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1.
Summary Characteristics of the native and reconstituted H+-ATPase from the plasma membrane of red beet (Beta vulgaris L.) were examined. The partially purified, reconstituted H+-ATPase retained characteristics similar to those of the native plasma membrane H+-ATPase following reconstitution into proteoliposomes. ATPase activity and H+ transport of both enzymes were inhibited by vanadate, DCCD, DES and mersalyl. Slight inhibition of ATPase activity associated with native plasma membranes by oligomycin, azide, molybdate or NO
3
–
was eliminated during solubilization and reconstitution, indicating the loss of contaminating ATPase activities. Both native and reconstituted ATPase activities and H+ transport showed a pH optimum of 6.5, required a divalent cation (Co2+>Mg2+>Mn2+>Zn2+>Ca2+), and preferred ATP as substrate. The Mg:ATP kinetics of the two ATPase activities were similar, showing simple Michaelis-Menten kinetics. Saturation occurred between 3 and 5mM Mg: ATP, with aK
m
of 0.33 and 0.46mM Mg: ATP for the native and reconstituted enzymes, respectively. The temperature optimum for the ATPase was shifted from 45 to 35°C following reconstitution. Both native and reconstituted H+-ATPases were stimulated by monovalent ions. Native plasma membrane H+-ATPase showed an order of cation preference of K+>NH
4
+
>Rb+>Na+>Cs+>Li+>choline+. This basic order was unchanged following reconstitution, with K+, NH
4
+
, Rb+ and Cs+ being the preferred cations. Both enzymes were also stimulated by anions although to a lesser degree. The order of anion preference differed between the two enzymes. Salt stimulation of ATPase activity was enhanced greatly following reconstitution. Stimulation by KCl was 26% for native ATPase activity, increasing to 228% for reconstituted ATPase activity. In terms of H+ transport, both enzymes required a cation such as K+ for maximal transport activity, but were stimulated preferentially by Cl– even in the presence of valinomycin. This suggests that the stimulatory effect of anions on enzyme activity is not simply as a permeant anion, dissipating a positive interior membrane potential, but may involve a direct anion activation of the plasma membrane H+-ATPase. 相似文献
2.
B.S. Jena B.K. Patnaik 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1995,111(4):545-552
Hepatic Na+-K+-ATPase and Mg2+-ATPase activities of male green lizards declined during the maturation phase (juvenile to 1-year-old) and stabilized thereafter. On the other hand, the Ca2+-ATPase activity of the liver declined during the later half of the life span (1-year-old to 2–4-year-old). Starvation stress induced a decline in hepatic Na+-K+-ATPase and Mg2+-ATPase activities of juvenile lizards and caused an increase in 1-year-old and 2–4-year-old counterparts. The Ca2+-ATPase activity declined only in starved 1-year-old lizards. Following cold stress, the hepatic Na+-K+-ATPase activity of juvenile lizards showed a higher degree of decline than 2–4-year-old counterparts. The Mg2+-ATPase activity declined in cold-stressed juvenile lizards, but the parameter was elevated in similarly treated 1-year-old lizards. On the other hand, the increase in Ca2+-ATPase activity in response to cold stress was confined only to 2–4-year-old lizards. 相似文献
3.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1981,649(3):533-540
