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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The membrane-bound coupling factor from Mycobacterium phlei was solubilized from membrane vesicles by washing with low ionic strength buffer or 0.25 M sucrose. The solubilized enzyme exhibited coupling factor, latent ATPase, and succinate oxidation-stimulating activity. Purification by affinity chromatography using Sepharose coupled to ADP yielded a homogeneous preparation of latent ATPase which was purified about 200-fold with an 84% yield in a single step. Purified latent ATPase exhibited coupling factor activity but no succinate oxidation-stimulating activity. The molecular weight of latent ATPase was determined to be 250,000 +/- 10,000 by Sephadex G-200 chromatography. The ATPase was unmasked by trypsin treatment and activated by Mg2+ ion. However, trypsin treatment inactivated the coupling factor activity in the purified enzyme, indicating that the catalytic sites for ATPase and coupling activity are different. Unlike mitochondrial ATPase, latent ATPase from M. phlei was not cold-labile. Of the nucleoside triphosphates, UTP, ITP, and epsilon-ATP (1-N6-ethenoadenosine triphosphate) were hydrolyzed to a lesser extent compared to ATP. Kinetic data showed that ADP acted as a competitive inhibitor of latent ATPase activity with a Ki of 5 x 10(-3) M. Uncouplers of oxidative phosphorylation and respiratory inhibitors did not affect the latent ATPase activity, while sodium azide (0.1 mM) inhibited the latent ATPase activity.
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PMID:Energy-transducing membrane-bound coupling factor-ATPase from Mycobacterium phlei. I. Purification, homogeneity, and properties. 12 54

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.
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PMID:Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase. 12 62

We have partially purified active delta and epsilon subunits of the E. coli membrane-bound Mg2+-ATPase (ECF1). Treating purified ECF1 with 50% pyridine precipitates the major subunits (alpha, beta, and gamma) of the enzyme, but the two minor subunits (delta and epsilon), which are present in relatively small amounts, remain in solution. The delta and epsilon subunits were then resolved from one another by anion exchange chromatography. The partially purified epsilon strongly inhibits the hydrolytic activity of ECF1. The epsilon fraction inhibits both the highly purified five-subunit ATPase and the enzyme deficient in the delta subunit. The latter result indicates that the delta subunit is not required for inhibition by epsilon. By contrast, two-subunit enzyme, consisting chiefly of the alpha and beta subunits, was insensitive to the ATPase inhibitor, suggesting that the gamma subunit may be required for inhibition by epsilon. The partially purified delta subunit restored the capacity of ATPase deficient in delta to recombine with ATPase-depleted membranes and to reconstitute ATP-dependent transhydrogenase. Previously we reported (Biochem, Biophys. Res. Commun. 62:764 [1975]) that a fraction containing both the delta and epsilon subunits of ECF1 restored the capacity of ATPase missing delta to recombine with depleted membranes and to function as a coupling factor in oxidative phosphorylation and for the energized transhydrogenase. These reconstitution experiments using isolated subunits provide rather substantial evidence that the delta subunit is essential for attaching the ATPase to the membrane and that the epsilon subunit has a regulatory function as an inhibitor of the ATPase activity of ECF1.
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PMID:Partial purification of active delta and epsilon subunits of the membrane ATPase from escherichia coli. 12 87

