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1.
Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum. 总被引:3,自引:0,他引:3
L R Jones 《Biochimica et biophysica acta》1979,557(1):230-242
ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme. 相似文献
2.
The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans 总被引:1,自引:0,他引:1
The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed. 相似文献
3.
The (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum catalyzes the hydrolysis of acetyl phosphate in the presence of Mg2+ and EGTA and is stimulated by Ca2+. The Mg2(+)-dependent hydrolysis of acetyl phosphate measured in the presence of 6 mM acetyl phosphate, 5 mM MgCl2, and 2 mM EGTA is increased 2-fold by 20% dimethyl sulfoxide. This activity is further stimulated 1.6-fold by the addition of 30 mM KCl. In this condition addition of Ca2+ causes no further increase in the rate of hydrolysis and Ca2+ uptake is reduced to a low level. In leaky vesicles, hydrolysis continues to be back-inhibited by Ca2+ in the millimolar range. Unlike ATP, acetyl phosphate does not inhibit phosphorylation by Pi unless dimethyl sulfoxide is present. The presence of dimethyl sulfoxide also makes it possible to detect Pi inhibition of the Mg2(+)-dependent acetyl phosphate hydrolysis. These results suggest that dimethyl sulfoxide stabilizes a Pi-reactive form of the enzyme in a conformation that exhibits comparable affinities for acetyl phosphate and Pi. In this conformation the enzyme is transformed from a Ca2(+)- and Mg2(+)-dependent ATPase into a (K+ + Mg2+)-ATPase. 相似文献
4.
Some properties of membrane-bound, solubilized and reconstituted into liposomes H+-ATPase of vacuoles of Saccharomyces carlsbergensis 总被引:1,自引:0,他引:1
Vacuoles of yeast grown in peptone medium possessed high ATPase activity (up to 1 mumol X mg protein-1 X min-1). Membrane-bound and solubilized ATPase activities were insensitive to vanadate and azide, but were inhibited by NO-3 . K+ and cyclic AMP stimulated both membrane-bound and solubilized ATPase activities. Dio-9 activated the membrane form of vacuolar ATPase 1.5-2-fold and did not affect the solubilized enzyme. Solubilized and partially purified vacuolar ATPase was reconstituted with soy-bean phospholipids by a freeze-thaw procedure. ATPase activities in native vacuoles and proteoliposomes were stimulated effectively by Dio-9, the protonophore FCCP and ionophores valinomycin and nigericin. ATP-dependent H+ transport into proteoliposomes was also shown by quenching of ACMA fluorescence. Vacuolar and partially purified ATPase preparations possessed also GTPase activity. Unlike ATPase, however, GTPase was not incorporated as a proton pump into liposomes. 相似文献
5.
The resolution-reconstitution approach has been employed in order to gain information as to the subunit composition of the Neurospora plasma membrane H+-ATPase. Proteoliposomes prepared from sonicated asolectin and a highly purified, radiolabeled preparation of the 105,000-dalton hydrolytic moiety of the H+-ATPase by a freeze-thaw procedure catalyze ATP hydrolysis-dependent proton translocation as indicated by the extensive 9-amino-6-chloro-2-methoxyacridine fluorescence quenching that occurs upon the addition of MgATP to the proteoliposomes, and the reversal of this quenching induced by the H+-ATPase inhibitor, vanadate, and the proton conductors, carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. ATP hydrolysis is tightly coupled to proton translocation into the liposomes as indicated by the marked stimulation of ATP hydrolysis by carbonyl cyanide m-chlorophenylhydrazone and nigericin plus K+. The maximum stimulation of ATPase activity by proton conductors is about 3-fold, which indicates that at least two-thirds of the hydrolytically active ATPase molecules present in the reconstituted preparation are capable of translocating protons into the liposomes. Furthermore, as estimated by the extent of protection of the reconstituted 105,000-dalton hydrolytic moiety against tryptic degradation by vanadate in the presence of Mg2+ and ATP, the fraction of the total population of ATPase molecules that are hydrolytically active is at least 91%. Taken together, these data indicate that at least 61% of the ATPase molecules present in the reconstituted preparation are able to catalyze proton translocation. This information allows an estimation of the amount of any polypeptide in the preparation that must be present in order for that polypeptide to qualify as a subunit that is required for proton translocation in addition to the 105,000-dalton hydrolytic moiety, and an analysis of the radiolabeled ATPase preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and urea rules out the involvement of any such polypeptides larger than 2,500 daltons. This indicates that the Neurospora plasma membrane H+-ATPase has no subunits even vaguely resembling any that have been found to be associated with other transport ATPases and that if this enzyme has any subunits at all other than the 105,000-dalton hydrolytic moiety, they must be very small. 相似文献
6.