ATP and adenylylimidodiphosphate (AdoPP[NH]P) bind to (Na+ + K+)-ATPase in the absence of Mg2+ (EDTA present) with a homogeneous but 15-fold different affinity, the Kd values being 0.13 μM and 1.9 μM, respectively. The binding capacities of the two nucleotides are nearly equal and amount to 3.9 and 4 nmol/mg protein or 1.7 and 1.8 mol/mol (Na+ + K+)-ATPase, respectively. The Kd value for ATP is equal to the Km for phosphorylation by ATP (0.05–0.25 μM) and the binding capacity is equivalent to the phosphorylation capacity of 1.8 mol/mol (Na+ + K+)-ATPase. Hence, the enzyme contains two high-affinity nucleotide binding and phosphorylating sites per molecule, or one per α-subunit. Additional low-affinity nucleotide binding sites are elicited in the presence of Mg2+, as shown by binding studies with the non-phosphorylating (AdoPP[NH]P). The Kd and binding capacity for AdoPP[NH]P at these sites is dependent on the Mg2+ concentration. The Kd increases from 0.06 mM at 0.5 mM Mg2+ to a maximum of 0.26 mM at 2 mM Mg2+ and the binding capacity from 1.5 nmol/mg protein at 0.5 mM Mg2+ to 3.3 nmol/mg protein at 4 mM Mg2+. Extrapolation of a double reciprocal plot of binding capacity vs. total Mg2+ concentration yields a maximal binding capacity at infinite Mg2+ concentration of 3.8 nmol/mg protein or 1.7 mol/mol (Na+ + K+)-ATPase. The Kd for Mg2+ at the sites, where it exerts this effect, is 0.8 mM. The Kd for the high-affinity sites increases from 1.5–1.9 μM in the absence of Mg2+ to a maximum of 4.2 μM at 2 mM Mg2+ concentration. The binding capacity of these sites (1.8 mol/mol enzyme) is independent of the Mg2+ concentration. Hence, Mg2+ induces two low-affinity non-phosphorylating nucleotide binding sites per molecule (Na+ + K+)-ATPase in addition to the two high-affinity, phosphorylating nucleotide binding sites. 相似文献
4.
Summary The plasma membrane (Mg2+)-dependent adenosine triphosphatase ((Mg2+)-ATPase) from human erythrocytes has been tested for its ability to transport ions. Using a preparation of inside-out vesicles loaded with the pH-sensitive fluorescence probe 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS), we have demonstrated the absence of proton movement during (Mg2+)-ATPase activity. From the rate of ATP hydrolysis and the passive proton permeability of these vesicles, an upper limit of 0.03 H+ transported per ATP hydrolyzed was calculated. To verify that proton pumping could be detected in this system, the intravesicular pH was monitored during (Ca2+)-dependent adenosine triphosphatase ((Ca2+)-ATPase) activity. Proton efflux associated with (Ca2+)-ATPase activity was observed (in agreement with a recent report of proton pumping by a reconstituted erythrocyte (Ca2+)-ATPase (Niggli, V., Sigel, E., Carafoli, E. (1982)J. Biol. Chem.
257:2350–2356)) and was shown to be stimulated by calmodulin. The ability of the (Mg2+)-ATPase to pump28Mg2+,35SO
4
2–
and86Rb+ was also tested, with the results leading to the conclusion that the human erythrocyte enzyme does not function as an ion transport system. 相似文献
5.
《Analytical biochemistry》2002,300(2):113
In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4°C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na+/K+-ATPase and Mg2+-ATPase, retained 70–80% activity after the adsorption. In addition, adsorption stabilizes Na+/K+-ATPase and Mg2+-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na+/K+-ATPase and Mg2+-ATPase and their sensitivity to and mechanism of Cd2+- or Hg2+-induced inhibition. The only exception is the “high affinity” Mg2+-ATPase moiety, whose affinity for ATP and sensitivity toward Cd2+ were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations. 相似文献
6.
猪心线粒体Fo的纯化、重建及其质子转运功能 总被引:1,自引:0,他引:1
比较了猪心线粒体FoF1-ATPase膜部分Fo的四种纯化方法.结果表明,用NaBr从亚线粒体除去FoF1-ATPase的水溶性部分F1-ATPase后,再以CHAPS增溶,并经蔗糖梯度离心,可获得高纯度的Fo.SDS-聚丙烯酰胺凝胶电泳鉴定表明,纯化的Fo含有b、OSCP(寡霉素敏感授予蛋白)、d、a、e、F6、IF1、A6L和c等9种亚基.用去污剂稀释法将纯化的Fo在脂质体上重建后,重建Fo表现较高的被动转运质子活性.这为在体外深入研究Fo的活性、构象与膜脂的关系,以及Fo与F1-ATPase的组装等提供了很好的实验模型. 相似文献
7.
