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1.
Homogenates of Tritrichomonas foetus exhibited a Mg2+-dependent adenosine triphosphatase (ATPase) activity, with a pH optimum in Tris buffers of 8.2 to 8.3. The activity was not sensitive to oxygen. At high concentrations, quercetin and 4-chloro-7-nitrobenzofurazan inhibited ATPase activity in the cytoplasmic extract by 20 and 70%, respectively, whereas oligomycin, venturicidin, triethyltin, leucinostatin, dibutylchloromethyltin chloride, spegazzinine, efrapeptin, citreoviridin and sodium azide had no effect and N,N'-dicyclohexylcarbodi-imide stimulated the activity somewhat. The activity was localized in a population of small cytoplasmic particles which also contained an acid phosphatase. There was no indication of an association of ATPase with hydrogenosomes. The ATPase activity (or activities) in this aerotolerant anaerobe is different from the ATPases characteristic of mitochondria or of anaerobic bacteria.  相似文献   

2.
1. The specific activity of mitochondrial ATPase (adenosine triphosphatase) in extracts of Schizosaccharomyces pombe decreased 2.5-fold as the glucose concentration in the growth medium decreased from 50mM to 15mM. 2. During the late exponential phase of growth, ATPase activity doubled. 3. Sensitivity to oligomycin and Dio-9 as measured by values for I50(mug of inhibitor/mg of protein giving 50% inhibition) at pH 6.8 increased sixfold and ninefold respectively during the initial decrease in ATPase activity, and this degree of sensitivity was maintained for the remainder of the growth cycle. 4. Increased sensitivity to NN'-dicyclohexylcarbodi-imide, triethyltin and venturicidin was also observed during the early stage of glucose de-repression. 5. Smaller increases in sensitivity to efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diaz-le, quercetin and spegazzinine also occurred. 6. The ATPase of glycerol-grown cells was less sensitive to inhibitors than that of glucose-repressed cells; change in values for I50 were not so marked during the growth cycle of cells growing with glycerol. 7. When submitochondrial particles from glycerol-grown cells were tested by passage through Sephadex G-50, a fourfold increase in activity was accompanied by increased inhibitor resistance. 8. Gel filtration of submitochondrial particles from glucose-de-repressed cells gave similar results, whereas loss of ATPase occurred in submitochondrial particles from glucose-repressed cells. 9. It is proposed that alterations in sensitivity to inhibitors at different stages of glucose derepression may be partly controlled by a naturally occuring inhibitor of ATPase. 10. The inhibitors tested may be classififed into two groups on the basis of alterations of sensitivity of the ATPase during physiological modification: (a) oligomycin, Dio-9, NN'-dicyclohexylcarbodi-imide, venturicidin and triethyltin, and (b) efrapeptin, aurovertin, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, quercetin and spegazzinine.  相似文献   

3.
1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[(3)H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F(1)-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[(113)Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8-9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000-8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.  相似文献   

4.
The effects of spegazzinine, a dihydroindole alkaloid, on various energy-transducing functions of chloroplasts were studied. The following observations were made, (i) Spegazzinine inhibited both cyclic and noncyclic photophosphorylation in isolated spinach chloroplast. The I50 value was about 80 μm. Over a concentration range which gave marked inhibition of ÀTP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids, while the fraction of electron transport stimulated by coupled phosphorylation was reduced to the basal level by spegazzinine. (ii) The regulatory effect of low concentrations of ATP on proton movements and electron transport was diminished by the alkaloid, (iii) Spegazzinine also inhibited with similar efficiency the ATPase activities of membrane-bound coupling factor 1 (CF1) and of purified CF1. One mole of spegazzinine per mole of CF1 seemed to be required to inhibit the ATPase activity, (iv) The allosteric effect of ADP on ATPase activity was not affected by spegazzinine. (v) On the basis of these results it is concluded that spegazzinine acts as an energy transfer inhibitor of hotophosphorylation and that its site of action may be at or near the catalytic site of ATPase.  相似文献   