Plasma membrane vesicles were reconstituted by freezing and thawing of purified plasma membrane fraction from the yeast Metschnikowia reukaufii and phosphatidylcholine (type II-S from Sigma). The reconstituted plasma membrane vesicles generated a proton gradient (acidic inside) upon addition of ATP in presence of alkali cations. delta pH generation was most efficient when K+ was present both outside and inside the plasma membrane vesicles. Both ATPase activity and proton translocation in plasma membrane vesicles were inhibited by orthovanadate (50% inhibition at 100 microM). Plasma membrane vesicles reconstituted without added phosphatidylcholine generated in addition to delta pH, also an electrical potential difference delta psi (inside positive). Delta psi generation exhibited no K+ specificity. 50 microM dicyclohexylcarbodiimide inhibited completely delta psi generation whereas the K+-channel blocker quinine (5 microM) caused an 8-fold increase of delta psi. The proton gradient was much less affected by the agents. Taking into account the K+-dependent stimulation of the plasma membrane ATPase of M. reukaufii, these results further support the conclusion that the ATPase operates as a partially electrogenic H+/K+ exchanger, as was also suggested for other yeast plasma membrane ATPases. 相似文献
7.
The Mg2+-dependent, K+-stimulated ATPase of microsomes from pig gastric mucosa has been studied in relation to observed active H+ transport into vesicular space. Uptake of fluorescent dyes (acridine orange and 9-aminoacridine) was used to monitor the generated pH gradient. Freeze-fracture electron microscopy showed that the vesicular gastric microsomes have an asymmetric distribution of intramembraneous particles (P-face was particulate; E-face was relatively smooth. Valinomycin stimulated both dye uptake and K+-ATPase (valinomycin-stimulated K+-ATPase); stimulation by valinomycin was due to increased K+ entry to some intravesicular activating site, which in turn depends upon the accompanying anion. Using the valinomycin-stimulated K+-ATPase and H+ accumulation as an index, the sequence for anion permeation was NO-3 greater than Br- greater than Cl- greater than I- greater than acetate approximately isethionate. When permeability to both K+ and H+ was increased (e.g using valinomycin plus a protonophore or nigericin), stimulation of K+-ATPase was much less dependent on the anion and the observed dissipation of the vesicular pH gradient was consistent with an 'uncoupling' of ATP hydrolysis from H+ accumulation. Thiocyanate interacts with valinomycin inhibiting the typical action of the K+ ionophore. But stimulation of ATPase activity was seen by adding 10 mM SCN- to membranes preincubated with valinomycin. From the relative activation of the valinomycin-stimulated K+-ATPase, it appears that SCN- is a very permeant anion which can be placed before NO-3 in the sequence of permeation. Valinomycin-stimulated ATPase and H+ uptake showed similar dependent correlations, including: dependence on [ATP] and [K+], pH optima, temperature activation, and selective inhibition by SH- or NH2-group reagents. These results are consistent with a pump-leak model for the gastric microsomal K+-ATPase which was simulated using Nernst-Planck conditions for passive pathways and simple kinetics for the pump. The pump is a K+/H+ exchange pump requiring K+ at an internal site. Rate of K+ entry would depend on permeability to K+ as well as the counterion, either (1) the anion to accompany K+ or (2) the H+ efflux path as an exchange ion. The former leads to net accumulation of H+ and anion, while the latter results in non-productive stimulation of ATP hydrolysis. 相似文献
8.
