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1.
Comparison of the rat microsomal Mg-ATPase of various tissues 总被引:1,自引:0,他引:1
The microsomal Mg-ATPase from various rat tissues was compared. After fractionating the microsomal vesicles by sucrose gradient centrifugation, the highest specific activity of the Mg-ATPase was found in the low-density vesicles which contained plasma membrane. A large fraction (25-90%) of the microsomal Ca-independent Mg-ATPase found in each tissue had the following properties: (1) the Km for ATP was 0.2 mM; (2) the rate of ATP hydrolysis by the Mg-ATPase was nonlinear due to an ATP-stimulated inactivation of the enzyme; (3) wheat germ agglutinin, concanavalin A, glutaraldehyde, and antiserum prevented inactivation induced by ATP or AdoPP[NH]P; (4) detergents at relatively low detergent:protein ratios increased the rate of inactivation with little change in the initial rate of ATP hydrolysis; (5) the Mg-ATPase was inactivated by irradiation in the presence of 8-azido ATP. (6) in addition to ATP, the Mg-ATPase was able to hydrolyze CTP, GTP, UTP, ITP, and GTP but was unable to hydrolyze any of the 10 nonnucleotide phosphocompounds which were tested; (7) the bivalent cation requirement of the Mg-ATPase could be provided by Mg2+, Ca2+, Mn2+, Zn2+, or Co2+ but the enzyme was inactive in the presence of Cu2+, Sr2+, Ba2+, or Be2+; (8) the Mg-ATPase activity was not altered by ionophores or inhibitors of the Na,K-ATPase, the Ca,Mg-ATPase or the mitochondrial F1ATPase. These data suggest that a major portion of the microsomal, basal Mg-ATPase activity is due to one unique enzyme found in most if not all tissues. 相似文献
2.
J D Robinson 《Archives of biochemistry and biophysics》1976,176(1):366-374
ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl2 was added in the presence of a “saturating” concentration of MgCl2 (4 mm). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at ?15 °C and with an increased stimulation in the presence of CaCl2. SrCl2 was more effective than CaCl2, but BaCl2 was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The Km for ATP was 0.1 mm, a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal 45Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity. 相似文献
3.
Parimal C. Sen Zhou Meng Ai Tushar K. Ray 《Archives of biochemistry and biophysics》1980,205(2):340-351
The transversal distribution of the free NH2 groups associated with phosphatidyl ethanolamine and the intrinsic membrane proteins of the purified pig gastric microsomes was quantitated and their relations to the function of the gastric K+-stimulated ATPase was investigated. Three different chemical probes such as 2,4,6-trinitrobenzene sulfonic acid (TNBS), 1-fluoro-2,4-dinitrobenzene (FDNB), and 2-methoxy-2,4-diphenyl-3(2H)-furanone (MDPF) were used for the study. The structure-function relationship of the membrane NH2 groups was studied after modification with the probes under various conditions and relating the inhibition of the K+-stimulated ATPase to the ATPase-dependent H+ accumulation by the gastric microsomal vesicles. TNBS (2 mm) inhibits nearly completely the K+-stimulated ATPase and the vesicular dye accumulation, both in presence and absence of valinomycin plus K+. Both the K+-ATPase and dye uptake were largely (about 50%) protected against TNBS inhibition if the treatment with TNBS was carried out in presence of 2 mm ATP. TNBS and FDNB labeled 70% of the total microsomal PE; the intra- and extravesicular orientation being 48 and 22%, respectively. The presence or absence of ATP did not have any effect on the TNBS labeling of microsomal PE. ATP, however, significantly (P < 0.05) reduced the labeling of protein-bound NH2 groups of gastric microsomes by TNBS. The intra- and extravesicular orientation of the protein NH2 groups were 60 and 40%, respectively. Eighteen percent of the total protein-NH2 appeared to be associated with the K+-stimulated ATPase; the rest being associated with non-ATPase proteins of the microsomes. About half (50%) of the total free NH2 groups of the K+-stimulated ATPase were exposed to the vesicle exterior and were found to play critical roles in gastric ATPase function. The generation of florescence after MDPF conjugation of gastric microsomes was largely (50%) inhibited by ATP. ATP also protected completely the MDPF inhibition of gastric K+-stimulated ATPase and dye uptake. 相似文献
4.
