EGTA and by the calmodulin antagonists compound 48/80 and chlorpromazine.     

Germ Tube Growth Inhibitor from Cronartium comandrae Aeciospores

The reduction of nucleic acid by an endogenous polynucleotide phosphorylase and ribonuclease in cells of Brevibacterium JM98A (ATCC 29895) was studied. A simple process was developed for the activation of the endogenous RNA-degrading enzyme(s). RNA degradation was activated by the presence of Pi with 14.2 mumol of ribonucleoside 5'-monophosphate per g of cell mass accumulating extracellularly. The optimum pH for degradation of RNA was 10.5 and the optimum temperature was 55 to 60 degrees C. Enzymatic activity was inhibited by the presence of Ca2+, Zn2+, or Mg2+. Although some of the RNA-degrading enzymatic activity was associated with the ribosomal fraction, most was soluble. Both polynucleotide phosphorylase and ribonuclease activities were identified.