Martínez F Uribe A Milán R Teresa Espinosa-García M Gracía-Pérez C Flores-Herrera O 《The international journal of biochemistry & cell biology》2002,34(8):1004-1016
This study evaluates the effect of Mg2+ on the extramitochondrial hydrolysis of ATP and ADP by human term placental mitochondria (HPM) and submitochondrial particle (SMP). Extramitochondrial ATPase and ADPase activities were evaluated in the presence or absence of K+, and different oxidizable substrates. Mg2+ increased both ATP and ADP hydrolysis according to the experimental conditions, and this stimulation was related to the mitochondrial intactness. The ADPase activity in intact mitochondria is 100-fold higher in presence of K+, succinate and 1mM Mg2+ while this activity is only increased by two-fold on the SMP when compared to the sample without Mg2+. It is clearly demonstrated that up-regulation of these enzyme activities occur in intact mitochondria and not on the enzyme itself. The results suggest that the regulation of ATP and ADP hydrolysis is complex, and Mg2+ plays an important role in the modulation of the extramitochondrial ATPase and ADPase activities in HPM 相似文献
9.
A nonelectrogenic H+ pump in plasma membranes of hog stomach. 总被引:12,自引:0,他引:12
G Sachs H H Chang E Rabon R Schackman M Lewin G Saccomani 《The Journal of biological chemistry》1976,251(23):7690-7698
Differential and density gradient centrifugation were used to prepare a vesicular membrane fraction from hog gastric mucosa enriched 17-fold with respect to cation-activated ATPase and 5'-AMPase. Fractionation of the gradient material by free flow electrophoresis resulted in a fraction 35-fold enriched in cation-activated ATPase and essentially free of 5'-AMPase and Mg2+ATPase. The addition of ATP to either fraction resulted in H+ uptake and Rb+ efflux. The ionophoric and osmotic sensitivity showed that these ion movements were due to transport rather than binding. The cation selectivity sequences, substrate specificities and action of inhibitors indicated that the transport was a function of K+ATPase activity. The characteristics of the ATP-dependent enhancement of SCN- uptake and 8-anilinonapthalene-1-sulfonate fluorescence in the presence of valinomycin and the action of ionophores and lipid-permeable ions suggested that the energy dependent K+:H+ exchange was effectively nonelectrogenic. Thus these vesicles contain a nonelectrogenic (H+ + K+)-ATPase, hence acid secretion by the stomach is probably due to an ATP-dependent H+ + K+ exchange. 相似文献
10.
An energy-dependent efflux system for potassium ions in yeast 总被引:3,自引:0,他引:3
An efflux of potassium ions was demonstrated in mutants of yeast cells lacking a functional high affinity carrier system for monovalent cations. This efflux showed the following characteristics: (a) It was stimulated by the presence of a substrate, either glucose or ethanol. (b) It was stimulated by several cationic organic molecules, such as ethidium bromide, dihydrostreptomycin, diethylaminoethyldextran, and also by trivalent cations, such as Al3+ and lanthanides; this stimulation also depended on the presence of a substrate. (c) K+ efflux was decreased in yeast mutants with decreased ATPase activity, which generated a lower membrane potential. (d) Although the efflux appeared to be of an electrogenic nature, producing hyperpolarization of cells, it was accompanied by the efflux of phosphate, probably as an anion partially compensating for the large amount of cations leaving the cell. (e) K+ efflux was also accompanied by an uptake of protons. (f) The efflux appeared more clearly in cells grown in YPD medium, and not in more complex media nor in the same YPD medium if supplemented with Ca2+ or Mg2+. Efflux of monovalent cations produced by Tb3+ and organic cationic agents was also demonstrated in wild type strains. This efflux system appears to be, at least partially, electrogenic, but seems to be also an exchange system for protons and to function as a symport with phosphate; it may be involved in the regulation of the internal pH of the cell, and appears to be regulated by its link to the energetic status of the cell, probably through the membrane potential. 相似文献
11.
