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1.
The cardiac Na+/Ca2+ exchanger (NCX) is the major Ca2+ efflux pathway on the sarcolemma, counterbalancing Ca2+ influx via L-type Ca2+ current during excitation-contraction coupling. Altered NCX activity modulates the sarcoplastic reticulum Ca2+ load and can contribute to abnormal Ca2+ handling and arrhythmias. NADH/NAD+ is the main redox couple controlling mitochondrial energy production, glycolysis, and other redox reactions. Here, we tested whether cytosolic NADH/NAD+ redox potential regulates NCX activity in adult cardiomyocytes. NCX current (INCX), measured with whole cell patch clamp, was inhibited in response to cytosolic NADH loaded directly via pipette or increased by extracellular lactate perfusion, whereas an increase of mitochondrial NADH had no effect. Reactive oxygen species (ROS) accumulation was enhanced by increasing cytosolic NADH, and NADH-induced INCX inhibition was abolished by the H2O2 scavenger catalase. NADH-induced ROS accumulation was independent of mitochondrial respiration (rotenone-insensitive) but was inhibited by the flavoenzyme blocker diphenylene iodonium. NADPH oxidase was ruled out as the effector because INCX was insensitive to cytosolic NADPH, and NADH-induced ROS and INCX inhibition were not abrogated by the specific NADPH oxidase inhibitor gp91ds-tat. This study reveals a novel mechanism of NCX regulation by cytosolic NADH/NAD+ redox potential through a ROS-generating NADH-driven flavoprotein oxidase. The mechanism is likely to play a key role in Ca2+ homeostasis and the response to alterations in the cytosolic pyridine nucleotide redox state during ischemia-reperfusion or other cardiovascular diseases. 相似文献
2.
Stephanie C. Agius Natalia V. Bykova Abir U. Igamberdiev Ian M. Møller 《Physiologia plantarum》1998,104(3):329-336
Inside-out submitochondrial particles from both potato (Solanum tuberosum L. cv. Bintje) tubers and pea (Pisum sativum L. cv. Oregon) leaves possess three distinct dehydrogenase activities: Complex I catalyzes the rotenone-sensitive oxidation of deamino-NADH, NDin(NADPH) catalyzes the rotenone-insensitive and Ca2+-dependent oxidation of NADPH and NDin(NADH) catalyzes the rotenone-insensitive and Ca2+-independent oxidation of NADH. Diphenylene iodonium (DPI) inhibits complex I, NDin(NADPH) and NDin (NADH) activity with a Ki of 3.7, 0.17 and 63 µM, respectively, and the 400-fold difference in Ki between the two NDin made possible the use of DPI inhibition to estimate NDin (NADPH) contribution to malate oxidation by intact mitochondria. The oxidation of malate in the presence of rotenone by intact mitochondria from both species was inhibited by 5 µM DPI. The maximum decrease in rate was 10–20 nmol O2 mg?1 min?1. The reduction level of NAD(P) was manipulated by measuring malate oxidation in state 3 at pH 7.2 and 6.8 and in the presence and absence of an oxaloacetate-removing system. The inhibition by DPI was largest under conditions of high NAD(P) reduction. Control experiments showed that 125 µM DPI had no effect on the activities of malate dehydrogenase (with NADH or NADPH) or malic enzyme (with NAD+ or NADP+) in a matrix extract from either species. Malate dehydrogenase was unable to use NADP+ in the forward reaction. DPI at 125 µM did not have any effect on succinate oxidation by intact mitochondria of either species. We conclude that the inhibition caused by DPI in the presence of rotenone in plant mitochondria oxidizing malate is due to inhibition of NDin(NADPH) oxidizing NADPH. Thus, NADP turnover contributes to malate oxidation by plant mitochondria. 相似文献
3.
