胰蛋白酶处理对质膜H~ -ATPase钒酸钠抑制效应的影响

采用经蔗糖密度梯度法纯化的大豆 (GlycinemaxL .)下胚轴质膜微囊为材料 ,分析了胰蛋白酶处理对质膜H ATPase钒酸钠抑制效应的影响。实验结果显示 ,温和胰蛋白酶处理显著提高H ATPase的ATP水解活力。并且发现酶切处理降低了钒酸钠对ATPase的抑制效应 ,当钒酸钠浓度为 2mmol/L时 ,ATPase活力仅被抑制 5 3.49% ,而未经酶切的对照组则被抑制 6 4.13%。ATP水解动力学分析表明 ,胰蛋白酶酶切处理既不影响ATP水解的Km 值也不影响钒酸钠的抑制类型 ,酶切前后的Km 值都等于 0 .34mmol/L ,并且都属于反竞争抑制。以上结果显示胰蛋白酶酶切处理可能改变了磷酸酶结构域的结构而影响了钒酸钠的抑制效应 ,暗示C_末端调节着磷酸酶结构域的结构和功能     

Changes in Inhibitory Effect of Vanadate on the Plasma Membrane H+-ATPase Under Trypsin Treatment

The changes in inhibitory effect of vanadate on the plasma membrane H + ATPase were studied with mild trypsin treatment using plasma membrane vesicles purified from soybean （Glycine max L.）hypocotyles by sucrose gradient centrifugation. Results showed that under mild trypsin treatment the ATPase ATP hydrolysis activity was increased significantly. It was also found that the inhibitory effect of vandate was reduced after proteolysis. In the presence of 2 mmol/L vanadate, the ATP hydrolysis activity of the cleaved ATPase was inhibited by only 53.49%,while that of the un-cleaved ATPase was inhibited by 64.13%. Kinetic studies indicated that both the Km values and the inhibition type of vanadate were not affected by trypsin treatment. Upon proteolysis, Km remained as 0.34 mmol/L,while vanadate was still an uncompetitive inhibitor. Taking together, the structure and activity of the ATPase phosphatase domain were affected by trypsin treatment, implying that this domain might be regulated by the C-terminal end of the plasma membrane H+ ATPase.     

The effects of several ligands on the potassium-vanadate interaction in the inhibition of the (Na+ + K+)-ATPase and the Na+, K+ pump

Inhibition by vanadate of the K+-dependent p-nitrophenylphosphatase activity catalyzed by the (Na+ + K+)-ATPase partially purified from pig kidney showed competitive behavior with the substrate, K+ and Mg2+ acted as cofactors in promoting that inhibition. Ligands which inhibited the K+-dependent p-nitrophenyl phosphate hydrolysis (Na+, nucleotide polyphosphates, inorganic phosphate) protected against inhibition by vanadate. The magnitude of that protection was proportional to the inhibition produced in the absence of vanadate. In the presence of only p-nitrophenyl phosphate and Mg2+, or when the protective ligands were tested alone, the activation of p-nitrophenyl phosphate hydrolysis by K+ followed a sigmoid curve in the presence as well in the absence of vanadate. However, the combination of 100 mM NaCl and 3 mM ATP resulted in a biphasic effect of K+ on the p-nitrophenyl phosphate hydrolysis in the presence of vanadate. After an initial rise at low K+ concentration, the p-nitrophenylphosphatase activity declined at high K+ concentrations; this decline became more pronounced as the vanadate concentration was increased. This biphasic response was not seen when a nonphosphorylating ATP analog was combined with Na+ (which favors the nucleotide binding) or with inorganic phosphate (a requirement for K+ - K+ exchange). Experiments with inside-out resealed vesicles from human red cells showed that in the absence of Na+ plus ATP, K+ promoted vanadate inhibition of p-nitrophenylphosphatase activity in a nonbiphasic manner, acting at cytoplasmic sites. On the other hand, in the presence of Na+ plus ATP, the biphasic response of p-nitrophenyl phosphate hydrolysis is due to K+ acting on extracellular sites. In vanadate-poisoned intact red blood cells, the biphasic response of the ouabain-sensitive Rb+ influx as a function of the external Rb+ concentration failed to develop when there was no Na+ in the extracellular media. In addition, in the absence of extracellular Na+, external Rb+ did not influence the magnitude of inhibition. The present findings indicate that external K+ favors vanadate inhibition by displacing Na+ from unspecified extracellular membrane sites.     