5.
1. The mitochondrial ATPase of Acanthamoeba castellanii accumulated discontinuously in synchronous cultures prepared by a minimally perturbing size-selection technique. 2. Enzyme activity per ml of culture doubled overall during one cell cycle time of 8 h, but oscillated to give seven maxima during this period. Similar oscillations were observed in the specific activities of ATPase and of the naturally occurring inhibitor protein. 3. These variations in enzyme activity reflected changes in amount of enzyme protein as assayed by an immunological technique. 4. Large variations in I50 values (micrograms of inhibitor/mg of protein necessary for 50% inhibition of inhibitor-sensitive activity) for inhibition of ATPase activity by seven different inhibitors of energy conservation were observed. Activity was more sensitive to inhibition by oligomycin, efrapeptin, citreoviridin and quercetin when values were highest. 5. The results are discussed in relation to the phased organization of biosynthesis and degradation of cellular components known to occur during the cell cycle of this organization.  相似文献   

6.
1. The mitochondrial adenosine triphosphatase (ATPase) of Acanthamoeba castellanii is Mg2+-requiring (optimum cation: ATP ratio of 1.5) and has two pH optima of activity (at pH 6.6 and 8.1). 2. ATPase activity of submitochondrial particles is effectively inhibited by twelve different inhibitors of energy conservation suggesting similarities in inhibitor-binding sites to other previously characterized complexes. 3. Gel filtration by passage through Sephadex G-50 increases ATPase activity of submitochondrial particles between 1.5 and 3.5 fold indicating the presence of a low molecular weight inhibitor protein. 4. After removal of the inhibitor protein, sensitivity to inhibitors of energy conservation decreases by between 1.5 and 14 fold. Crude F1-inhibitor preparations from A. castellanii, Schizosaccharomyces pombe, Tetrahymena pyriformis and bovine heart also inhibit ATPase activity. 5. Large variations in ATPase activity, F1-inhibitor protein activity, and amounts of immunologically-determined ATPase protein were observed during exponential growth, and the correlation between changes in these measurements is discussed. 6. The results are also discussed highlighting the similarities between the mitochondrial ATPase of A. castellanii and other mitochondrial ATPases.  相似文献   

7.
A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. N,N'-Dicyclohexylcarbodiimide, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, N-1-napthylphthalamate, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H+ transport across their respective membranes.  相似文献   

8.
The limiting membranes of pituitary growth hormone and prolactin secretory granules contain a Mg2+-ATPase sensitive to anions. This enzyme is in many ways similar to mitochondrial ATPase. The enzyme was potently inhibited by oligomycin (Ki 6.5 X 10(-9) M), and was much more sensitive to the inhibitor than pituitary mitochondrial ATPase (Ki 2.7 X 10(-7) M). In contrast, the enzyme activity of intact secretory granules was only sparingly inhibited by oligomycin (maximal inhibition close to 30% at 5 X 10(-4) M). However, oligomycin (5 microM) did diminish to basal levels the enhanced granule ATPase activity observed in the presence of a stimulatory anion (25 mM sodium sulfite). Other compounds known to inhibit the proton translocating mitochondrial ATPase were also tested for their ability to inhibit the secretory granule ATPase. A similar pattern of limited inhibition in granules and greater sensitivity in isolated membranes was seen with the inhibitors N,N-dicyclohexylcarbodiimide and efrapeptin. In contrast, tri-n-butyltin chloride was a potent inhibitor of the ATPase of intact granules, and the susceptibility of the enzyme to inhibition by this compound was less after isolation of membranes. These observations suggest that pituitary secretory granule membrane ATPase may have a proton pumping function similar to that of the mitochondrial enzyme. In addition, the data imply that the inhibitor binding site(s) may be masked, inaccessible, or ineffective in intact granules, but exposed (or activated) in isolated membranes. The greater sensitivity of granule ATPase to tri-n-butyltin chloride, in contrast to the greater sensitivity of membrane ATPase to the other inhibitors, indicates that the tin compound may be effective at a membrane site(s) distinct from the others, or that the mechanism of inhibition is different.  相似文献   