Gethyn J. Allen Francisco Morais Shelagh Muir R. Gareth Wyn Jones Roger A. Leigh 《Physiologia plantarum》1995,94(3):506-514
Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca2+ than Mg2+. Here we characterise the Ca2+-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca2+-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg2+. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca2+-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca2+ on the plasma membrane K+, Mg2+-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca2+-dependent activity is stimulated by free Ca2+ with a Km of 8.1 μM in the absence of Mg2+ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca2+-dependent than Mg2+-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca2+ and Mg2+ The Ca2+-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg2+-dependent activity functioned as a bafilomycin sensitive H+-pump in vacuolar membrane vesicles. Ca2+-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K+. Possible functions of Ca2+-dependent hydrolysis as a H+-pump or a Ca2+-pump was investigated using vacuolar membrane vesicles. No H+ or Ca2+ translocating activity was observed under conditions when the Ca2+-dependent ATP hydrolysis was active. 相似文献
5.
[14C]DDT was used as a probe to determine the subcellular localization of DDT in the ventral nerve cord (VNC) of the cockroach, Periplaneta americana (L.). Male cockroaches were injected intra-abdominally with [14C]DDT and their VNCs removed at 1 h post-injection. The VNCs were then subjected to homogenization and differential centrifugation to isolate plasma membrane, mitochondrial, and microsomal fractions. Results indicate that the plasma membrane fraction contained the greatest amount of [14C]DDT, with the mitochondrial and microsomal fractions containing significantly less. Calculations and a comparison with I50 values for oligomycin-sensitive (OS)Mg-ATPase from the literature support the prediction that an insufficient amount of DDT reaches the ventral nerve cord mitochondria of a cockroach to effect an I50 level of inhibition of the (OS)Mg-ATPase. 相似文献
6.
Senescence-Related Changes in ATP-Dependent Uptake of Calcium into Microsomal Vesicles from Carnation Petals 总被引:3,自引:3,他引:0
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Microsomal membrane vesicles isolated from the petals of young carnation (Dianthus caryophyllus L. cv White Sim) flowers accumulate Ca2+ in the presence of ATP. The specific activity of ATP-dependent uptake is ~20 nanomoles per milligram of protein per 30 minutes. The membranes also hydrolyze ATP, but Ca2+ stimulation of ATP hydrolysis was not discernible above the high background of Ca2+-insensitive ATPase activity. The initial velocity of uptake showed a sigmoidal rise with increasing Ca2+ concentration, suggesting that Ca2+ serves both as substrate and activator for the enzyme complex mediating its uptake. The concentration of Ca2+ at half maximal velocity of uptake (S0.5) was 12.5 micromolar and the Hill coefficient (nH) was 2.5. The addition of calmodulin to membrane preparations that had been isolated in the presence of chelators did not promote ATP-dependent accumulation of Ca2+, although this may reflect the fact that the treatment with chelators did not fully remove endogenous calmodulin. Transport of Ca2+ into membrane vesicles was unaffected by 50 micromolar ruthenium red and 5 micromolar sodium azide, indicating that uptake is primarily into vesicles of non-mitochondrial origin. By subfractionating the microsomes on a linear sucrose gradient, it was established that the ATP-dependent Ca2+ transport activity comigrates with endoplasmic reticulum and plasma membrane. During post-harvest development of cut flowers, ATP-dependent uptake of Ca2+ into microsomal vesicles declined by ~70%. This occurred before the appearance of petal-inrolling and the climacteric-like rise in ethylene production, parameters that denote the onset of senescence. There were no significant changes during this period in S0.5 or nH, but Vmax for ATP-dependent Ca2+ uptake decreased by ~40%. A similar decline in ATP-dependent uptake of Ca2+ into microsomal vesicles was induced by treating young flowers with physiological levels of exogenous ethylene. 相似文献
7.
H-ATPase Activity from Storage Tissue of Beta vulgaris: II. H/ATP Stoichiometry of an Anion-Sensitive H-ATPase 总被引:6,自引:5,他引:1
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The H+/ATP stoichiometry was determined for an anion-sensitive H+-ATPase in membrane vesicles believed to be derived from tonoplast. Initial rates of proton influx were measured by monitoring the alkalinization of a weakly buffered medium (pH 6.13) following the addition of ATP to a suspension of membrane vesicles of Beta vulgaris L. Initial rates of ATP hydrolysis were measured in an assay where ATP hydrolysis is coupled to NADH oxidation and monitored spectrophotometrically (A340) or by monitoring the release of 32P from [γ-32P]ATP. Inasmuch as this anion-sensitive H+-ATPase is strongly inhibited by NO3−, initial rates of H+ influx and ATP hydrolysis were measured in the absence and presence of NO3− to account for ATPase activity not involved in H+ transport. The NO3−-sensitive activities were calculated and used to estimate the ratio of H+ transported to ATP hydrolyzed. These measurements resulted in an estimate of the H+/ATP stoichiometry of 1.96 ± 0.14 suggesting that the actual stoichiometry is 2 H+ transported per ATP hydrolyzed. When compared with the reported values of the electrochemical potential gradient for H+ across the tonoplast measured in vivo, our result suggests that the H+-ATPase does not operate near equilibrium but is regulated by cellular factors other than energy supply. 相似文献
8.