The properties of arginine transport in vacuolar membrane vesicles of Neurospora crassa

We have measured the uptake of arginine into vacuolar membrane vesicles from Neurospora crassa. Arginine transport was found to be dependent on ATP hydrolysis, Mg2+, time, and vesicle protein with transported arginine remaining unmodified after entry into the vesicles. The Mg2+ concentration required for optimal arginine transport varied with the ATP concentration so that maximal transport occurred when the MgATP2- concentration was at a maximum and the concentrations of free ATP and Mg2+ were at a minimum. Arginine transport exhibited Michaelis-Menten kinetics when the arginine concentration was varied (Km = 0.4 mM). In contrast, arginine transport did not follow Michaelis-Menten kinetics when the MgATP2-concentration was varied (S0.5 = 0.12 mM). There was no inhibition of arginine transport when glutamine, ornithine, or lysine were included in the assay mixture. In contrast, arginine transport was inhibited 43% when D-arginine was present at a concentration 16-fold higher than that of L-arginine. Measurements of the internal vesicle volume established that arginine is concentrated 14-fold relative to the external concentration. Arginine transport was inhibited by dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl-hydrazone, and potassium nitrate (an inhibitor of vacuolar ATPase activity). Inhibitors of the plasma membrane or mitochondrial ATPase such as sodium vanadate or sodium azide did not affect arginine transport activity. In addition, arginine transport had a nucleoside triphosphate specificity similar to that of the vacuolar ATPase. These results suggest that arginine transport is dependent on vacuolar ATPase activity and an intact proton channel and proton gradient.     

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo.F1-ATP synthase

Fo.F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP.Mg2+ -induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319-12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 x 10(-6) M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo.F1-ATP synthase.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

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.     

Proton pumping kinetics and origin of nitrate inhibition of tonoplast-type H+-ATPase

A tonoplast-type vesicle preparation, substantially free from other subcellular membranes, was obtained from corn roots by equilibrium sucrose density gradient centrifugation. At pH 6.5 and in the presence of chloride ions, the tonoplast-type ATPase activity as measured by Pi release, was inhibited by nitrate ions. The ATPase activity was insensitive to molybdate and vanadate, indicating a minimum nonspecific phosphatase and plasma membrane contamination. The vesicles exhibited an ATP hydrolysis-supported proton uptake which was measured by the absorption change of acridine orange. The ATP hydrolysis supported uptake and the subsequent perturbant-induced release of protons (decay) was described by a kinetic model which was previously developed to evaluate the coupling between proton pumping and the primary energy yielding process for other biomembranes. The proton pumping activity was more sensitive to nitrate ions then was ATP hydrolysis. The differential effect and the kinetic analysis of nitrate inhibition led us to suggest that (i) the coupling between Pi release and proton pumping was indirect in nature and (ii) the primary inhibitory effect of nitrate ion was originated from an interaction with a protogenic protein domain which is functionally linked to the ATPase in the tonoplast-type membrane.     

Purification and properties of the h-translocating ATPase from the plasma membrane of tomato roots

The proton-translocating, plasma membrane ATPase was purified from tomato roots. At the final stage of purification approximately 80% of the protein was found in a single band with an apparent molecular weight of 90 kilodaltons. Cross-linking studies indicated that the ATPase normally exists as a trimer of catalytic subunits. No evidence was found for any additional subunits. The pH optimum for ATP hydrolysis by the purified protein was 6.5. Activity was stimulated by K⁺, especially at low pH, and inhibited by vanadate, N,N′-dicyclohexylcarbodiimide, and diethylstilbestrol; nitrate was weakly inhibitory. Activity was stimulated by lysolecithin but inhibited by sonicated phospholipids. The inhibition by lipids could be prevented if octylglucoside was added with the lipids; the combination of octylglucoside and lipids actually stimulated activity. The purified protein could be reconstituted into liposomes and catalyzed ATP-dependent, vanadate-sensitive proton translocation.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