Oxidation of External NAD(P)H by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria : A Kinetic and Inhibitor Study 总被引:2,自引:2,他引:0
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The functional interaction between the externally located NAD(P)H dehydrogenase and the Q-pool acceptor site(s) in Percoll-purified mitochondria from Jerusalem artichoke (Helianthus tuberosus L. cv OB1) mitochondria has been investigated. Oxidation of exogenous NADH is stimulated by ubiquinone (UQ1) with a parallel decrease of the apparent Km for NADH. In the presence of saturating amounts of UQ1 as electron acceptor, the Km (NADH) is not affected by variations of the ionic strength. Conversely, the Km for UQ1 is decreased by the screening effect of negative charges on the outer membrane surface. Under low-ionic strength, the hydroxyflavone platanetin progressively inhibits NADH oxidation with a mean inhibition dose of approximately 3 nanomoles of inhibitor per milligram of protein. Interestingly, under high-ionic strength, oxidation of NADH proceeds through two platanetin binding sites, one of which has a lower affinity for the inhibitor (mean inhibition dose = 20 nanomoles per milligram protein), because it is located near the outer surface of the membrane. This latter site is the one involved in the oxidation of external NADPH and, possibly, also affected by spermine and spermidine. Similarly to NADH, oxidation of NADPH is fully sensitive to micromolar concentrations of free Ca2+ ions; in addition, similar concentrations of the sulfhydryl reagent mersalyl are required to inhibit both NADH and NADPH oxidative activities. The results are interpreted as evidence for the presence of a single nonspecific NAD(P)H dehydrogenase. 相似文献
4.
A plasmalemma-bound NADH-dependent redox system has been identified in protoplasts isolated from cell suspensions of sugarcane. This system oxidized NADH as well as NADPH, increased O2 consumption 3-fold, and increased the pH of the external medium while the cytoplasmic pH was decreased. In the presence of NADH, ferricyanide was rapidly reduced and the external medium was acidified. The uptake rates of K+, 3-O-methylglucose, leucine, and arginine were all decreased in the presence of NADH. 相似文献
5.
Effects of Polyamines on the Oxidation of Exogenous NADH by Jerusalem Artichoke (Helianthus tuberosus) Mitochondria 总被引:1,自引:1,他引:0
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The effect of polyamines (putrescine, spermine, and spermidine) on the oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L. cv. OB1) mitochondria, have been studied. Addition of spermine and/or spermidine to a suspension of mitochondria in a low-cation medium (2 millimolar-K+) caused a decrease in the apparent Km and an increase in the apparent Vmax for the oxidation of exogenous NADH. These polycations released by screening effect the mitochondrially induced quenching of 9-aminoacridine fluorescence, their efficiency being dependent on the valency of the cation (C4+ > C3+). Conversely, putrescine only slightly affected both kinetic parameters of exogenous NADH oxidation and the number of fixed charges on the membranes. Spermine and spermidine, but not putrescine, decreased the apparent Km for Ca2+ from about 1 to about 0.2 micromolar, required to activate external NADH oxidation in a high-cation medium, containing physiological concentrations of Pi, Mg2+ and K+. The results are interpreted as evidence for a role of spermine and spermidine in the modulation of exogenous NADH oxidation by plant mitochondria in vivo. 相似文献
6.