《BBA》2022,1863(5):148544
Proton-translocating FOF1 ATP synthase (F-ATPase) couples ATP synthesis or hydrolysis to transmembrane proton transport in bacteria, chloroplasts, and mitochondria. The primary function of the mitochondrial FOF1 is ATP synthesis driven by protonmotive force (pmf) generated by the respiratory chain. However, when pmf is low or absent (e.g. during anoxia), FOF1 consumes ATP and functions as a proton-pumping ATPase.Several regulatory mechanisms suppress the ATPase activity of FOF1 at low pmf. In yeast mitochondria they include special inhibitory proteins Inh1p and Stf1p, and non-competitive inhibition of ATP hydrolysis by MgADP (ADP-inhibition). Presumably, these mechanisms help the cell to preserve the ATP pool upon membrane de-energization. However, no direct evidence was presented to support this hypothesis so far.Here we report that a point mutation Q263L in subunit beta of Saccharomyces cerevisiae ATP synthase significantly attenuated ADP-inhibition of the enzyme without major effect on the rate of ATP production by mitochondria. The mutation also decreased the sensitivity of the enzyme ATPase activity to azide. Similar effects of the corresponding mutations were observed in earlier studies in bacterial enzymes. This observation indicates that the molecular mechanism of ADP-inhibition is probably the same in mitochondrial and in bacterial FOF1.The mutant yeast strain had lower growth rate and had a longer lag period preceding exponential growth phase when starved cells were transferred to fresh growth medium. However, upon the loss of mitochondrial DNA (ρ0) the βQ263L mutation effect was reversed: the βQ263L ρ0 mutant grew faster than the wild-type ρ0 yeast. The results suggest that ADP-inhibition might play a role in prevention of wasteful ATP hydrolysis in the mitochondrial matrix. 相似文献
9.
Life by a new decarboxylation-dependent energy conservation mechanism with Na as coupling ion 总被引:17,自引:1,他引:16
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We report here a new mode of ATP synthesis in living cells. The anaerobic bacterium Propionigenium modestum gains its total energy for growth from the conversion of succinate to propionate according to: succinate + H2O → propionate + HCO3- (Go' = -20.6 kJ/mol). The small free energy change of this reaction does not allow a substrate-linked phosphorylation mechanism, and no electron transport phosphorylation takes place. Succinate was degraded by cell-free extracts to propionate and CO2 via succinyl-CoA, methyl-malonyl-CoA and propionyl-CoA. This pathway involves a membrane-bound methylmalonyl-CoA decarboxylase which couples the exergonic decarboxylation with a Na+ ion transport across the membrane. The organism also contained a membrane-bound ATPase which was specifically activated by Na+ ions and catalyzed and transport of Na+ ions into inverted bacterial vesicles upon ATP hydrolysis. The transport was abolished by monensin but not by the uncoupler carbonylcyanide-p-trifluoromethoxy phenylhydrazone. Isolated membrane vesicles catalyzed the synthesis of ATP from ADP and inorganic phosphate when malonyl-CoA was decarboxylated and malonyl-CoA synthesis from acetyl-CoA when ATP was hydrolyzed. These syntheses were sensitive to monensin which indicates that Na+ functions as the coupling ion. We conclude from these results that ATP synthesis in P. modestum is driven by a Na+ ion gradient which is generated upon decarboxylation of methylmalonyl-CoA. 相似文献
10.
