Magnesium-dependent ATPase Activity Research Articles

The Hsp60 Protein of Helicobacter Pylori Exhibits Chaperone and ATPase Activities at Elevated Temperatures

The heat-shock protein, Hsp60, is one of the most abundant proteins in Helicobacter pylori. Given its sequence homology to the Escherichia coli Hsp60 or GroEL, Hsp60 from H. pylori would be expected to function as a molecular chaperone in this organism. H. pylori is a type of bacteria that grows on the gastric epithelium, where the pH can fluctuate between neutral and 4.5, and the intracellular pH can be as low as 5.0. We previously showed that Hsp60 functions as a chaperone under acidic conditions. However, no reports have been made on the ability of Hsp60 to function as a molecular chaperone under other stressful conditions, such as heat stress or elevated temperatures. We report here that Hsp60 could suppress the heat-induced aggregation of the enzymes rhodanese, malate dehydrogenase, citrate synthase, and lactate dehydrogenase. Moreover, Hsp60 was found to have a potassium and magnesium-dependent ATPase activity that was stimulated at elevated temperatures. Although, Hsp60 was found to bind GTP, the hydrolysis of this nucleotide could not be observed. Our results show that Hsp60 from H. pylori can function as a molecular chaperone under conditions of heat stress.

Structural Analysis of the Regulatory Dithiol-containing Domain of the Chloroplast ATP Synthase γ Subunit

The gamma subunit of the F1 portion of the chloroplast ATP synthase contains a critically placed dithiol that provides a redox switch converting the enzyme from a latent to an active ATPase. The switch prevents depletion of intracellular ATP pools in the dark when photophosphorylation is inactive. The dithiol is located in a special regulatory segment of about 40 amino acids that is absent from the gamma subunits of the eubacterial and mitochondrial enzymes. Site-directed mutagenesis was used to probe the relationship between the structure of the gamma regulatory segment and its function in ATPase regulation via its interaction with the inhibitory epsilon subunit. Mutations were designed using a homology model of the chloroplast gamma subunit based on the analogous structures of the bacterial and mitochondrial homologues. The mutations included (a) substituting both of the disulfide-forming cysteines (Cys199 and Cys205) for alanines, (b) deleting nine residues containing the dithiol, (c) deleting the region distal to the dithiol (residues 224-240), and (d) deleting the entire segment between residues 196 and 241 with the exception of a small spacer element, and (e) deleting pieces from a small loop segment predicted by the model to interact with the dithiol domain. Deletions within the dithiol domain and within parts of the loop segment resulted in loss of redox control of the ATPase activity of the F1 enzyme. Deleting the distal segment, the whole regulatory domain, or parts of the loop segment had the additional effect of reducing the maximum extent of inhibition obtained upon adding the epsilon subunit but did not abolish epsilon binding. The results suggest a mechanism by which the gamma and epsilon subunits interact with each other to induce the latent state of the enzyme.

Magnesium-dependent ATPase Activity and Cooperativity of Magnesium Chelatase from Synechocystis sp. PCC6803

The first committed step in chlorophyll biosynthesis is catalyzed by magnesium chelatase, a complex enzyme with at least three substrates, cooperative Mg(2+) activation, and free energy coupling between ATP hydrolysis and metal-ion chelation. A detailed functional study of the behavior of the intact magnesium chelatase has been performed, including characterization of magnesium cooperativity and the stoichiometry of ATP consumption in relation to the magnesium porphyrin produced. It is demonstrated that, in vitro, this catalyzed reaction requires hydrolysis of approximately 15 MgATP(2-) and that the chelation partial reaction is energetically unfavorable, under our assay conditions, with a DeltaG degrees ' of 25-33 kJ mol(-1). Given the likely metabolite concentrations in vivo, this results in the chelatase reaction operating far from equilibrium. We have also determined the steady-state kinetic behavior of the intact enzyme and have compared the kinetic parameters obtained with those observed for the partial reactions of individual subunits. K(DIX) (where D(IX) represents deuteroporphyrin IX) is estimated to be 3.20 microm, and K(MgATP)(2-) is 0.45 mm. k(cat) for chelation is estimated to be 0.8 min(-1), suggesting that the ATP hydrolysis catalyzed by the isolated ChlI subunit is substantially slower in the intact chelatase. The magnesium-rich form of the chelatase is a more effective catalyst of the chelation reaction; magnesium activation of the chelatase increases V, as well as the specificity constant for the reaction of MgATP(2-) and D(IX), possibly as a result of a magnesium-triggered conformational change.