Armen Trchounian Yelena Ohanjanyan Karine Bagramyan Vitya Vardanian Eleonora Zakharyan Anait Vassilian Misak Davtian 《Bioscience reports》1998,18(3):143-154
K+ uptake by the Escherichia coli TrkA system is unusual in that it requires both ATP and
; a relation withH+ circulation through the membrane is thereforesuggested. The relationship of this system with theF0F1-ATPase was studied in intact cells grownunder different conditions. A significant increase of theN,N-dicyclohexylcarbodiimide(DCCD)-inhibitedH+ efflux through the F0F1 by 5 mMK+, but not by Na+ added into thepotassium-free medium was revealed only in fermenting wild-type orparent cells, that were grown under anaerobic conditions withoutanaerobic or aerobic respiration and with the production ofH2. Such an increase disappeared in the unc or the trkA mutants that have alteredF0F1 or defective TrkA, respectively.This finding indicates a closed relationship between TrkA andF0F1, with these transport systems beingassociated in a single mechanism that functions as an ATP-drivenH+–K+-exchanging pump. ADCCD-inhibited H+–K+-exchangethrough these systems with the fixed stoichiometry of H+and K+ fluxes(2H+/K+) and a higherK+ gradient between the cytoplasm and the externalmedium were also found in these bacteria. They were not observed incells cultured under anaerobic conditions in the presence of nitrate orunder aerobic conditions with respiration and without production ofH2. The role of anaerobic or aerobic respiration as adeterminant of the relationship of the TrkA with theF0F1 is postulated. Moreover, an increase ofDCCD-inhibited H+ efflux by added K+, aswell as the characteristics of DCCD-sensitiveH+–K+-exchange found in a parentstrain, were lost in the arcA mutant with a defectiveArc system, suggesting a repression of enzymes in respiratorypathways. In addition, K+ influx in the latest mutantwas not markedly changed by valinomycin or with temperature. ThearcA gene product or the Arc system is proposed to beimplicated in the regulation of the relationship between TrkAand F0F1. 相似文献
8.
K.Damodar Reddy A Chaudhuri K Thangavelu 《Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology》1997,116(4):459-466
Tissue-specific age-dependent changes were observed in Na+K+-, Ca2+-, and Mg2+-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na+K+-, Ca2+-, and Mg2+-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na+K+-, Ca2+-, and Mg2+-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na+K+- and Ca2+-ATPase in testes and only Ca2+-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na+K+-, Ca2+- and Mg2+-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system. 相似文献
9.
Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group 总被引:1,自引:0,他引:1
The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca2+–Mg2+)-ATPase in the plasma membranes of rat liver was investigated. (Ca2+–Mg2+)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca2+-Mg2+)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca2+–Mg2+)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca2+–Mg2+)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca2+–Mg2+)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme. 相似文献
10.
Frasch WD 《Journal of bioenergetics and biomembranes》2000,32(5):539-546
The Mg2+ dependent asymmetry of the F1-ATPase catalytic sites leads to the differences in affinity for nucleotides and is an essential component of the binding-change mechanism. Changes in metal ligands during the catalytic cycle responsible for this asymmetry were characterized by vanadyl (V
IV + O)2+, a functional surrogate for Mg2+. The 51V-hyperfine parameters derived from EPR spectra of VO2+ bound to specific sites on F1 provide a direct probe of the metal ligands. Site-directed mutations of metal ligand residues cause measurable changes in the 51V-hyperfine parameters of the bound VO2+, thereby providing a means to identification. Initial binding of the metal–nucleotide to the low-affinity catalytic site conformation results in metal coordination by hydroxyl groups from the P-loop threonine and catch-loop threonine. Upon conversion to the high-affinity conformation, carboxyl groups from the Walker homology B aspartate and MF1E197 become ligands in lieu of the hydroxyl groups. 相似文献
11.