The gastric [H,K]ATPase:H+/ATP stoichiometry 总被引:2,自引:0,他引:2
An H+/ATP ratio of 2 for H+ transport was determined from initial rate measurements at pH 6.1 in a purified gastric microsomal fraction containing the [H,K]ATPase. This ratio was independent of external KCl, though the apparent K0.5 for ATP was increased from 10.78 +/- 0.51 (n = 3) to 64.6 +/- 11.9 (n = 3) microM ATP and from 5.13 +/- 0.64 (n = 3) to 65.2 +/- 0.64 (n = 3) microM ATP for H+ transport and the K+-stimulated ATPase, respectively, as K+external was increased from 12 to 150 mM. The H+/ATP ratio was also relatively independent of ATP concentration. Maximum initial rates obtained in KCl-equilibrated vesicles were independent of added valinomycin, though net H+ transport was increased 29.3 +/- 1.03% (n = 6) by the addition of ionophore. Maximum net H+ transport in this vesicle preparation was 185 +/- 2.1 (n = 14) nmol mg-1 of protein. Initial rate measurements of ATPase represent a burst of K+-dependent activity of approximately 10-15 s duration. The H+/ATP stoichiometry was calculated based on the K+-stimulated component of hydrolysis. Under most conditions, the Mg2+-dependent component of hydrolysis was less than 10% of the (Mg2+ + K+) component of hydrolysis. 相似文献
12.
Solubilization and reconstitution of the gastric H,K-ATPase 总被引:3,自引:0,他引:3
E Rabon R D Gunther A Soumarmon S Bassilian M Lewin G Sachs 《The Journal of biological chemistry》1985,260(18):10200-10207
Proteoliposomes containing the hog gastric H+,K+-ATPase were prepared from cholate and n-octyl glucoside extracts of native microsomes. Experiments were presented which show reconstitution-dependent selective purification of a 94-kDa peptide capable of Rb+/Rb+ exchange and active H+ transport. The absence of selective enrichment of residual protein contamination in this material suggests but does not prove that those transport reactions are attributable only to the 94-kDa peptide. Transport demonstrated inhibitor sensitivity and cation specificity comparable to the microsomal gastric ATPase. In K2SO4 media the H+ transport reaction was protonophore insensitive and correlated with MgATP-dependent 86Rb+ extrusion. This and other evidence suggested that active transport occurs via electroneutral H+in for K+out exchange. 86Rb+ exchange (uptake) in the proteoliposomes demonstrated both saturable and nonsaturable components. At a K0.5 = 1.5 mM, saturable 86Rb+ uptake accounted for about 90% of Rb+ influx. The vanadate-sensitive cation exchange indicated that the ATPase was reconstituted asymmetrically into the proteoliposomes (70% cis-/30% trans-vanadate site). 86Rb+ exchange was inhibited by ATP and stimulated about 2-fold by low Mg2+ and 5 mM phosphate. These ligand effects and the demonstration of comparable rates of passive exchange and active Rb+ efflux suggest that passive K+ exchange is not severely limited by a K+-occluded enzyme form in the H,K-ATPase. A model compatible with this hypothesis is suggested. 相似文献
13.
W B Im D P Blakeman J E Bleasdale J P Davis 《The Journal of biological chemistry》1987,262(20):9865-9871
Rat stimulated heavy gastric membranes enriched with (H+-K+)-ATPase, a marker for the apical membrane of the parietal cell, displayed a 32P-histone-dephosphorylating activity which appeared to be physically copurified with, but functionally independent of, the ATPase. The protein phosphatase activity was optimal at pH 7.5 and was inhibited by fluoride (50 mM), inorganic phosphate (50 mM), and p-chloromercuribenzoate (0.1 mM), but was insensitive to vanadate (1 mM). The 32P-phosphoproteins in the heavy gastric membranes were also dephosphorylated, apparently by their own membrane-bound phosphatase in the presence of Mg2+ at millimolar concentrations, which is likely to enhance membrane-membrane interaction. Heavy gastric membrane vesicles incubated with Mg2+ (2 mM) exhibited no alterations in K+-dependent ATP-hydrolyzing activity, Cl permeability, and protein and lipid compositions, but irreversibly lost the ATP, K+-dependent H+-pumping activity. Since valinomycin, a K+-specific ionophore, restored the intravesicular acidifying activity and an inhibitor of the protein phosphatase, inorganic phosphate, largely blocked the Mg2+-induced change in the membrane transport function, it is reasonable to propose that the phosphatase action on certain membrane proteins, possibly the putative K+ transporter or regulatory proteins, selectively decreases K+-conductance in the apical membranes of gastric parietal cells. 相似文献
14.