Calcium permeability and ATPase activities of red blood cells of magnesium deficient rats

Decreased intracellular magnesium and potassium and increased sodium levels were shown in the erythrocytes of weanling rats fed a low magnesium diet for 8 days. Magnesium-dependent ATPase activity was significantly increased in membranes obtained from Mg-deficient cells and the (Ca 2+-and (Na + + K +)-stimulated activities were similar to control values. Calcium flux measurements clearly show that passive permeability was significantly increased in red blood cells from Mg-deficient rats. Moreover, initial rates of calcium influx and to a lesser degree calcium efflux were increased in ghosts prepared from Mg-deficient erythrocytes. In agreement with our previous findings on the physico-chemical parameters of these membranes, the present results strongly suggest that permeability properties rather than the pump activities are primarily affected by dietary magnesium deficiency.

The immunoglobulin-binding protein in vitro autophosphorylation site maps to a threonine within the ATP binding cleft but is not a detectable site of in vivo phosphorylation

In vitro incubation of immunoprecipitated immunoglobulin-binding protein (BiP) complexes with calcium and [gamma-32P]ATP resulted in the phosphorylation of BiP on a threonine residue. This autophosphorylation activity did not occur in the presence of magnesium but had the same pH optimum as reported for its magnesium-dependent ATPase activity. This suggested the possibility that both activities could occur through ATP hydrolysis at the same site. In support of this, mutation of either Thr-37 or Thr-229 to a glycine eliminated both autophosphorylation and ATPase activities, and mutation of either residue to a serine significantly reduced both activities. Glutamic acid 175 in HSC71 has been hypothesized to flank the divalent cation complexed with ATP. Mutation of the analogous glutamic acid, Glu-201, in BiP abolished ATPase activity but still supported some autophosphorylation. The in vitro phosphorylation site was mapped to Thr-229 by mutational analysis. This threonine has been hypothesized to interact with the gamma-phosphate of ATP through a polarized water molecule and would be in a position to act as a phosphate acceptor in the ATP hydrolysis reaction. These data imply that both ATPase and autophosphorylation result from ATP hydrolysis at the same site and that the cation associated with BiP determines which activity is observed. Comparison of partial protease digestion or cyanogen bromide cleavage products of in vitro and in vivo phosphorylated BiP demonstrated that Thr-229 is not a detectable site of phosphorylation in cells. Therefore, whatever functional role phosphorylation may have in vivo, it cannot be attributed to autophosphorylation of Thr-229.