Using isolated chloroplasts or purified thylakoids from photoautotrophically grown cells of the chromophytic alga Pleurochloris meiringensis (Xanthophyceae) we were able to demonstrate a membrane bound NAD(P)H dehydrogenase activity. NAD(P)H oxidation was detectable with menadione, coenzyme Q0, decylplastoquinone and decylubiquinone as acceptors in an in vitro assay. K
m-values for both pyridine nucleotides were in the molar range (K
m[NADH]=9.8 M, K
m[NADPH]=3.2 M calculated according to Lineweaver-Burk). NADH oxidation was optimal at pH 9 while pH dependence of NADPH oxidation showed a main peak at 9.8 and a smaller optimum at pH 7.5–8. NADH oxidation could be completely inhibited with rotenone, an inhibitor of mitochondrial complex I dehydrogenase, while NADPH oxidation revealed the typical inhibition pattern upon addition of oxidized pyridine nucleotides reported for ferredoxin: NADP+ reductase. Partly-denaturing gel electrophoresis followed by NAD(P)H dehydrogenase activity staining showed that NADPH and NADH oxidizing proteins had different electrophoretic mobilities. As revealed by denaturing electrophoresis, the NADH oxidizing enzyme had one main subunit of 22 kDa and two further polypeptides of 29 and 44 kDa, whereas separation of the NADPH depending protein yielded five bands of different molecular weight. Measurement of oxygen consumption due to PS I mediated methylviologen reduction upon complete inhibition of PS II showed that the NAD(P)H dehydrogenase is able to catalyze an input of electrons from NADH to the photosynthetic electron transport chain in case of an oxidized plastoquinone-pool. We suggest ferredoxin: NADP+ reductase to be the main NADPH oxidizing activity while a thylakoidal NAD(P)H: plastoquinone oxidoreductase involved in the chlororespiratory pathway in the dark acts mainly as an NADH oxidizing enzyme.Abbreviations Coenzyme
Q0-2,3-dimethoxy-5-methyl-1,4-benzoquinone
- FNR
ferredoxin: NADP+ reductase
- MD
menadione
- MV
methylviologen
- NDH
NAD(P)H dehydrogenase
- PQ
plastoquinone
- PQ10
decylplastoquinone
- SDH
succinate dehydrogenase
- UQ10
decylubiquinone (2,3-dimethoxy-5-methyl-6-decyl-1,4-benzoquinone) 相似文献
7.
Ian M. Møller Allan G. Rasmusson Kenneth M. Fredlund 《Journal of bioenergetics and biomembranes》1993,25(4):377-384
Plant (and fungal) mitochondria contain multiple NAD(P)H dehydrogenases in the inner membrane all of which are connected to the respiratory chain via ubiquinone. On the outer surface, facing the intermembrane space and the cytoplasm, NADH and NADPH are oxidized by what is probably a single low-molecular-weight, nonproton-pumping, unspecific rotenone-insensitive NAD(P)H dehydrogenase. Exogenous NADH oxidation is completely dependent on the presence of free Ca2+ with aK
0.5 of about 1 µM. On the inner surface facing the matrix there are two dehydrogenases: (1) the proton-pumping rotenone-sensitive multisubunit Complex I with properties similar to those of Complex I in mammalian and fungal mitochondria. (2) a rotenone-insensitive NAD(P)H dehydrogenase with equal activity with NADH and NADPH and no proton-pumping activity. The NADPH-oxidizing activity of this enzyme is completely dependent on Ca2+ with aK
0.5 of 3 µM. The enzyme consists of a single subunit of 26 kDa and has a native size of 76 kDa, which means that it may form a trimer. 相似文献
8.
This study aims at characterizing NAD(P)H dehydrogenases on the inside and outside of the inner membrane of mitochondria of one phosphoenolpyruvate carboxykinase??crassulacean acid metabolism plant, Hoya carnosa. In crassulacean acid metabolism plants, NADH is produced by malate decarboxylation inside and outside mitochondria. The relative importance of mitochondrial alternative NADH dehydrogenases and their association was determined in intact??and alamethicin??permeabilized mitochondria of H. carnosa to discriminate between internal and external activities. The major findings in H. carnosa mitochondria are: (i) external NADPH oxidation is totally inhibited by DPI and totally dependent on Ca2+, (ii) external NADH oxidation is partially inhibited by DPI and mainly dependent on Ca2+, (iii) total NADH oxidation measured in permeabilized mitochondria is partially inhibited by rotenone and also by DPI, (iv) total NADPH oxidation measured in permeabilized mitochondria is partially dependent on Ca2+ and totally inhibited by DPI. The results suggest that complex I, external NAD(P)H dehydrogenases, and internal NAD(P)H dehydrogenases are all linked to the electron transport chain. Also, the total measurable NAD(P)H dehydrogenases activity was less than the total measurable complex I activity, and both of these enzymes could donate their electrons not only to the cytochrome pathway but also to the alternative pathway. The finding indicated that the H. carnosa mitochondrial electron transport chain is operating in a classical way, partitioning to both Complex I and alternative Alt. NAD(P)H dehydrogenases. 相似文献
9.