Gastric microsomal vesicles isolated from dog fundic mucosa were shown to be relatively ion tight and have a low level of proton permeability. The H+ translocase, basal ATPase and K+-activated ATPase activities of these vesicles were measured and the H+/ATP stoichiometry calculated using either the total K+-ATPase or the K+-stimulatable component (total K+-ATPase—basal ATPase). The former estimations consistently gave stoichiometric of approximately one, whereas the use of only the K+-stimulatable component gave widely differing values. Measurement of the dephosphorylation of the enzyme under basal conditions revealed both a labile and a stable phosphoenzyme component. The rate of decay of the labile component completely accounted for the basal ATPase activity observed. We conclude that the basal ATPase associated with our preparations is a spontaneous dephosphorylation of the phosphoenzyme occurring in the absence of K+ and that the H+/ATP stoichiometry of the gastric ATPase is one. 相似文献
11.
THE LOCALIZATION OF Mg-Na-K-ACTIVATED ADENOSINE TRIPHOSPHATASE ON RED CELL GHOST MEMBRANES 总被引:32,自引:17,他引:15
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The lead salt method introduced by Wachstein and Meisel (12) for the cytochemical demonstration of ATPase activity was modified and used to determine sites of activity on red cell ghost membranes. Preliminary studies showed that aldehyde fixation and standard concentrations of the capture reagent Pb(NO3)2 resulted in marked inhibition of the ATPase activity of these membranes. By lowering the concentration of Pb2+ and incubating unfixed red cell ghosts, over 50% of the total ATPase activity, which included an ouabain-sensitive, Na-K-activated component, could be demonstrated by quantitative biochemical assay. Cytochemical tests, carried out under the same conditions, gave a reaction product localized exclusively along the inner surfaces of the ghost membranes for both Mg-ATPase and Na-K-ATPase. These findings indicate that the ATPase activity of red cell ghosts results in the release of Pi on the inside of the ghost membrane at sites scattered over its inner aspect. There were no deposits of reaction product on the outer surface of the ghost membrane, hence no indication that upon ATP hydrolysis Pi is released outside the ghosts. Nor was there any clear difference in the localization of reaction product of Mg-ATPase as opposed to that of Na-K-ATPase. 相似文献
12.
Determination of H/ATP Stoichiometry for the Plasma Membrane H-ATPase from Red Beet (Beta vulgaris L.) Storage Tissue 总被引:1,自引:1,他引:0
The H+/ATP stoichiometry was determined for the plasma membrane H+-ATPase from red beet (Beta vulgaris L., var Detroit Dark Red) storage tissue associated with native vesicles. The determination of H+/ATP stoichiometry utilized a kinetic approach where rates of H+ influx, estimated by three different methods, were compared to rates of ATP hydrolysis measured by the coupled enzyme assay under identical conditions. These methods for estimating H+ influx were based upon either determining the initial rate of alkalinization of the external medium from pH 6.13, measuring the rate of vesicle H+ leakage from a steadystate pH gradient after stopping the H+-ATPase or utilizing a mathematical model which describes the net transport of H+ at any given point in time. When the rate of H+ influx estimated by each of these methods was compared to the rate of ATP hydrolysis, a H+/ATP stoichiometry of about 1 was observed. In consideration of the maximum free energy available from ATP hydrolysis (ΔGatp), this value for H+/ATP stoichiometry is sufficient to account for the magnitude of the proton electrochemical gradient observed across the plasma membrane in vivo. 相似文献
13.
《Plant science》1988,54(3):177-184
A member fraction from corn roots which contains a vanadate-sensitive ATPase activity has been prepared. The specific activity at 38°C is between 3 and mol 12 μmol · min−1 · mg−1, depending on the age of roots. Addition of ATP promotes a very rapid quenching of the fluorescence of 9-amino-6-chloro-3-methoxy-acridin (ACMA). Proton pumping exhibits a delayed sensitivity to vanadate but is strongly and instantaneously inhibited by the new inhibitor SW 26. Both proton pumping, measured by the initial quenching rate, and ATP hydrolysis show maximum activities at ATP concentrations in the millimolar range, but the apparent Km-value for hydrolysis is higher than that observed for proton pumping. This is interpreted as being due to the presence of two populations of ATPases, one of them hydrolyzing ATP without creating a pH-gradient. The vanadate-sensitive ATP hydrolysis and H+-pumping activity may be solubilized with lysolecithin and reconstituted into liposomes either by a freeze-thawing-sonication or an octylglucoside dilution procedure. Both methods yield proteoliposomes exhibiting very effecient proton pumping, which is more sensitive to vanadate (I50 = 2 μM) or to SW 26 (I50 = 0.5 μM) than that of the original membrane fractions. 相似文献
14.