The ATPase activity of CF1 isolated from pea chloroplasts with epsilon-ATP, the fluorescent analog of ATP and ATP used as substrates, in the presence of Mg2+, Ca2+ and sodium sulfite (stimulator of the ATPase activity) was studied. The rate of epsilon-ATP hydrolysis in the presence of Mg2+ is nearly two times as low as that of ATP; an addition of sodium sulfite to the reaction mixture increases the reaction rate without changing the above ratio. The rate of Ca2+-dependent hydrolysis of epsilon-ATP is rather low as compared to that in the presence of Mg2+. epsilon-ADP is a competitive inhibitor of Mg2+-dependent ATPase reaction and inhibits this process in the presence of Ca2+, the inhibition being of a mixed type. Modification of CF1 by covalent binding of epsilon-ADP results in a 70-80% decrease of the Mg2+-dependent ATPase activity, the Ca2+-dependent ATPase activity is changed only insignificantly thereby. The differences in the activation of ATP and epsilon-ATP hydrolyses by Ca2+ and Mg2+ can be accounted for by the existence of two sites in the active center of CF1, which are specific for Mg2+ and Ca2+, respectively. It is concluded that the binding of epsilon-ADP occurs in the Mg2+-dependent ATPase site of the active center. 相似文献
15.
Chemical modification of sarcoplasmic reticulum with methylbenzimidate. Stimulation of Ca2+ efflux.
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V Shoshan-Barmatz 《The Biochemical journal》1987,243(1):165-173
Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca2+ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5'-[beta, gamma-imido]triphosphate greater than or equal to ATP greater than UTP greater than ADP greater than AMP. Divalent cations such as Ca2+, Mg2+ and Mn2+, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca2+ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca2+ binding or ATP-Pi exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca2+ released about 80% of their Ca2+ content within 3 s. Control membranes released only 3% of their Ca2+ during the same time period. MBI modification inhibited Ca2+ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that MBI in the presence of ATP stimulates Ca2+ release by modifying a protein factor(s) other than the (Ca2+ + Mg2+)-ATPase. 相似文献
16.
The K+/H+ exchange activity of the inner mitochondrial membrane was investigated in the yeast Saccharomyces cerevisiae. Swelling experiments in potassium acetate indicated that the K+/H+ exchange was active without any additional treatment after the mitochondria isolation, such as a Mg2+ depletion. As in mammalian mitochondria, the activity of yeast mitochondria was stimulated by increasing pH and was inhibited by the amphiphilic amines quinine and propranolol and by the carboxyl reagent dicyclohexylcarbodiimide. However, the activity was poorly inhibited by Mg2+ and consequently was only slightly stimulated by the Mg2+/H+ exchanger A23187. On the other hand, Zn2+ was very efficient for inhibiting the exchange and consequently the activity was strongly stimulated by the permeant metal-chelator o-phenanthroline. The [86Rb]Rb+ accumulation in mitochondria and mitoplasts was only partially inhibited by quinine and propranolol suggesting that part of the accumulation monitored under these conditions was due to cation leak through the inner membrane together with adsorption on the membrane. The DCCD-sensitive activity could be reconstituted from mitochondria and from mitoplasts solubilized with Triton X-100; this activity, measured by [86Rb]Rb+ accumulation, was quinine- and propranolol-sensitive. A spectrophotometric method, based on the capacity of negatively charged proteoliposomes to swell, was then developed in order to continuously follow the reconstituted activity. 相似文献
17.