9.
Seven different Chinese hamster cell lines were found to vary greatly in their sensitivity to inhibitors of the mitochondrial ATPase. In plating-efficiency experiments, Chinese hamster lung V79 and bone marrow M3-1 cells were approximately 10,000-fold more resistant to oligomycin, 100-fold more resistant to efrapeptin, and 10-fold more resistant to ossamycin and leucinostatin than were ovary CHO or peritoneal B14 cells. In vitro experiments indicated that the increased resistance of V79 versus CHO cells to these inhibitors was due to an increased resistance of the mitochondrial ATPase. Heat-inactivation experiments indicated that there was a difference in the structure of the mitochondrial ATPase of V79 and CHO cells. Genetic experiments indicated that the difference in the sensitivity of V79 and CHO cells to inhibitors of the ATPase and the difference in the structure of the mitochondrial ATPase of V79 and CHO cells was due to a difference in both a nuclear and a cytoplasmic gene.  相似文献   

10.
1. The cell-membrane ATP phosphohydrolase of vegetatively grown Clostridium pasteurianum was specifically Mg2+-dependent, but demonstrated significant activity with GTP, CTP and UTP. It displayed approximate Michaelis-Menten kinetics only in the presence of certain effectors (e.g. phosphoenolpyruvate, fructose 1,6-bis-phosphate) which decreased the Km for ATP (to below 2 mM) but also V, whilst extending to pH 5.8 the effective pH range of activity of the enzyme. 2. ATP phosphohydrolase activity of the membrane ATPase (BF0F1) was inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan, efrapeptin, leucinostatin and quercetin, and to a lesser degree by aurovertin and citreoviridin. The enzyme was not inhibited by oligomycin, spegazzinine, tributyl tin, triethyl tin or venturicidin. The soluble ATPase (BF1) component differed in not being inhibited by N,N'-dicyclohexylcarbodiimide, butyricin 7423 or leucinostatin. 3. The ATPase (BF0F1) complex and its soluble (BF1) component were separately purified. 4. Dodecylsulphate/polyacrylamide gel electrophoresis separated only four polypeptide components in the purified ATPase (BF0F1), with approximate molecular weights (+/- 10%) as follows: subunit a, 65 500; subunit c, 57 500; subunit da, 43 000; subunit fa, 15 000. The soluble (BF1 component contained only the three polypeptide subunits a, c and da. These were present in the BF0F1 preparation in the ratio 2 : 1 : 2; the contribution of subunit fa could not satisfactorily be quantified. 5. Subunit a was identified as the component binding 4-chloro-7-nitrobenzofurazan and subunit fa as the component binding N,N'-dicyclohexylcarbodiimide. The ATP phosphohydrolase activity of the membrane ATPase was not activated by trypsin treatment and the ATPase (BF0F1) contained no trypsin-sensitive inhibitor protein subunit. 6. Purified ATPase (BF0F1) was incorporated into artificial proteoliposomes which demonstrated ATP-dependent enhancement of 8-anilinonaphthalene-1-sulphonate fluorescence and ATP-dependent proton influx. These reactions were abolished by proton conductors (e.g. carbonylcyanide m-chlorophenylhydrazone) by valinomycin in the presence of a high external concentration of K+, or by N,N'-dicyclohexylcarbodiimide, butyricin 7423, Dio-9, 4-chloro-7-nitrobenzofurazan or leucinostatin. Oligomycin, tributyl tin, triethyl tin and venturicidin were not inhibitory. 7. When stripped of the soluble BF1 component, such ATPase-proteoliposomes demonstrated nil ATP phosphohydrolase activity and did not display ATP-dependent enhancement of 8-anilino-naphthalene-1-sulphonate fluorescence or ATP-dependent protein influx. All of these activities were restored by incubation of the BF1-depleted proteoliposomes with a purified preparation of the soluble BF1 component.  相似文献   