Selective production of sealed plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue

Modification of our previous procedure for the isolation of microsomal membrane vesicles from red beet (Beta vulgaris L.) storage tissue allowed the recovery of sealed membrane vesicles displaying proton transport activity sensitive to both nitrate and orthovanadate. In the absence of a high salt concentration in the homogenization medium, contributions of nitrate-sensitive (tonoplast) and vanadate-sensitive (plasma membrane) proton transport were roughly equal. The addition of 0.25 M KCl to the homogenization medium increased the relative amount of nitrate-inhibited proton transport activity while the addition of 0.25 M KI resulted in proton pumping vesicles displaying inhibition by vanadate but stimulation by nitrate. These effects appeared to result from selective sealing of either plasma membrane or tonoplast membrane vesicles during homogenization in the presence of the two salts. Following centrifugation on linear sucrose gradients it was shown that the nitrate-sensitive, proton-transporting vesicles banded at low density and comigrated with nitrate-sensitive ATPase activity while the vanadate-sensitive, proton-transporting vesicles banded at a much higher density and comigrated with vanadate-sensitive ATPase. The properties of the vanadate-sensitive proton pumping vesicles were further characterized in microsomal membrane fractions produced by homogenization in the presence of 0.25 M KI and centrifugation on discontinuous sucrose density gradients. Proton transport was substrate specific for ATP, displayed a sharp pH optimum at 6.5, and was insensitive to azide but inhibited by N'-N-dicyclohexylcarbodiimide, diethylstilbestrol, and fluoride. The Km of proton transport for Mg:ATP was 0.67 mM and the K0.5 for vanadate inhibition was at about 50 microM. These properties are identical to those displayed by the plasma membrane ATPase and confirm a plasma membrane origin for the vesicles.     

The H+-translocating ATP synthase in Halobacterium halobium differs from F0F1-ATPase/synthase

Cell envelope vesicles of Halobacterium halobium synthesize ATP by utilizing base-acid transition (an outside acidic pH jump) under optimal conditions (1 M NaCl, 80 mM MgCl2, pH 6.8) even in the presence of azide (a specific inhibitor of F0F1-ATPase) (Mukohata & Yoshida (1987) J. Biochem. 101, 311-318). An azide-insensitive ATPase was isolated from the inner face of the vesicle membrane, and shown to hydrolyze ATP under very specific conditions (1.5 M Na2SO4, 10 mM MnCl2, pH 5.8) (Nanba & Mukohata (1987) J. Biochem. 102, 591-598). This ATPase activity could also be detected when the vesicle components were solubilized by detergent. The relationship between ATP synthesis and the membrane-bound ATPase was investigated by modification of the vesicles with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) or N-ethylmaleimide (NEM). The inhibition pattern of ATP synthesis in the modified vesicles and that of ATP hydrolysis of the solubilized modified vesicles were compared under the individual optimum conditions. The inhibition patterns were almost identical, suggesting that the ATP synthesis and hydrolysis are catalyzed by a single enzyme complex. The ATP synthase includes the above ATPase (300-320 kDa), which is composed of two pairs of 86 and 64 kDa subunits. This is a novel H+-translocating ATP synthase functioning in the extremely halophilic archaebacterium. This "archae-ATP-synthase" differs from F0F1-ATPase/synthase, which had been thought to be ubiquitous among all respiring organisms on our biosphere.     