Ana Maria Landeira-Fernandez Maricilia S. Costa Leopolde de Meis 《Bioscience reports》1996,16(6):439-451
Vesicles derived from maize roots retain a membrane bound H+-ATPase that is able to pump H+ at the expense of ATP hydrolysis. In this work it is shown that heparin, fucose-branched chondroitin sulfate and dextran sulfate 8000 promote a shift of the H+-ATPase optimum pH from 6.0 to 7.0. This shift is a result of a dual effect of the sulfated polysaccharides, inhibition at pH 6.0 and activation at pH 7.O. At pH 6.0 dextran 8000 promotes an increase of the apparent Km for ATP from 0.28 to 0.95 mM and a decrease of the Vmax from 14.5 to 7.1 mol Pi/mg · 30 min–1. At pH 7.0 dextran 8000 promotes an increase in Vmax from 6.7 to 11.7 mol Pi/mg · 30 min–1. In the presence of lysophosphatidylcholine the inhibitory effect of the sulfated polysaccharides observed at pH 6.0 was not altered but the activation of pH 7.0 decreased. It was found that in the presence of sulfated polysaccharides the ATPase became highly sensitive to K+ and Na+. Both the inhibition at pH 6.0 and the activation promoted by the polysaccharide were antagonized by monovalent cations (K+>Na+Li+).Abbreviations Mops
4-morpholinopropanesulfonic acid
- EDTA
ethylenediaminetetraacetic acid
- ACMA
9-amino-6-chloro-2-methoxyacridine
- FCCP
carbonyl cyanide p(trifluoromethoxy)-phenylhyrazone 相似文献
12.
James Hughes Saroj Joshi Katalin Torok D. Rao Sanadi 《Journal of bioenergetics and biomembranes》1982,14(5-6):287-295
The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H+-ATPase with high levels of Pi-ATP exchange activity (400–600 nmol × min–1 × mg–1). This activity is further enhanced (1400–1600 nmol × min–1 × mg–1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H+ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. Pi-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP). 相似文献
13.
Van Walraven HS Scholts MJ Zakharov SD Kraayenhof R Dilley RA 《Journal of bioenergetics and biomembranes》2002,34(6):455-464
It was shown before (Wooten, D. C., and Dilley, R. A. (1993) J. Bioenerg. Biomembr. 25, 557–567; Zakharov, S. D., Li, X., Red'ko, T. P., and Dilley, R. A. (1996) J. Bioenerg. Biomembr. 28, 483–493) that pH dependent reversible Ca2+ binding near the N- and C-terminal end of the 8 kDa subunit c modulates ATP synthesis driven by an applied pH jump in chloroplast and E. coli ATP synthase due to closing a proton gate proposed to exist in the F0 H+ channel of the F0F1 ATP synthase. This mechanism has further been investigated with the use of membrane vesicles from mutants of the cyanobacterium Synechocystis 6803. Vesicles from a mutant with serine at position 37 in the hydrophilic loop of the c-subunit replaced by the charged glutamic acid (strain plc 37) has a higher H+/ATP ratio than the wild type and therefore shows ATP synthesis at low values of
H
+. The presence of 1 mM CaCl2 during the preparation and storage of these vesicles blocked acid–base jump ATP formation when the pH of the acid side (inside) was between pH 5.6 and 7.1, even though the pH of the acid–base jump was thermodynamically in excess of the necessary energy to drive ATP formation at an external pH above 8.28. That is, in the absence of added CaCl2, ATP formation did occur under those conditions. However, when the base stage pH was 7.16 and the acid stage below pH 5.2, ATP was formed when Ca2+ was present. This is consistent with Ca2+ being displaced by H+ ions from the F0 on the inside of the thylakoid membrane at pH values below about 5.5. Vesicles