Sarcoplasmic reticulum function in the “stunned” myocardium

Transient ischemia does not induce myocardial necrosis but may be associated with prolonged contractile dysfunction (“stunned” myocardium). It has been suggested that alteration of the excitation-contraction coupling system (sarcoplasmic reticulum) could be responsible for this phenomenon. We tested this hypothesis by characterizing sarcoplasmic reticulum (SR) function in an isolated rat heart model of “stunned” myocardium (hearts reperfused after 10 min of normothermic global ischemia). At the end of the ischemic period oxalatesupported Ca-uptake was depressed either in the whole homogenate or in isolated SR (to 47% and 22% of control values, respectively). During reperfusion Ca-uptake of the whole heart homogenate recovered almost completely whereas slight but significant depression persisted in isolated SR (48 ± 2 vs 67 ± 4 nmol/min × mg, P < 0.01). In the presence of ruthenium red or ryanodine, two inhibitors of SR Ca-release channels, Ca-uptake was stimulated. Both in the whole heart homogenate and in isolated SR, such stimulation was remarkably smaller after reperfusion than in control conditions ( P < 0.001) suggesting reduced conductivity state of the SR Ca-release channels. Ca-stimulated, magnesium-dependent ATPase activity was remarkably reduced during ischemia and postischemic reperfusion induced only incomplete recovery (93 ± 18 vs 169 ± 14 nmol ATP/min × mg protein, P < 0.05). We conclude that complex modifications of SR function occur in the “stunned” myocardium and could contribute to the contractile impairment found in this condition.

Calcium-stimulated ATPase activity in plasma membrane vesicles from pancreatic acinar cells

Plasma membrane vesicles were prepared from rat pancreatic acinar cells using nitrogen cavitation and magnesium precipitation. The vesicles exhibited ATPase activity that was stimulated by submicromolar concentrations of free calcium and was dependent upon the presence of magnesium. This enzyme activity was localized to the cytoplasmic surface of the plasma membrane by two criteria. First, no activity was observed when intact cells replaced the membrane vesicles in the assay. Second, right side-out and inside-out vesicles were separated using concanavalin A sepharose-B. The calcium-stimulated, magnesium-dependent ATPase activity per mg protein in the inside-out fraction was 60% greater than that occurring in the mixed vesicle preparation. These results indicate that the plasma membrane of pancreatic acinar cells has a calcium-stimulated, magnesium-dependent ATPase located on its cytoplasmic surface and that this enzyme is stimulated by submicromolar concentrations of free calcium.

Plasma membrane ATPase of sugarbeet

A membrane fraction enriched with magnesium-dependent ATPase activity was isolated from sugarbeet ( Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI and sucrose density gradient centrifugation. This activity was inhibited by vanadate, N, N′-dicyclohexylcarbodiimide and diethylstilbestrol, but was insensitive to molybdate, azide, oligomycin, ouabain, and nitrate, suggesting enrichment in plasma membrane ATPase. The enzyme was substrate specific for ATP, had a pH optimum of 7.0, but showed little stimulation by 50 mM KCl. The sugarbeet ATPase preparation contained endogenous protein kinase activity which could be reduced by extraction of the membranes with 0.1% (w/v) sodium deoxycholate. Reduction of protein kinase activity allowed the demonstration of a rapidly turning over phosphorylated intermediate on a M r 105000 polypeptide, most likely representing the catalytic subunit of the ATPase. Phosphorylation was magnesium dependent, sensitive to diethylstilbestrol and vanadate but insensitive to oligomycin and azide. Neither the ATPase activity nor phosphoenzyme level were affected by combinations of sodium and potassium in the assay. These results argue against the presence of a synergistically stimulated NaK-ATPase at the plasma membrane of sugarbeet.

Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle.

The extent of oxygen exchange between phosphate and water has been measured for the calcium-regulated magnesium-dependent ATPase activity of chemically skinned fibers from rabbit skeletal muscle. The oxygen exchange was determined for isometrically held fibers by measuring with a mass spectrometer the distribution of 18O atoms in the product inorganic phosphate when ATP hydrolysis was carried out in H2(18)O. The extent of exchange was much greater in relaxed muscle (free Ca2+ less than 10(-8) M) than in calcium-activated muscle (free Ca2+ approximately equal to 3 X 10(-5) M). Activated fibers had an ATPase activity at least 30-fold greater than the relaxed fibers. These results correlate well with the extents of oxygen exchange accompanying magnesium-dependent myosin and unregulated actomyosin ATPase activities, respectively. In relaxed fibers, comparison of the amount of exchange with the ATPase activity suggests that the rate constant for the reformation of myosin-bound ATP from the myosin products complex is about 10 s-1 at 20 degrees C and pH 7.1. In each experiment the distribution of 18O in the Pi formed was incompatible with a single pathway for ATP hydrolysis. In the case of the calcium-activated fibers, the multiple pathways for ATP hydrolysis appeared to be an intrinsic property of the actomyosin ATPase in the fiber. These results indicate that in muscle fibers, as in isolated actomyosin, cleavage of protein-bound ATP is readily reversible and that association of the myosin products complex with actin promotes Pi release.