The Mg2+-dependency of Ca2+-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg2+- and Ca2+-buffering ligands. ATP hydrolysis is strongly stimulated by Mg2+ with a Km of 13 μ M in the absence or presence of 1 μ M free Ca2+. At free Mg2+ concentrations of 1 μ M and lower, ATP hydrolysis is Mg2+ -independent, but is strongly stimulated by submicromolar Ca2+ concentrations Km 0.25 μM, Vmax 24 μmol Pi/h per mg protein). The Ca2+-stimulated ATP hydrolysis strongly decreases at higher Mg2+ concentrations. The Ca2+-stimulated Mg2+-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg2+ concentrations calmodulin and trifluoperazine affect the high affinity Ca2+-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca2+-pumping in renal basolaterals and on Ca2+-ATPase in erythrocyte ghosts suggest that the Ca2+-pumping enzyme requires Mg2+. In contrast, a role of the Ca2+-stimulated Mg2+-independent ATP hydrolysis in active Ca2+ transport across basolateral membranes is rather unlikely. 相似文献
15.
A smooth microsomal fraction isolated from homogenates of Pbaseolus vulgaris root tissue has been found to possesss a highly active basal ATPase (measured in the absence of added cations). The microsomal membranes also feature a cation-sensitive ATPase which responds to Mg2+, Na+ and K+, but in a manner that is highly variable with pH. In contrast, membrane fragments prepared by a technique designed to yield purified plasma membrane were capable of little or no hydrolysis of ATP either in the presence or absence of added cations. This suggests that the microsomal activity is a reflection of membrane-bound ATPase which has been derived from cytoplasmic membranes, possibly the tonoplast, rather than plasma membrane. 相似文献
16.
3-Hydroxy-3-methylglutaryl-coenzyme A reductase. A comparison of the modulation in vitro by phosphorylation and dephosphorylation to modulation of enzyme activity by feeding cholesterol- or cholestyramine-supplemented diets
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Konstantinos A. Mitropoulos Brian L. Knight Bernard E. A. Reeves 《The Biochemical journal》1980,185(2):435-441
The activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hydroxymethylglutaryl-CoA reductase) was considerably inhibited during incubation with ATP+Mg2+. The inactivated enzyme was reactivated on further incubation with partially purified cytosolic phosphoprotein phosphatase. The inactivation was associated with a decrease in the apparent Km of the reductase for hydroxymethylglutaryl-CoA, and this was reversed on reactivation. The slight increase in activity observed during incubation of microsomal fraction without ATP was not associated with a change in apparent Km and, unlike the effect of the phosphatase, was not inhibited by NaF. Liver microsomal fraction from rats given cholesterol exhibited a low activity of hydroxymethylglutaryl-CoA reductase with a low apparent Km for hydroxymethylglutaryl-CoA. Mícrosomal fraction from rats fed cholestyramine exhibited a high activity with a high Km. To discover whether these changes had resulted from phosphorylation and dephosphorylation of the reductase, microsomal fraction from rats fed the supplemented diets and the standard diet were inactivated with ATP and reactivated with phosphoprotein phosphatase. Inactivation reduced the maximal activity of the reductase in each microsomal preparation and also reduced the apparent Km for hydroxymethylglutaryl-CoA. There was no difference between the preparations in the degree of inactivation produced by ATP. Treatment with phosphatase restored both the maximal activity and the apparent Km of each preparation, but never significantly increased the activity above that observed with untreated microsomal fraction. It is concluded that hydroxymethylglutaryl-CoA reductase in microsomal fraction prepared by standard procedures is almost entirely in the dephosphorylated form, and that the difference in kinetic properties in untreated microsomal fraction from rats fed the three diets cannot be explained by differences in the degree of phosphorylation of the enzyme. 相似文献
17.
The properties and kinetics of ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activities of plasma membrane H+-ATPase from the two reed ecot ypes, swamp reed (SR) and dune reed (DR), were investigated. The pH optimum of the plasma membrane H+-ATPase in both reed ecotypes was similar but the sensitivity of the enzyme to the reaction medium pH seemed to be higher in DR than that in SR. Compared to SR, the DR exhibited a higher Vmax value for ATP hydrolysis whereas the Km value was almost similar in both reed ecotypes. The PNPP hydrolysis of the plasma membrane H+-ATPase was also studied in both reed ecotypes at increasing PNPP concentrations. Km and Vmax for PNPP hydrolysis showed great differences in the two reed ecotypes and in DR the Km and Vmax values were 2- and 10-fold, respectively, higher than those in SR. The ATP hydrolysis activity of the plasma membrane was markedly inhibited by hydroxylamine in both reed ecotypes, and the percentage inhibition of ATP hydrolysis rate seemed higher in DR than that in SR. In addition, the structure or property of the C-terminal end of the plasma membrane H+-ATPase were also different in the two reed ecotypes. These data suggest that different isoforms of the plasma membrane H+-ATPase might be developed and involved in the adaptation of the plant to the long-term drought-prone habitat.This research was supported by Natural Science Foundation of China (No. 30270238 & No. 30470274) and the National Key Basic Research Special Funds of China (G1999011705). 相似文献
18.