Chlorotetracycline inhibits the uncoupled oxidation of exogenous NADH by Jerusalem artichoke (Helianthus tuberosus L.) mitochondria extensively (over 80%) and rapidly (inhibition complete in 10 s) in the presence of added Ca2+. Half-maximal inhibition is observed at 15 μM chlorotetracycline in the presence of 2 mM Ca2+. The oxidation of succinate is only affected marginally by chlorotetracycline plus Ca2+. The inhibition of NADH oxidation and the fluorescence of CTC are well correlated. Mn2+ is the only other cation which shows an (increased) inhibition in the presence of chlorotetracycline. The inhibition by Ca2+ and chlorotetracycline disappears at acid pH, and the pH optimum in their presence is 6.4. The inhibition caused by other lipid-soluble Ca2+-chelators is not reversible or is enhanced by the addition of excess Ca2+. In contrast, inhibition caused by relatively water-soluble chelators is completely reversed by added Ca2+. It is suggested that a neutral 1:2 complex is formed between Ca2+ and chlorotetracycline which can substitute for Ca2+ bound at sites in the lipophilic phase of the inner mitochondrial membrane, which are essential for the activity of the external NADH dehydrogenase. 相似文献
10.
Properties of substantially chlorophyll-free pea leaf mitochondria prepared by sucrose density gradient separation 总被引:5,自引:4,他引:1
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Mitochondria isolated from pea leaves (Pisum sativum L. var Massey Gem) and purified on a linear sucrose density gradient were substantially free of contamination by Chl and peroxisomes. They showed high respiratory rates and good respiratory control and ADP/O ratios. Malate, glutamate, succinate, glycine, pyruvate, α-ketoglutarate, NADH, and NADPH were oxidized but little or no oxidation of citrate, isocitrate, or proline was detected. The oxidation of NADPH by the purified mitochondria did not occur via a transhydrogenase or phosphatase converting it to NADH. NADPH oxidation had an absolute requirement for added Ca2+, whereas NADH oxidation proceeded in its absence. In addition, oxidation of the two substrates showed different sensitivities to chelators and sulfhydryl reagents, and faster rates of O2 uptake were observed with both substrates than with either alone. This indicates that the NADPH dehydrogenase is distinct from the exogenous NADH dehydrogenase. 相似文献
11.
Pyrroline-5-carboxylic acid reductase from soybean leaves 总被引:1,自引:0,他引:1
Pyrroline-5-carboxylic acid reductase was purified 40-fold from soybean leaves (Glycine max L. var Corsoy). The enzyme was fairly unstable, had a broad pH optimum, and was inactivated by heat and acid; NADH and NADPH both served as cofactors. It had a higher activity with NADH (about 4 ×) compared to NADPH, but a lower Km for NADPH. NADP+ inhibited both the NADH- and NADPH-dependent activity. Sulfhydryl group blocking agents reduced the activity as did the carbonyl blocking agent, NH2OH. Thiazolidine-4-carboxylic acid and phosphate inhibited the enzyme and proline inhibited only at high concentrations. ATP, GTP, and CTP were all effective inhibitors of both the NADH- and NADPH-dependent activity. Phosphorylated nucleotide inhibition was reversed by Mg2+ ions. 相似文献
12.