Distance measurement between the active site and cysteine-177 of the alkali one light chain of subfragment 1 from rabbit skeletal muscle.

Förster energy-transfer techniques have been applied to labeled myosin subfragment 1 from rabbit skeletal muscle to determine an intramolecular distance and whether this distance changes during magnesium-dependent ATPase activity. The alkali one light chain was labeled at Cys-177 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-IAEDANS) and then exchanged into subfragment 1. High specificity of labeling was indicated by high-performance liquid chromatography analysis of a tryptic digest of the labeled light chain. 2'(3')-O-(2,4,6-Trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) was bound to the labeled protein at the ATPase active site. The efficiency of energy transfer between the probes was 0.09 when measured by both steady-state and time-resolved fluorescence. Anisotropy measurements of the bound AEDANS indicated considerable freedom of motion of the probe. The probable distance between the probes was 57 A. This distance was unchanged during triphosphatase activity. Two further sites of TNP-ADP interaction with subfragment 1 were found. The effect of these interactions on the energy-transfer measurements was reduced to a minimum by careful choice of reaction conditions.

Evidence for the existence of a monovalent cation-stimulated, magnesium-dependent adenosine triphosphatase activity in the isolated plasma membranes of amoebas of the slime mold Dictyostelium discoideum

Evidence for a magnesium-dependent ATPase activity that can be stimulated by Na + and K +, or equally by Na + or K + alone, has been found in the plasma membranes isolated from amoebas of the slime mold Dictyostelium discoideum when the membranes are isolated from cultures grown up to the stationary phase. This ATPase activity is scarcely inhibitable by ouabain or phlorizin, but is very sensitive to low concentrations of azide or thimerosal. When the plasma membranes are isolated from amoebas growing in logarithmic phase, this monovalent cation-stimulated Mg 2+-dependent activity is barely detectable.

Effects of Sodium and Potassium on Binding of Ouabain to the Transport Adenosine Triphosphatase

Abstract The effects of sodium and potassium on the rate of ouabain binding to sodium and potassium ion-activated adenosine triphosphatase, isolated from the outer medulla of canine kidney, were examined in the presence of a magnesium ATP complex (37°; pH 7.4). In the presence of high sodium (≥ 10 mm), sodium accelerated and potassium retarded the rate of binding through a process kinetically defined by competition between sodium and potassium for a common site. The slow rate of binding in the presence of magnesium·ATP was also elevated by low sodium and retarded by potassium. The sensitivity of the latter reaction to potassium appeared to be an inverse function of the magnesium·ATP concentration and the ability of potassium to inhibit ouabain binding in the absence of sodium correlated with its ability to inhibit a ouabain-sensitive, magnesium-dependent ATPase activity, which was observed in the absence of sodium. These findings are consistent with glycoside binding to a receptor conformation that exist within the enzyme turnover cycle, which can operate under three conditions: high sodium and high potassium = fast rate; low sodium and zero potassium = intermediate rate; zero sodium and zero potassium = very slow rate. The antagonism between magnesium·ATP and potassium may reflect conversion of the enzyme from one state to another by magnesium·ATP which counteracts a potassium-induced stabilization of the other state. The kinetic data are also consistent with the existence of two sodium-dependent glycoside binding sites. If the latter were the case, analysis of the reaction rates at varying concentrations of sodium suggested two apparent sites for sodium-enzyme interaction, one with Ka of 16 mm and the other with Ka of 0.63 mm.