1. Insulin stimulates the activity of membrane-bound ATPase isolated from frog skeletal muscle and from rat brain. The increase in activity of the membrane-bound ATPase system isolated from frog ranged from 9-8 to 53% at concentrations of Na+ (25 mM), K+ (10 mM), and ATP (2 mM) similar to those in in vivo experiments conducted previously (Moore, 1973). The increased activity of the membrane-bound ATPase is, therefore, at least as great as the insulin-induced increase in Na efflux (10-38%) from intact cells (Moore, 1973). If the concentration of Na+ is lowered to 4 mM and that of ATP lowered to 0-5 mM albumin, and 10(6) M, the increase in ouabain-inhibitable ATPase activity can reach as high as 400%. 2. Ouabain, at a concentration (10(-3) M) sufficient to inhibit stimulation of the frog ATPase by increasing Na from 4 to 25 mM, completely blocked the stimulation of ATPase activity due to insulin. 3. At 2 mM-ATP, 100 mM-Na+, and 20 mM-K+, conditions which maximally activate the (Na+ + K+)-ATPase, insulin did not increase the ATPase, activity. Stimulation was consistently seen at 10 mM-K+, 0-5 mM-ATP, and either 4 mM or 25 mM-Na+. 4. The finding that insulin does not stimulate the ATPase activity in conditions in which the (Na+ + K+)-ATPase component is maximally activated and especially the fact that ouabain can reproducibly inhibit insulin stimulation of the membrane-bound ATPase activity strongly suggest that interaction of insulin with its receptor upon the plasma membrane somehow stimulates the (Na+ + K+)-ATPase system (ouabain sensitive; ATP phosphohydrolase, EC (3.6.1.3). These results are consistent with previous studies of the effect of insulin upon Na efflux from intact cells (Moore, 1973) and support the previous conclusion that the component of Na efflux stimulated by insulin is active. The evidence suggests that insulin probably does not affect Vmax of the (Na+ + K+)-ATPase system, but may increase the affinity of the enzyme system to one or more effectors, most likely Na+, ATP, and perhaps K+. 5. Oxidized glutathione (2-7 X 10(-6) M), 10(-6) M, 10(-7) M, and 10(-8) M cyclic AMP did not affect the ATPase activity 10(-6)Malbumin, and . 6. The results are consistent with the view that the Na pump, (Na+ + K+)-ATPase, is intimately involved with the physiological action of insulin and may be transducer between the binding of insulin to its receptor on the plasma membrane and the cellular actions of insulin.
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PMID:Effect of insulin upon membrane-bound (Na+ + K+)-ATPase extracted from frog skeletal muscle. 12 36

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.
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PMID:Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. Kinetic properties of the basal and trypsin-stimulated activities. 12 30

At very low concentrations (less than 1 muM) triphenyltin chloride inhibits ATP formation and coupled electron transport in isolated spinach chloroplasts. Basal (-Pi) and uncoupled electron transport are not affected by triphenyltin. The membrane-bount ATP in equilibrium Pi exchange and Mg2+-dependent ATPase activities of chloroplasts are also completely sensitive to triphenyltin, although the Ca2+-dependent and Mg2+-dependent ATPase activities of the isolated coupling factor protein are insensitive to triphenyltin. The light-driven proton pump in chloroplasts is stimulated (up to 60%) by low levels of triphenyltin. Indeed, the amount of triphenyltin necessary to inhibit ATP formation or stimulate proton uptake is dependent upon the amount of chloroplasts present in the reaction mixture, with an apparent stoichiometry of 2-2.5 triphenyltin molecules/100 chlorophyll molecules at 50% inhibition of ATP formation and half-maximal stimulation of proton uptake. Chloroplasts partially stripped of coupling factor by an EDTA was are no longer able to accumulate protons in the light. However, low levels of triphenyltin can effectively restore this ability. The amount of triphenyltin required for the restoration of net proton uptake is also dependent upon the amount of chloroplasts, with a stoichiometry of 4-5 triphenyltin molecules/100 chlorophyll molecules at 50% reconstitution. On the basis of this and other evidence it is concluded that triphenyltin chloride inhibits phosphorylation, ATP + Pi exchange and membrane-bound ATPase activities in chloroplasts by specifically blocking the transport of protons through a membrane-bound carrier or channel located in a hydrophobic region of the membrane at or near the functional binding site for the coupling factor.
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PMID:Inhibition by triphenyltin chloride of a tightly-bound membrane component involved in photophosphorylation. 13 Oct 35