《BBA》1987,893(2):275-288
The membrane-bound ATP synthase from chloroplasts can occur in different redox and activation states. In the absence of reductants the enzyme usually is oxidized and inactive, Eoxi. Illumination in the presence of dithiothreitol leads to an active, reduced enzyme, Ereda. If this form is stored in the dark in the presence of dithiothreitol an inactive, reduced enzyme Eredi is formed. The rates of ATP synthesis and ATP hydrolysis catalyzed by the different enzyme species are measured as a function of ΔpH (Δψ = 0 mV). The ΔpH was generated with an acid-base transition using a rapid-mixing quenched flow apparatus. The following results were obtained. (1) The oxidized ATP synthase catalyzes high rates of ATP synthesis, voxmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 3.4. (2) The active, reduced ATP synthase catalyzes high rates of ATP synthesis, vredmax = 400 ATP per CF0F1 per s. The half-maximal rate is obtained at ΔpH = 2.7. It catalyzes also high rates of ATP hydrolysis vredmax = −90 ATP per CF0F per s at ΔpH = 0. (3) The inactive species (both oxidized and reduced) catalyze neither ATP synthesis nor ATP hydrolysis. The activation/inactivation of the reduced enzyme is completely reversible. (4) The activation of the reduced, inactive enzyme is measured as a function of ΔpH by measuring the rate of ATP hydrolysis catalyzed by the active species. Half-maximal activation is observed at ΔpH = 2.2. (5) On the basis of these results a reaction scheme is proposed relating the redox reaction, the activation and the catalytic reaction of the chloroplast ATP synthase. 相似文献
19.
Gerhard Obermeyer Rosa Kriechbaumer Doris Strasser Angela Maschessnig Friedrich-Wilhelm Bentrup 《Physiologia plantarum》1996,98(2):281-290
The stimulation of the plasma membrane (PM) H+-ATPase by boric acid was studied on a microsomal fraction (MF) obtained from ungerminated, boron-dependent pollen grains of Lilium longiflorum Thunb. which usually need boron for germination and tube growth. ATP hydrolysis and H+ transport activity increased by 14 and 18%, respectively, after addition of 2-4 mM boric acid. The optimum of boron stimulation was at pH 6.5-8.5 for ATP hydrolysis and at pH 6.5-7.5 for H+ transport. No boron stimulation was detected when vanadate was added to the MF, whereas an increase of 10-20% in ATP hydrolysis and H+ transport was still measured in the presence of inhibitors specific for V -type ATPase (nitrate and bafilomycin) and F-type ATPase (azide), respectively. A vanadate-sensitive increase in ATP hydrolysis activity was also observed in partially permeabilized vesicles (0.001%[w/v] Triton X-100) suggesting a direct interaction between borate and the PM H+-ATPase rather than a weak acid-induced stimulation. Additionally, we measured the effect of boron on membrane voltage (Vm) of ungerminated pollen grains and observed small hyperpolarizations in 48% of all experiments. Exposing pollen grains to a more acidic pH of 4 caused a depolarization, followed in some experiments by a repolarization (21%). In the presence of 2 mM boron such hyperpolarizations, perhaps caused by an enhanced activity of the H+-ATPase, were measured in 58% of all tested pollen grains. The effects of boron on Vm may be reduced by additional stimulation of a K+ inward current of opposite direction to the H+-ATPase. All experiments indicate that boron stimulates an electrogenic transport system in the plasma membrane which is sensitive to vanadate and has a pH optimum around 7, i.e. the plasma membrane H+-ATPase. A boron-increased PM H+-ATPase activity in turn may stimulate germination and growth of pollen tubes. 相似文献
20.
Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn 总被引:7,自引:6,他引:1
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The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca2+ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive 45Ca2+ transport into mitochondria of corn roots. Ca2+ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca2+ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca2+ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca2+ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca2+ uptake. However, ATP-dependent Ca2+ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([14C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn. 相似文献