11.
Inhibition by methylmercury and mercuric chloride of Mg,Ca ATPase and Na,K ATPase activities in human erythrocyte ghosts was correlated with the binding capacity of ghosts for the mercurial. Full inhibition was always reached below saturation of binding capacity, and half-inhibition at levels as low as 10% saturation. Under such conditions, concentrations of free inhibitor were negligibly low, and existing mathematical models of inhibition were not applicable. New inhibitor partition equations were introduced to model the mechanisms of action of mercurials. Up to 7 methylmercury groups were calculated to bind to one Na,K ATPase molecule at non-inhibitory sites, while only one reacted with the inhibitory site. Mg,Ca ATPase showed simple one-hit inhibition (one mercurial per enzyme); further washing of ghosts, however, unmasked a second binding site (cooperative two-hit inhibition). Affinities of mercurials to sites of inhibition were calculated relative to other ligands in erythrocyte membranes: the ratios ranged from 3 : 1 to 50 : 1. The results demonstrated the use of binding capacity assays and inhibitor partition equations to measure and compare the susceptibilities of membrane-bound enzymes to poisoning by mercurials.  相似文献   

12.
(1) F0.F1ATPase (EC 3.6.1.3) from Micrococcus luteus ATCC 4698 was solubilized from plasma membranes by the non-ionic detergent Triton X-100 in the presence of 0.05 M MgCl2. (2) The antibiotics rutamycin, Dio-9, quercetin, oligomycin, botrycidin, efrapeptin, leucinostatin, valinomycin, and venturicidin as well as N,N'-dicyclohexylcarbodiimide and dinitrophenol are potent inhibitors of F0.F1ATPase activity.(3) F0.F1ATPase activity is completely inhibited by anti-F1ATPase antibodies. The inhibition is non-competitive. (4) Crossed immunoelectrophoresis reveals a reaction of immunological identity of F0.F1ATPase and F1ATPase indicating that both enzymes have in common antigenic sites.  相似文献   

13.
The properties of a new type of oligomycin-resistant Chinese hamster ovary (CHO) cell line (Olir 2.2) are described in this paper. Olir 2.2 cells were approximately 50,000-fold more resistant to oligomycin than were wild-type CHO cells when tested in glucose-containing medium, but only 10- to 100-fold more resistant when tested in galactose-containing medium. Olir 2.2 cells grew with a doubling time similar to that of wild-type cells both in the presence or absence of oligomycin. Oligomycin resistance in Olir 2.2 cells was stable in the absence of drug. In vitro assays indicated that there was approximately a 25-fold increase in the resistance of the mitochondrial ATPase to inhibition by oligomycin in Olir 2.2 cells, with little change in the total ATPase activity. The electron transport chain was shown to be functional in Olir 2.2 cells. Olir 2.2 cells were cross-resistant to other inhibitors of the mitochondrial ATPase (such as rutamycin, ossamycin, peliomycin, venturicidin, leucinostatin, and efrapeptin) and to other inhibitors of mitochondrial functions (such as chloramphenicol, rotenone, and antimycin). Oligomycin resistance was expressed codominantly in hybrids between Olir 2.2 cells and wild-type cells. Cross-resistance to ossamycin, peliomycin, chloramphenicol, antimycin, venturicidin, leucinostatin, and efrapeptin was also expressed codominantly in hybrids. Fusions of enucleated Olir 2.2 cells with wild-type cells and characterization of the resulting cybrid clones indicated that resistance to oligomycin and ossamycin results from a mutation in both a nuclear gene and a cytoplasmic gene. Cross-resistance to efrapeptin, leucinostatin, venturicidin, and antimycin results from a mutation in only a nuclear gene.  相似文献   

14.
1. Changes in activity of ATPase (adenosine triphosphatase) during the cell cycle of Schizosaccharomyces pombe were analysed in cell-free extracts of cells harvested from different stages of growth of synchronous cultures and also after cell-cycle fractionation. 2. Oligomycin-sensitive ATPase oscillates in both glucose-repressed synchronous cultures and shows four maxima of activity approximately equally spaced through the cell cycle. The amplitude of the oscillations accounts for between 13 and 80% of the total activity at different times in the cell cycle. 3. Oligomycin sensitivity varies over a fourfold range at different stages of the cell cycle. 4. The periodicity of maximum oligomycin sensitivity is one-quarter of a cell cycle. 5. These results were confirmed for the first three-quarters of the cell cycle by cell-cycle fractionation. 6. In cells growing synchronously with glycerol, ATPase activity increases in a stepwise pattern, with two steps per cell cycle; the first of these occurs at 0.54 of the cell cycle and the second at 0.95. 7. These results are discussed in relation to previously obtained data on the development of mitochondrial activities during the cell cycle.  相似文献   