from a mutant with the serine of position 3 replaced by a cysteine apparently already contain some bound Ca2+ to F0. Addition of 1 mM EGTA during preparation and storage of those vesicles shifted the otherwise already low internal pH needed for onset of ATP synthesis to higher values when the external pH was above 8. With both strains it was shown that the Ca2+ binding effect on acid–base induced ATP synthesis occurs above an internal pH of about 5.5. These results were corroborated by 45Ca2+- ligand blot assays on organic solvent soluble preparations containing the 8 kDa F0 subunit c from the S-3-C mutant ATP synthase, which showed 45Ca2+ binding as occurs with the pea chloroplast subunit III. The phosphorylation efficiency (P/2e), at strong light intensity, of Ca2+ and EGTA treated vesicles from both strains were almost equal showing that Ca2+ or EGTA have no other effect on the ATP synthase such as a change in the proton to ATP ratio. The results indicate that the Ca2+ binding to the F0 H+ channel can block H+ flux through the channel at pH values above about 5.5, but below that pH protons apparently displace the bound Ca2+, opening the CF0 H+ channel between the thylakoid lumen and H+ conductive channel. 相似文献
14.
- 1. (1) VO3− combines with high affinity to the Ca2+-ATPase and fully inhibits Ca2+-ATPase and Ca2+-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca2+-dependent phosphoenzyme.
- 2. (2) VO3− blocks hydrolysis of ATP at the catalytic site. The sites for VO3− also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca2+-ATPase.
- 3. (3) The sites for VO3− show positive interactions in affinity with sites for Mg2+ and K+. This accounts for the dependence on Mg2+ and K+ of the inhibition by VO3−. Although, with less effectiveness, Na+ substitutes for K+ whereas Li+ does not. The apparent affinities for Mg2+ and K+ for inhibition by VO3− seem to be less than those for activation of the Ca2+-ATPase.
- 4. (4) Inhibition by VO3− is independent of Ca2+ at concentrations up to 50 μM. Higher concentrations of Ca2+ lead to a progressive release of the inhibitory effect of VO3−.
Keywords: Ca2+-ATPase; Vanadate inhibition; K+; Li+; (Red cell membrane) 相似文献
15.
Rat C6 glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na+,K+-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K+ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K+. The specific activity of Na+,K+-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10–4 or 10–5 M noradrenaline (NA) remained without any effect on NA+,K+-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na+,K+-ATPase of C6 glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na+,K+-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10–7 M concentration. In spite of the fact that Na+,K+-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na+,K+-ATPase may contribute to the previously reported inhibition by vanadate of Na+,K+-ATPase of the whole brain tissue. 相似文献
16.
Divalent cation and lipid-protein interactions of biomembranes 总被引:1,自引:0,他引:1
Divalent cations play an important role in the functions of biomembranes. This review deals with three topics: (1) Mg2+-mediated change in physical state of phospholipid induces conformation and activity change of reconstituted mitochondrial H+-ATPase, (2) a proper transmembrane Ca2+ gradient is essential for the higher enzymatic activity of adenylate cyclase, and (3) role of transmembrane Ca2+ gradient in the modulation of reconstituted sarcoplasmic reticulm Ca2+-ATPase activity. 相似文献
17.