ATPase (ATP phosphohydrolase, EC 3.6.1.3) was detected in the membrane fraction of the strict anaerobic bacterium, Clostridium pasteurianum. About 70% of the total activity was found in the particulate fraction. The enzyme was Mg2+ dependent; Co2+ and Mn2+ but not Ca2+ could replace Mg2+ to some extent; the activation by Mg2+ was slightly antagonized by Ca2+. Even in the presence of Mg2+, Na+ or K+ had no stimulatory effect. The ATPase reaction was effectively inhibited by one of its products, ADP, and only slightly by the other product, inorganic phosphate. Of the nucleoside triphosphates tested ATP was hydrolyzed with highest affinity ([S]0.5 v = 1.3 mM) and maximal activity (120 U/g). The ATPase activity could be nearly completely solubilized by treatment of the membranes with 2 M LiCl in the absence of Mg2+. Solubilization, however, led to instability of the enzyme. The clostridial solubilized and membrane-bound ATPase showed different properties similar to the "allotopic" properties of mitochondrial and other bacterial ATPases. The membrane-bound ATPase in contrast to the soluble ATPase was sensitive to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD). DCCD, at 10(-4) M, led to 80% inhibition of the membrane-bound enzyme; oligomycin ouabain, or NaN3 had no effect. The membrane-bound ATPase could not be stimulated by trypsin pretreatment. Since none of the mono- or divalent cations had any truly stimulatory effect, and since a pH gradient (interior alkaline), which was sensitive to the ATPase inhibitor DCCD, was maintained during growth of C. pasteurianum, it was concluded that the function of the clostridial ATPase was the same as that of the rather similar mitochondrial enzyme, namely H+ translocation. A H+-translocating, ATP-consuming ATPase appears to be intrinsic equipment of all prolaryotic cells and as such to be phylogenetically very old; in the course of evolution the enzyme might have been developed to a H+-(re)translocating, ATP-forming ATPase as probably realized in aerobic bacteria, mitochondria and chloroplasts.
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PMID:Properties and function of clostridial membrane ATPase. 13 64

The kinetics of Ca2+ activation of membrane-bound (Ca2+ + Mg2+)-dependent ATPase (ATP phosphohydrolase EC 3.6.1.3) from human erythrocytes was studied. The ATPase from membrane prepared in the presence of 0.7-500muM Ca2+ showed positively cooperative behaviour and a Km for Ca2+ of between 1 and 4 muM. If the membranes were prepared in the absence of Ca2+ the Km increased, and an enzyme model with at least four calcium-binding sites accounted for the kinetic change assuming that one calcium-binding site decreased its affinity. Mg2+ or Mg-ATP could not replace Ca2+. Continuous-flow centrifugation involving a shear stress on membranes was necessary to obtain the high affinity ATPase activity. Using ordinary centrifugation the Ca2+-prepared membranes behaved as membranes prepared in the absence of Ca2+. The Ca2+-stimulated ATPase from membranes prepared without Ca2+ showed reduced maximum activity, but dialyzed, membrane-free hemolysates, whether prepared with Ca2+ present or not, recovered the activity when the hemolysate was present during the ATPase assay. It is suggested that the different Ca2+-affinities of the Ca2+-stimulated ATPase correspond to two different states of the calcium-pump.
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PMID:Ca2+ activation of membrane-bound (Ca2++Mg2+)-dependent ATPase from human erythrocytes prepared in the presence or absence of Ca2+. 13 27

Conditions are reported under which ATP protects membrane-bound coupling factor 1 against sodium bromide inactivation. The presence of Mg2+ was found to be obligatory for this protection. ADP and GTP also protected the enzyme against salt inactivation but to a much smaller extent. Other nucleotides tested were ineffective. At low ATP concentrations ADP prevented the effect of ATP and modified the saturation curve for ATP from hyperbolic to sigmoidal. Treatment of chloroplasts with 0.4 M MgCl2 or 2 M LiCl resulted in inactivation of photophosphorylation. In contrast to NaBr-depleted particles the MgCl2 or LiCl-depleted chloroplasts can be reconstituted by purified coupling factor 1. A binding site for Mg2+ and two different sites for ATP upon the coupling factor 1 are suggested to explain the mechanism of their protection against salt inactivation.
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PMID:Salt inactivation as a mechanistic probe of membrane-bound chloroplast coupling factor 1. 13 44

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300,000. An inhibitor protein in bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised condtions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Ppi is incorporated into the beta and gamma positions of the bound nucleotides, but beta-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.
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PMID:Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system. 13 62


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