Characteristics of adenosinetriphosphatase of ram ejaculated spermatozoa and isolated sperm tails.

Characteristics of adenosinetriphosphatase of ejaculated spermatozoa from three rams have been studied. Ram spermatozoa were found to hydrolyse ATP in preference to ADP as evidenced by the insignificant amounts of AMP recovered from the incubation medium containing both ATP and ADP as substrates. However, ram spermatozoa could dephosphorylate ADP alone at a 20% lower rate than ATP. With ATP as substrate the Km was 3.6 X 1Oand inhibition was observed when the substrate concentration ex- ceeded that of magnesium; this was attributed to possible binding of free ATP without splitting at the active sites. The magnesium dependent ATPase activity of sperm tails, isolated by ultrasonic disintegration and fractional centrifugation, was stimulated by sodium and potassium more at 25-36 C than at 5-25 C presumably as a result of alterations in the configura- tion of the enzyme. However, above 36 C or after preincubation at 45 C monovalent cations inhibited the magnesium dependent ATPase activity which was ascribed to alterations of the liquid-crystalline structure of lipids associated with the enzyme. The ATPase activity of whole sperm increased with total cation concentrations of up to 30 mM but was depressed by 100 mae. Magnesium was a less efficient activator than calcium. The pH optimum of divalent cation activated ATPase of isolated sperm tails in the presence of 0.1 M KCI was about 8.0. When the KCI concentration was increased to 0.5 ae and 1.0 M the ATPase activity was markedly reduced and the pH optimum shifted to 7.2. Under these conditions, activation of the enzyme appeared to be de- pendent on the total divalent cation concentration rather than the nature of the cation. In view of these and other observations it is suggested that divalent cations modify the environment of the active centres by occupying fixed binding sites which may then influence orientation of the substrate on the enzyme.

Comparison of adenosinetriphosphatase activity in testicular and ejaculated spermatozoa of the ram.

been compared under various conditions. The magnesium-activated and sodium-potassiumstimulated ATPase activity was found to be less in testicular than in ejaculated spermatozoa despite the fact that the former cell type contained 50% more protein than the latter. The pH optimum (pH 8.4) of the magnesium-dependent ATPase activity was the same for both types of spermatozoa. However, maximal stimulation of the enzyme by monovalent cations occurred at pH 8.1 in testicular and pH 6.7 in ejaculated spermatozoa and coincided approximately with optimal conditions for motility. Above pH 8.6 monovalent cations inhibited rather than stimulated ATPase activities. The addition of increasing amounts of EDTA to the reaction mixtures greatly reduced the enzymic activity. In the presence of calcium ions, however, ATPase activities were sustained, but when the concentration of the added EDTA was increased to that of calcium the enzymic activity was significantly reduced; the resulting reduction in ATPase activity was ascribed to the chelating properties of EDTA. Determination of the enzyme and protein distribution in the head, mid-piece, tail, and an unidentified lighter fraction of testicular and ejaculated spermatozoa revealed that cation-dependent ATPase activities were lowest in the heads although the fractions accounted for more than 42% of the total protein. With the exception of the mid-piece of testicular spermatozoa, which possessed only 18% of the total enzymic activity, ATPase activities were about equally distributed in the remaining fractions and were always less in testicular than in ejaculated spermatozoa. The implications of these findings with respect to differences in motility between testicular and ejaculated spermatozoa have been considered.

An anion stimulated particulate ATP phosphohydrolase (ATPase) from bakers' yeast

A magnesium-dependent ATPase activity associated with a particulate fraction of bakers' yeast was investigated. This enzyme was markedly stimulated by various salts of bicarbonate and chloride ions. The stimulation seemed to be dependent upon the anion moieties rather than the cation moieties of the salts. The possible role of this ATPase in anion transports is discussed.