M. G. Alekseeva S. M. Elizarov O. B. Bekker I. K. Lubimova V. N. Danilenko 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2009,3(1):16-23
Inverted membrane vesicles of Gram-positive actinobacteria Streptomyces fradiae, S. lividans, and S. avermitilis have been prepared and membrane-bound F0F1 ATP synthase has been biochemically characterized. It has been shown that the ATPase activity of membrane-bound F0F1 complex is Mg2+-dependent and moderately stimulated by high concentrations of Ca2+ ions (10–20 mM). The ATPase activity is inhibited by N,N′-dicyclohexylcarbodiimide and oligomycin A, typical F0F1 ATPase inhibitors that react with the membrane-bound F0 complex. The assay of biochemical properties of the F0F1 ATPases of Streptomycetes in all cases showed the presence of ATPase populations highly susceptible and insensitive to oligomycin A. The in vitro labeling and inhibitory assay showed that the inverted phospholipid vesicles of S. fradiae contained active membrane-bound Ser/Thr protein kinase(s) phosphorylating the proteins of the F0F1 complex. Inhibition of phosphorylation leads to decrease of the ATPase activity and increase of its susceptibility to oligomycin. The in vivo assay confirmed the enhancement of actinobacteria cell sensitivity to oligomycin after inhibition of endogenous phosphorylation. The sequencing of the S. fradiae genes encoding oligomycin-binding A and C subunits of F0F1 ATP synthase revealed their close phylogenetic relation to the genes of S. lividans and S. avermitilis. 相似文献
18.
Georgina Rodríguez de Lores Arnaiz 《Neurochemical research》1990,15(3):289-294
Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na+, K+-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na+, K+-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na+, K+- and Mg2+-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na+, K+-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg2+-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na+, K+-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source. 相似文献
19.
Plant vacuoles were isolated from cotyledons of germinatingAcacia mangium seeds, which had been treated with or withoutcolchicine, to measure vacuolar membrane pyrophosphate (PPi)- andATP-dependent H+ transport activities, and enzymaticactivities of H+-pyrophosphatase(H+-PPase) and H+-ATPase. Innon-colchicine-treated seeds, activities of the two enzymes increasedrapidly after seed germination to almost a maximal level on the seventhday. A linear function relationship exists in magnitude between PPi- orATP-dependent H+transport activity and its correspondingenzymatic activity. The former regression equation is: PPi-dependentH+ transport activity(%A.min–1.g–1) =–0.039 + H+-PPase activity(units.mg–1) × 1.574, the latter is:ATP-dependent H+ transport activity(%A.min–1.g–1) =–0.003 + H+-ATPase activity(units.mg–1) × 0.549. In colchicine-treatedseeds, activities of the two enzymes increased very slowly during 8 daysof germination and the relationship to their respectiveH+ transport activities was not in agreement with theabove-mentioned regression equations. PPi- and ATP-dependentH+ transport activities were lower than thecorresponding values calculated from H+-PPase activityand H+-ATPase activity according to the two regressionequations, respectively. However, when sucrose, indole butyric acid(IBA), or 6-benzyladenine (6-BA) were applied exogenously to the seedsfollowing colchicine treatment for 3 days, activities ofH+-PPase, H+-ATPase, PPi- andATP-dependent H+ transport in the 6-day-old seedlingsall increased. By statistical analysis, it was concluded that colchicineinhibits cotyledon vacuolar membrane H+-PPase,H+-ATPase activities, PPi- and ATP-dependentH+ transport activities during seed germination andearly seedling growth of Acacia mangium. The inhibitory effectsof colchicine could be overcome by IBA, 6-BA and sucrose to varyingdegrees. 相似文献
20.
The regulatory effects of malate on chloroplast Mg2+-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF1 Mg2+-ATPase activity. The subunit of CF1 may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg2+-ATPase was also controlled by the Mg2+/ATP ratio in the reaction medium. The enhancing effect of malate on Mg2+-ATPase activity depended on the presence of high concentrations of Mg2+ in the reaction mixture. Kinetic study showed that malate raised the Vmax of catalysis without affecting the Km for Mg2+ ATP. The experiments imply that the stimulation of Mg2+-ATPase by malate is probably correlated with the Pi binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF1
chloroplast coupling factor 1
- CF1 (-) and CF1 (-)
CF1 deficient in the and subunit
- MF1
mitochondria coupling factor 1
- NEM
N-ethylmaleimide
- PMS
phenazine methosulfate
- OG
n-octyl--d-glucopyranoside 相似文献