Dynamic features of Ca2+ interactions with transport and regulatory sites control the Ca2+-fluxes in mammalian Na+/Ca2+(NCX) exchangers bearing the Ca2+-binding regulatory domains on the cytosolic 5L6 loop. The crystal structure of Methanococcus jannaschii NCX (NCX_Mj) may serve as a template for studying ion-transport mechanisms since NCX_Mj does not contain the regulatory domains. The turnover rate of Na+/Ca2+ exchange (kcat = 0.5 ± 0.2s−1) in WT–NCX_Mj is 103–104 times slower than in mammalian NCX. In NCX_Mj, the intrinsic equilibrium (Kint) for bidirectional Ca2+ movements (defined as the ratio between the cytosolic and extracellular Km of Ca2+/Ca2+ exchange) is asymmetric, Kint = 0.15 ± 0.5. Therefore, the Ca2+ movement from the cytosol to the extracellular side is ∼7-times faster than in the opposite direction, thereby representing a stabilization of outward-facing (extracellular) access. This intrinsic asymmetry accounts for observed differences in the cytosolic and extracellulr Km values having a physiological relevance. Bidirectional Ca2+ movements are also asymmetric in mammalian NCX. Thus, the stabilization of the outward-facing access along the transport cycle is a common feature among NCX orthologs despite huge differences in the ion-transport kinetics. Elongation of the cytosolic 5L6 loop in NCX_Mj by 8 or 14 residues accelerates the ion transport rates (kcat) ∼10 fold, while increasing the Kint values 100–250-fold (Kint = 15–35). Therefore, 5L6 controls both the intrinsic equilibrium and rates of bidirectional Ca2+ movements in NCX proteins. Some additional structural elements may shape the kinetic variances among phylogenetically distant NCX variants, although the intrinsic asymmetry (Kint) of bidirectional Ca2+ movements seems to be comparable among evolutionary diverged NCX variants. 相似文献
13.
(1) The effects of calmodulin binding on the rates of Ca2+-dependent phosphorylation and dephosphorylation of the red-cell Ca2+ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg2+-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg2+-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca2+ + Mg2+)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca2+ affinity for sustaining ATP hydrolysis is raised at least 20-fold, Km(Ca) ? 10 μM (—calmodulin) and Km(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca2+ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (Km(Ca) ? 3–4 · 10-8 M). (Ca2+ + Mg2+)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca2+ + Mg2+)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca2+ + Mg2+)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (Km(MgATP) ? 360 μM), and CaATP is an inhibitor (Ki(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca2+- pump active site, producing a high (Ca2+ + Mg2+)-ATPase activity, high Ca2+ affinity and regulation of activity by MgATP. 相似文献
14.
Ca2+ inhibited the Mg2+-dependent and K+-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na+ + K+)-ATPase. In the absence of K+, however, a Mg2+-dependent and Ca2+-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K+-stimulated phosphatase. The Ca2+-stimulated phosphatase, like the K+-stimulated activity, was inhibited by either ouabain or Na+ or ATP. Ouabain sensitivity was decreased with increase in Ca2+, but the K0.5 values of the inhibitory effects of Na+ and ATP were independent of Ca2+ concentration. Optimal pH was 7.0 for Ca2+-stimulated activity, and 7.8–8.2 for the K+-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca2+-stimulated phosphatase is catalyzed by (Na+ + K+)-ATPase; (b) there is a site of Ca2+ action different from the site at which Ca2+ inhibits in competition with Mg2+; and (c) Ca2+ stimulation can not be explained easily by the action of Ca2+ at either the Na+ site or the K+ site. 相似文献
15.
When human red cells are incubated at 37°C with the artificial electron donor system ascorbate + phenazine methosulphate the fluxes of Rb+ (K+) through the cell membrane are increased. The effect of this donor system is much stronger in energy-depleted than in normal cells. The same effects are produced by HS-glutathione, NADH or NADPH loaded into resealed ghosts, but these electron donors were ineffective when added to the incubation medium. The Rb+ (K+) fluxes induced by electron donors resemble closely those induced by an increase of intracellular Ca2+ (Gardos effect). The electron donors require the presence of intracellular Ca2+ to be effective, but at levels that do not stimulate by themselves the fluxes of K+. Flavoenzyme inhibitors (atebrin and chlorpromazine), oligomycin and quinine prevented the effects of both electron donors and Ca2+ alone; antimycin, uncouplers and ethacrynic acid inhibited them partially; ouabain, furosemide, and rotenone had no effect.The results could be explained if the effect of electron donors is to bring about a change in the redox state of some membrane component(s) that makes intracellular Ca2+ more effective to elicit rapid K+ movements. Plasma membrane oxidoreductase activities could be engaged in this change. 相似文献
16.