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot: III. Effects of Fluoride on ATPase Activity and Transport

The effects of fluoride on the tonoplast type ATPase and transport activities associated with sealed membrane vesicles isolated from sugarbeet (Beta vulgaris L.) storage tissue were examined. This anion had two distinct effects upon the proton-pumping vesicles. When ATP hydrolysis was measured in the presence of gramicidin D, significant inhibition (approximately 50%) only occurred when the fluoride concentration approached 50 millimolar. In contrast, the same degree of inhibition of proton transport occurred when the fluoride concentration was about 24 millimolar. Effects on proton pumping at this concentration of fluoride could be attributed to an inhibition of chloride movement which serves to dissipate the vesicle membrane potential. Valinomycin could partially restore ATPase activity in sealed vesicles which were inhibited by fluoride and this restoration occurred with a reduction in the membrane potential. Fluoride demonstrated a competitive interaction with chloride-stimulation of proton transport and inhibited the uptake of radioactive chloride into sealed vesicles. When the vesicles were allowed to develop a pH gradient in the absence of KCl, and KCl was subsequently added, fluoride reduced enhancement of the existing pH gradient by KCl. The results are consistent with a chloride carrier that is inhibited by fluoride.     

Correlation between uncoupled ATP hydrolysis and heat production by the sarcoplasmic reticulum Ca2+-ATPase: coupling effect of fluoride.

The sarcoplasmic reticulum Ca(2+)-ATPase transports Ca(2+) using the chemical energy derived from ATP hydrolysis. Part of the chemical energy is used to translocate Ca(2+) through the membrane (work) and part is dissipated as heat. The amount of heat produced during catalysis increases after formation of the Ca(2+) gradient across the vesicle membrane. In the absence of gradient (leaky vesicles) the amount of heat produced/mol of ATP cleaved is half of that measured in the presence of the gradient. After formation of the gradient, part of the ATPase activity is not coupled to Ca(2+) transport. We now show that NaF can impair the uncoupled ATPase activity with discrete effect on the ATPase activity coupled to Ca(2+) transport. For the control vesicles not treated with NaF, after formation of the gradient only 20% of the ATP cleaved is coupled to Ca(2+) transport, and the caloric yield of the total ATPase activity (coupled plus uncoupled) is 22.8 kcal released/mol of ATP cleaved. In contrast, the vesicles treated with NaF consume only the ATP needed to maintain the gradient, and the caloric yield of ATP hydrolysis is 3.1 kcal/mol of ATP. The slow ATPase activity measured in vesicles treated with NaF has the same Ca(2+) dependence as the control vesicles. This demonstrates unambiguously that the uncoupled activity is an actual pathway of the Ca(2+)-ATPase rather than a contaminating phosphatase. We conclude that when ATP hydrolysis occurs without coupled biological work most of the chemical energy is dissipated as heat. Thus, uncoupled ATPase activity appears to be the mechanistic feature underlying the ability of the Ca(2+)-ATPase to modulated heat production.     

Energy-dependent transformation of F0.F1-ATPase in Paracoccus denitrificans plasma membranes

F(0).F(1)-ATP synthase in tightly coupled inside-out vesicles derived from Paracoccus denitrificans catalyzes rapid respiration-supported ATP synthesis, whereas their ATPase activity is very low. In the present study, the conditions required to reveal the Deltamu(H+)-generating ATP hydrolase activity of the bacterial enzyme have been elucidated. Energization of the membranes by respiration results in strong activation of the venturicidin-sensitive ATP hydrolysis, which is coupled with generation of Deltam?(H+). Partial uncoupling stimulates the proton-translocating ATP hydrolysis, whereas complete uncoupling results in inhibition of the ATPase activity. The presence of inorganic phosphate is indispensable for the steady-state turnover of the Deltam?(H+)-activated ATPase. The collapse of Deltam?(H+) brings about rapid deactivation of the enzyme, which has been subjected to pre-energization. The rate and extent of the deactivation depend on protein concentration, i.e. the more vesicles are present in the assay mixture, the higher the rate and extent of the deactivation is seen. Sulfite and the ADP-trapping system protect ATPase against the Deltam?(H+) collapse-induced deactivation, whereas phosphate delays the rate of deactivation. A low concentration of ADP (<1 microm) increases the rate of deactivation. Taken together, the results suggest that latent proton-translocating ATPase in P. denitrificans is kinetically equivalent to the previously characterized ADP(Mg2+)-inhibited, azide-trapped bovine heart mitochondrial F(0).F(1)-ATPase (Galkin, M. A., and Vinogradov, A. D. (1999) FEBS Lett. 448, 123-126). A Deltam?(H+)-sensitive mechanism operates in P. denitrificans that prevents physiologically wasteful consumption of ATP by F(0).F(1)-ATPase (synthase) complex when the latter is unable to maintain certain value of Deltam?(H+).     