Oligomycin-sensitive ATPase activity was studied in isolated yeast mitochondria. The protonophore CCCP, at a concentration which completely inhibited ATP synthesis, induced only a low rate of hydrolysis of externally added ATP, and the extent of hydrolysis was dependent upon phosphate (Pi) concentration. CCCP promoted hydrolysis of intramitochondrial ATP. However, hydrolysis of externally added ATP was total in a medium containing potassium phosphate plus valinomycin. Without ionophores, ATPase activity was only observed at high external pH or with detergent-treated mitochondria. Under state 4 conditions, external ATP had access to the catalytic nucleotide site of ATPase as shown by 32Pi-ATP exchange experiments. These results are discussed in terms of a limitation of the translocase-mediated ATP/ADP exchange in uncoupled mitochondria. 相似文献
18.
Meyer-Fernandes JR Lanz-Mendoza H Gondim KC Willott E Wells MA 《Archives of biochemistry and biophysics》2000,382(1):152-159
In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity. 相似文献
19.
The kinetic data of magnesium and inorganic phosphate inhibition of the (Na+,K+)-dependent ATP hydrolysis are consistent with a model where both ligands act independently and their release in the ATPase cycle is an ordered process where inorganic phosphate is released first. The effects of magnesium on the stimulation of the ATPase activity by Na+, K+ and ATP, and the inhibition of that activity by inorganic phosphate, are consistent with Mg2+ acting both as a 'product' and as a dead-end inhibitor. The dead-end Mg-enzyme complex would be produced with an enzyme form located downstream in the reaction sequence from the point where Mg2+ acts as a 'product' inhibitor. In the absence of K+, Mg2+ inhibition was reduced when either Na+ or ATP concentrations were increased well beyond those concentrations needed to saturate their high-affinity sites. This ATP effect suggests that the dead-end Mg-enzyme complex formation is affected by the speed of the E2-E1 conformational change. The present model is consistent with the formation of an Mg-phosphoenzyme complex insensitive to K+ which could become K+-sensitive in the presence of high Na+ concentrations. These Mg-enzyme complexes appear as intermediaries in the Na+-ATPase activity found in the absence of external Na+ and K+. These results can be interpreted on the basis of Mg2+ binding to a single site in the enzyme molecule. In addition, these experiments provide kinetic evidence indicating that the stimulation by external Na+ of the ATPase activity in the absence of K+ is due to a K+-like action of Na+ on the external K+ sites. 相似文献
20.
Properties of H+-translocating adenosine triphosphatase in vacuolar membranes of SAccharomyces cerevisiae 总被引:23,自引:0,他引:23
The properties of Mg2+-ATPase in the vacuole of Saccharomyces cerevisiae were studied, using purified intact vacuoles and right-side-out vacuolar membrane vesicles prepared by the method of Y. Ohsumi and Y. Anraku ((1981) J. Biol. Chem. 256, 2079). The enzyme requires Mg2+ ion but not Ca2+ in. Cu2+ and Zn2+ ions inhibit the activity. The optimal pH is at pH 7.0. The enzyme hydrolyzes ATP, GTP, UTP, and CTP in this order and the Km value for ATP was determined as 0.2 mM. It does not hydrolyze ADP, adenosyl-5'-yl imidodiphosphate, or p-nitrophenyl phosphate. ADP does not inhibit hydrolysis of ATP by the enzyme. The activities of intact vacuoles and of vacuolar membrane vesicles were stimulated 3- and 1.5-fold, respectively, by the protonophore uncoupler 3,5-di-tert-butyl-4-hydroxybenzilidenemalononitrile and the K+/H+ antiporter ionophore nigericin. Sodium azide at a concentration exerting an uncoupler effect also stimulated the activity. The activity was sensitive to the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, but not to sodium vanadate. The ATP-dependent formation of an electrochemical potential difference of protons, measured by the flow-dialysis method, was determined as 180 mV, with contribution of 1.7 pH units, interior acid, and of a membrane potential of 75 mV. It is concluded that the Mg2+-ATPase of vacuoles is a new marker enzyme for these organelles and is a N,N'-dicyclohexylcarbodiimide-sensitive, H+-translocating ATPase whose catalytic site is exposed to the cytoplasm. 相似文献