NADPH is the reducing agent for mitochondrial H2O2 detoxification systems. Nicotinamide nucleotide transhydrogenase (NNT), an integral protein located in the inner mitochondrial membrane, contributes to an elevated mitochondrial NADPH/NADP+ ratio. This enzyme catalyzes the reduction of NADP+ at the expense of NADH oxidation and H+ reentry to the mitochondrial matrix. A spontaneous Nnt mutation in C57BL/6J (B6J-NntMUT) mice arose nearly 3 decades ago but was only discovered in 2005. Here, we characterize the consequences of the Nnt mutation on the mitochondrial redox functions of B6J-NntMUT mice. Liver mitochondria were isolated both from an Nnt wild-type C57BL/6 substrain (B6JUnib-NntW) and from B6J-NntMUT mice. The functional evaluation of respiring mitochondria revealed major redox alterations in B6J-NntMUT mice, including an absence of transhydrogenation between NAD and NADP, higher rates of H2O2 release, the spontaneous oxidation of NADPH, the poor ability to metabolize organic peroxide, and a higher susceptibility to undergo Ca2+-induced mitochondrial permeability transition. In addition, the mitochondria of B6J-NntMUT mice exhibited increased oxidized/reduced glutathione ratios as compared to B6JUnib-NntW mice. Nonetheless, the maximal activity of NADP-dependent isocitrate dehydrogenase, which is a coexisting source of mitochondrial NADPH, was similar between both groups. Altogether, our data suggest that NNT functions as a high-capacity source of mitochondrial NADPH and that its functional loss due to the Nnt mutation results in mitochondrial redox abnormalities, most notably a poor ability to sustain NADP and glutathione in their reduced states. In light of these alterations, the potential drawbacks of using B6J-NntMUT mice in biomedical research should not be overlooked. 相似文献
17.
The Conversion of Nitrite to Nitrogen Oxide(s) by the Constitutive NAD(P)H-Nitrate Reductase Enzyme from Soybean 总被引:18,自引:1,他引:17
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A two-step purification protocol was used in an attempt to separate the constitutive NAD(P)H-nitrate reductase [NAD(P)H-NR, pH 6.5; EC 1.6.6.2] activity from the nitric oxide and nitrogen dioxide (NO(x)) evolution activity extracted from soybean (Glycine max [L.] Merr.) leaflets. Both of these activities were eluted with NADPH from Blue Sepharose columns loaded with extracts from either wild-type or LNR-5 and LNR-6 (lack constitutive NADH-NR [pH 6.5]) mutant soybean plants regardless of nutrient growth conditions. Fast protein liquid chromatography-anion exchange (Mono Q column) chromatography following Blue Sepharose affinity chromatography was also unable to separate the two activities. These data provide strong evidence that the constitutive NAD(P)H-NR (pH 6.5) in soybean is the enzyme responsible for NO(x) formation. The Blue Sepharose-purified soybean enzyme has a pH optimum of 6.75, an apparent Km for nitrite of 0.49 millimolar, and an apparent Km for NADPH and NADH of 7.2 and 7.4 micromolar, respectively, for the NO(x) evolution activity. In addition to NAD(P)H, reduced flavin mononucleotide (FMNH2) and reduced methyl viologen (MV) can serve as electron donors for NO(x) evolution activity. The NADPH-, FMNH2-, and reduced MV-NO(x) evolution activities were all inhibited by cyanide. The NADPH activity was also inhibited by p-hydroxymer-curibenzoate, whereas, the FMNH2 and MV activities were relatively insensitive to inhibition. These data indicate that the terminal molybdenum-containing portion of the enzyme is involved in the reduction of nitrite to NO(x). NADPH eluted both NR and NO(x) evolution activities from Blue Sepharose columns loaded with extracts of either nitrate- or zero N-grown winged bean (Psophocarpus tetragonolobus [L.]), whereas NADH did not elute either type of activity. Winged bean appears to contain only one type of NR enzyme that is similar to the constitutive NAD(P)H-NR (pH 6.5) enzyme of soybean. 相似文献
18.