Identification of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as beta-aspartyl phosphate

The chemical nature of the phosphoryl enzyme linkage of the electrogenic proton-translocating ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the plasma membrane of Neurospora has been identified as a mixed anhydride between phosphate and the beta-carboxyl group of an aspartic acid residue in the polypeptide chain. Incubation of isolated Neurospora plasma membrane vesicles containing 32P-labeled ATPase in buffers of increasing pH followed by analysis of the hydrolysis products yielded a pH versus hydrolysis profile characteristic of an acyl phosphate linkage. Reaction of labeled membranes with hydroxylamine at pH 5.3 also released [32P]i from the ATPase. Amino acid analyses of the Na[3H]BH4 reduction products obtained from membranes containing phosphorylated and dephosphorylated ATPase identified [3H]homoserine, the expected reduction product of beta-aspartyl phosphate, as the only additional tritiated reduction product in the samples from phosphorylated membranes. Tritium was not found in alpha-amino-delta-hydroxyvaleric acid, the reduction product of gamma-glutamyl phosphate, nor in proline, the degradation product of alpha-amino-delta-hydroxyvaleric acid. These results indicate that the phosphorylated intermediate of the Neurospora plasma membrane ATPase is a beta-aspartyl phosphate identical with that already known to exist in the Na+:K+- and Ca2+-translocating ATPases of animal cell origin. A common model for the mechanisms of all 3 ion-translocating ATPases is presented.     

Partial resolution and reconstitution of the subunits of the clathrin-coated vesicle proton ATPase responsible for Ca2+-activated ATP hydrolysis

The clathrin-coated vesicle proton-translocating complex is composed of a maximum of eight major polypeptides. Of these potential subunits, only the 17-kDa component, which is a proton pore, has been defined functionally (Sun, S.Z., Xie, X. S., and Stone, D. K. (1987) J. Biol. Chem. 262, 14790-14794). ATPase-and proton-pumping activities of the 200-fold purified proton-translocating complex are supported by Mg2+, whereas Ca2+ will only activate ATP hydrolysis. Like Mg2+-activated ATPase activity, Ca2+-supported ATP hydrolysis is inhibited by N-ethylmaleimide, NO3-, and an inhibitory antibody and is stimulated by Cl- and phosphatidylserine. Thus, Ca2+ prevents coupling of ATPase activity to vectoral proton movement, and Ca2+-activated ATPase activity is a partial reaction useful for analyzing the subunit structure required for ATP hydrolysis. The 530-kDa holoenzyme was dissociated with 3 M urea and subcomplexes, and isolated subunits were partially resolved by glycerol gradient centrifugation. No combination of these components yielded Mg2+-activated ATPase or proton pumping. Ca2+-activated ATP hydrolysis was not catalyzed by a subcomplex containing the 70- and 58-kDa subunits but was restored by recombination of the 70-, 58-, 40-, and 33-kDa polypeptides, indicating that these are subunits of the clathrin-coated vesicle proton pump which are necessary for ATP hydrolysis.     

Vanadate inhibition of ATP and p-nitrophenyl phosphate hydrolysis in the fragmented sarcoplasmic reticulum.