15.
The properties of Alcaligenes eutrophus ATPase (adenosine triphosphatase) were investigated by using subcellular fractions prepared from cells growing in exponential and synchronous cultures. Both the soluble and membrane-bound forms of the ATPase were inhibited non-competitively (K(i) 142mum) by Nbf-Cl (4-chloro-7-nitrobenzofurazan), whereas only the membrane-bound enzyme was inhibited (non-competitive; K(i) 750mum) by NN'-dicyclohexylcarbodi-imide. Neither the activity of the ATPase nor its sensitivity to these two inhibitors varied during exponential growth. However, marked variations in ATPase activity were observed during synchronous growth, which were characterized by maxima at approx. 0.4 and 0.9 of a cell cycle and minima at approx. 0.1 and 0.6 of a cycle. Sensitivity to Nbf-Cl and NN'-dicyclohexylcarbodi-imide also varied during the cell cycle; maximum inhibition by the former occurred at approx. 0.4 and 0.9 of a cell cycle, whereas maximum inhibition by the latter was located at approx. 0.1 and 0.6 of a cell cycle. Proton conductance by whole cells was also periodic during the cell cycle, the lowest rates occurring at approx. 0.15 and 0.55 of a cycle and the highest rates at approx. 0.4 and 0.9 of a cycle, but -->H(+)/O quotients for the oxidation of endogenous substrates remained relatively constant and indicated the presence of four proton-translocating respiratory segments throughout the cell cycle. These results are discussed in terms of ATPase and respiratory-chain structure and function during the cell cycle of Alcaligenes eutrophus.  相似文献   

16.
The effect of pH on the sensitivity of F1-ATPase as well as mitochondrial ATPase activity to nucleoside diand triphosphates and to inhibitory anions such as cyanate and thiocyanate, has been studied. The results obtained show that nucleotides could act as activators or inhibitors of the ATPase hydrolytic activity depending on pH, substrate concentration, and binding of the enzyme to the membrane. The effect of those nucleotides which activate the hydrolysis of ATP-Mg2+ was more pronounced beyon the optimum pH corresponding to each of the three catalytic sites of the enzyme, whereas those which are inhibitors had a lower effect above this value. The sensitivity to the inhibitory anions decreased with increasing pH values; the decrease in the inhibitory effect was sharper when approaching the optimum pH value. These data are in agreement with the existence in mitochondrial ATPase of two different regulatory sites, one being specific for binding nucleotides, and another for anions. Both of them showed a different response upon changes of pH.  相似文献   

17.
At low concentrations, diethylstilbestrol (DES) is shown to be a potent F0-directed inhibitor of the F0F1-ATPase of rat liver mitochondria. In analogy to other F0-directed inhibitors, DES inhibits both the ATPase and ATP-dependent proton-translocation activities of the purified and membrane bound enzyme. When added at low concentrations with dicyclohexylcarbodiimide (DCCD), a covalent inhibitor, DES acts synergistically to inhibit ATPase activity of the complex. At higher concentrations, DES restores DCCD-inhibited ATPase activity. However, there is no restoration of ATP-dependent proton translocation. Under these conditions DCCD remains covalently bound to the F0F1-ATPase complex and F1 remains bound to Fo. Significantly, when the F0F1-ATPase is inhibited by the Fo-directed inhibitor venturicidin rather than DCCD, DES is also able to restore ATPase activity. In contrast, DES is unable to restore ATPase activity to F0F1 preparations inhibited by the Fo-directed inhibitors oligomycin or tricyclohexyltin. However, combinations of [DES + DCCD] or [DES + venturicidin] can restore ATPase activity to F0F1 preparations inhibited by either oligomycin or tricyclohexyltin. Results presented here indicate that the F0 moiety of the rat liver mitochondrial proton ATPase contains a distinct binding site for DES. In addition, they suggest that at saturating concentrations simultaneous occupancy of the DES binding site and sites for either DCCD or venturicidin promote "uncoupled" ATP hydrolysis.  相似文献   