Kinetics of Ca/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris L 总被引:1,自引:0,他引:1
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Artificial pH gradients across tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were used to study the kinetics of a Ca2+/H+ antiport across this membrane. Ca2+-dependent H+ fluxes were measured by the pH-dependent fluorescence quenching of acridine orange. ΔpH-dependent Ca2+ influx was measured radiometrically. Both H+ efflux and Ca2+ influx displayed saturation kinetics and an identical dependence on external calcium with apparent Km values of 43.9 and 41.7 micromolar, respectively. Calcium influx was unaffected by an excess of Mg2+ but was inhibited by La3+ > Mn2+ > Cd2+. The apparent Km for external calcium was greatly affected (5-fold) by internal pH in the range of 6.0 to 6.5 and a transmembrane effect of internal proton binding on the affinity for external calcium is suggested. 相似文献
19.
Regulation of ribulose-1,5-bisphosphate carboxylase from tobacco: changes in pH response and affinity for CO2 and Mg2+ induced by chloroplast intermediates 总被引:10,自引:0,他引:10
Ribulose-1,5-bisphosphate caryboxylase-oxygenase is activated by CO2 and Mg2+ in a process distinct from catalysis. The effect of chloroplast metabolites as they separately influenced either activation or catalysis of tobacco carboxylase was examined. Of the 28 metabolites examined, 13 effected activation of the carboxylase. The strongest positive effectors were NADPH, gluconate-6-P, glycerate-2-P, and glycerate-3-P. Negative effectors included ribose-5-P, fructose-6-P, glucose-6-P, and pyrophosphate. The concentration of CO2 or Mg2+ necessary to produce half-maximal activation is defined as Kact. NADPH and gluconate-6-P decreased the Kact(CO2) from 43 to 7.4 and 3.5 μm, respectively (pH 8.0, 5 mm MgCl2). They also decreased the Kact(M.g2+), but had little affect on the affinity of the enzyme for CO2 during the catalytic process. Increasing Mg2+ concentration decreased the Kact(CO2) and increasing CO2 concentration decreased the Kact-(Mg2+). NADP+ and gluconate-6-P also affected the pH profile of activation, shifting it toward lower pH values. Changes in activation had no effect on the pH profile for catalysis of CO2 fixation. Effectors influenced ribulose-1,5-bisphosphate oxygenase in a manner analogous to the carboxylase. At air levels of O2 and CO2, the ratio of carboxylase to oxygenase activity was not changed by the presence of effectors, including hydroxylamine. 相似文献
20.
Purification and Partial Characterization of Two Soluble NAD(P)H Dehydrogenases from Arum maculatum Mitochondria 总被引:6,自引:6,他引:0
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Two enzyme systems carrying out the oxidation of NAD(P)H in the presence of various electron acceptors have been isolated and partially characterized from the supernatant of frozen-thawed mitochondria from Arum maculatum spadices. The two systems contain flavoproteins and differ by their ability to oxidize NADH or NADPH, optimum pH and pI values, sensitivity to Ca2+ and EGTA, denaturation by 4 molar urea, molecular mass, and number of subunits. These properties, together with methodological considerations, are compatible with the location of these enzyme activities on the outer surface of the inner mitochondrial membrane, and support the hypothesis of the existence of two separate dehydrogenases responsible for the mitochondrial oxidation of cytosolic NADH and NADPH. 相似文献