The vanadate inhibition of the Ca(2+)-ATPase activity was analysed both in intact sarcoplasmic reticulum vesicles and in the presence of low concentrations of Tween 20, using ATP and p-nitrophenyl phosphate as substrates. The saturation of the internal low-affinity calcium-binding sites protects the enzyme against vanadate inhibition, because: (1) p-nitrophenyl phosphate hydrolysis is not inhibited by vanadate in intact vesicles, but inhibition developed after solubilization with detergents; (2) the vanadate inhibition of the p-nitrophenyl phosphate hydrolysis in solubilized preparations is prevented by free Ca2+ concentrations higher than 10(-3) M and vanadate competes with calcium (10(-5)-10(-3) M); and (3) the vanadate inhibition of ATP hydrolysis is decreased with an increase in vesicular Ca2+ concentration. The presence of magnesium ions is indispensable for the vanadate effect. The vanadate inhibition is non-competitive with respect to Mg-p-nitrophenyl phosphate and uncompetitive with respect to Mg-ATP. However, in the presence of dimethyl sulfoxide, which facilitates phosphorylation of the enzyme, the inhibition is converted to a competitive one with respect to a substrate. The results suggest, that in the process of enzyme operation vanadate interacts with the unliganded E form of Ca(2+)-ATPase, occupying probably an intermediate position between the E2 and E1 forms, with the formation of an E2 Van complex, that imposes the inhibition on the Ca(2+)-ATPase activity.     

Purification and characterization of the proton translocating plasma membrane ATPase of red beet storage tissue

Plasma membranes were prepared from red beet (Beta vulgaris L.) storage tissue by partition in an aqueous two-phase system. A highly active proton-translocating ATPase was purified from these membranes by lysophosphatidylcholine extraction and glycerol density gradient centrifugation. The ATPase activity was inhibited by vanadate or dicyclohexyl carbodiimide, but was insensitive to azide, nitrate and molybdate at concentrations which inhibit the F1ATPase, the tonoplast ATPase, and acid phosphatase. Inhibition by vanadate was consistent with a non-competitive mechanism, with Ki = 10 microM. The Km for Mg-ATP was about 1 mM, magnesium ions were required, and the activity was stimulated by KCl and by lysophosphatidylcholine. The optimal pH was 6.5. The molecular mass by gel filtration in the presence of 2 g/liter octyl glucoside was 600 kDa, while dodecyl sulfate gel electrophoresis gave a polypeptide molecular mass of 100 kDa. After blotting onto nitrocellulose, the purified enzyme did not bind concanavalin A, although a concanavalin A-binding peptide of the plasma membrane runs to nearly the same position on the gel and showed some tendency to co-purify with the ATPase. Phospholipid vesicles into which the purified ATPase had been incorporated by the freeze-thaw technique showed vanadate-sensitive, ATP-dependent proton uptake. When the ATPase was reconstituted into lipid membranes at high protein to lipid ratios and incubated with ATP, two-dimensionally crystalline arrays of protein molecules were formed.     

Energy transduction in Escherichia coli. The role of the Mg2+ATPase.

Inverted membrane vesicles from strain 7, a wild type Escherichia coli K12 strain, actively transport calcium with energy supplied either by respiration or by ATP. These vesicles also have energy-linked quenching of quinacrine fluorescence. Membranes of strain 7, depleted of Mg2+ATPase by EDTA treatment, lack both activities. Membrane vesicles from strain NR70, a mutant lacking the Mg2+ATPase, show neither calcium transport nor energy-linked fluorescence quenching. Neither EDTA treatment nor genetic loss of the Mg2+atpase causes a reduction in respiration. Purified Mg2+ATPase from strain 7 can bind to EDTA-treated membrane vesicles from either strain 7 or NR70. This binding restored both calcium transport and fluorescence quenching, driven either by respiration or by ATP. Dicyclohexylcarbodiimide treatment mimics the effect of the Mg2+ATPase in the case of respiration-driven reactions. Treatment with EDTA, while not essential for the binding of the Mg2+ATPase to membrane vesicles of NR70, produced better restoration of both activities. The rate of restoration of fluorescence quenching showed a time lag which may indicate that binding of the Mg2+ATPase is a relatively slow process. Antiserum prepared against the Mg2+ATPase inhibited the quenching of quinacrine fluorescence when driven by ATP but not when driven by respiration. Addition of antiserum prior to addition of Mg2+ATPase prevented the restoration of fluorescence quenching, whether driven by respiration or ATP. These results clearly show that MG2+ATPase has an important role not only in catalyzing ATP synthesis and hydrolysis but also in maintaining the energized membrane state.