18.
The effects of spegazzinine, a dihydroindole alkaloid, on mitochondrial oxidative phosphorylation were studied.Spegazzinine inhibited coupled respiration and phosphorylation in rat liver mitochondria. The I50 was 120 μM. Uncouplers released the inhibition of coupled respiration. Arsenate-stimulated mitochondrial respiration was partially inhibited by spegazzinine. The stimulation of mitochondrial respiration by Ca2+ and the proton ejection associated with the ATP-dependent Ca2+ uptake were not affected by the alkaloid.Oxidative phosphorylation and the Pi-ATP exchange reaction of phosphorylating beef heart submitochondrial particles were strongly inhibited by spegazzinine (I50, 50 μM) while the ATP-dependent reactions, reduction of NAD+ by succinate and the pyridine nucleotides transhydrogenase were less sensitive (I50, 125 μM). Oxygen uptake by submitochondrial particles was not affected.The 2,4-dinitrophenol-stimulated ATPase activity of rat liver mitochondria was not affected by 300 μM spegazzinine, a concentration of alkaloid that completely inhibited phosphorylation. However, higher concentrations of spegazzinine did partially inhibit it. The ATPase activities of submitochondrial particles, insoluble and soluble ATPases were also partially inhibited by high concentrations of spegazzinine.The inhibitory properties of spegazzinine on energy transfer reactions are compared with those of oligomycin, aurovertin and dicyclohexylcarbodiimide. It is concluded that spegazzinine effects are very similar to the effects of aurovertin and that its site of action may be the same or near the site of aurovertin.  相似文献   

19.
A comparative study has been made of the effects of a variety of inhibitors on the plasma membrane ATPase and mitochondrial ATPase of Neurospora crassa. The most specific inhibitors proved to be vanadate and diethylstilbestrol for the plasma membrane ATPase and azide, oligomycin, venturicidin, and leucinostatin for mitochondrial ATPase. N,N′-Dicyclohexylcarbodiimide, octylguanidine, triphenylsulfonium chloride, and quercetin and related bioflavonoids inhibited both enzymes, although with different concentration dependences. Other compounds that were tested (phaseolin, fusicoccin, deoxycorticosterone, alachlor, salicyclic acid, N-1-napthylphthalamate, triiodobenzoic acid, cyclic AMP, cyclic GMP, theobromine, theophylline, and histamine) had no significant effect on either enzyme. Overall, the results indicate that the plasma membrane and mitochondrial ATPases are distinct enzymes, in spite of the fact that they may play related roles in H+ transport across their respective membranes.  相似文献   

20.
The regulatory behavior, inhibitor sensitivity, and properties of the whole cell chloride conductance observed in cells expressing the cDNA coding for a chloride conductance mediator isoform of the CLCA gene family, pCLCA1, have been studied. Common C-kinase consensus phosphorylation sites between pCLCA1 and the closely related human isoform hCLCA1 are consistent with a role for calcium in channel activation. Both channels are activated rapidly on exposure to the calcium ionophore ionomycin. Direct involvement of calcium in the activation of pCLCA1 was supported by the finding that treatment with the intracellular calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM reduced the rate of chloride efflux from NIH/3T3 cells expressing the pCLCA1 channel. No combination of A-kinase activators used was effective in activating chloride efflux via this channel despite the presence of a unique strong A-kinase consensus site in pCLCA1. Notable differences of pCLCA1 from the reported properties of CLCA family members include the failure of phorbol 12-myristate 13-acetate to activate chloride efflux in cells expressing pCLCA1 and a lack of inhibition of chloride efflux from these cells after treatment with DIDS or dithiothreitol. However, selected inhibitors of anionic conductance inhibited pCLCA1-dependent anion efflux. The electrogenic nature of the ionomycin-dependent efflux of chloride from cells expressing pCLCA1 was confirmed by detection of outwardly rectifying chloride current and inhibition of this current by chloride conductance inhibitors in a whole cell patch-clamp study.  相似文献   

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