Abbreviations: PMS, phenazine methosulphate; CMU, 1-(p-chlorophenyl)-3,3′-dimethylurea 相似文献
The activity exhibited an absolute requirement for Mg2+ in the neutral pH range, while Ca2+ was found able to activate ATPase at more alkaline pH. Optimal activity was observed at pH 7.5, with a Mg/ATP ratio of 0.5.
ADP was found to inhibit ATP hydrolysis and to transform the Michaelian ATP concentration dependence with a Km of 0.5 mM into a sigmoid curve with increasing Km and decreasing V.
In contrast ADP activated an ATP-ADP exchange process and this shift from hydrolysis to exchange was stimulated by high Mg2+ and by orthophosphate.
All nucleoside triphosphates tested interfered with ATP hydrolysis, all could be hydrolyzed and could donate their terminal phosphate group to ADP. The relative efficiencies of nucleoside triphosphates in these three processes varied in parallel with minor discrepancies.
ATP hydrolysis was inhibited by N,N′-dicyclohexylcarbodiimide (DCCD) Dio 9, NaN3 and pyrophosphate, the first two being ineffective against ATP-ADP exchange, the third being stimulatory and the last inhibitory.
ATP hydrolysis and ATP-ADP exchange are tentatively attributed to the terminal enzyme of oxidative phosphorylation. 相似文献
2. Freeze-drying of the bacterial membranes causes a selective detachment of DPNH dehydrogenase (DPNH: (acceptor) oxidoreductase, EC 1.6.99.3) from the membranes. This solubilization is accompanied by a decrease of Km(K3Fe(CN)6) from 2.0 to 0.25 mM, while no change is detected in Km(DPNH). This enzyme is not the DPNH diaphorase found in the bacteria.
3. DPNH dehydrogenase of E. coli is a metalloflavoprotein, containing non-heme iron, labile sulfide, FMN and FAD.
4. Reduction of the enzyme with DPNH in the absence of electron acceptor (ferricyanide or DCIP) causes a rapid and irreversible change to a less active state, Form II. Form II is characterized by a higher Km(DPNH) and slower vmax., while the Km(K3Fe(CN)6) remains unchanged.
5. The transformation of the enzyme to Form II is accompanied by the reduction of the non-heme iron component. The role of non-heme iron in the enzymic reaction is discussed. 相似文献
1. 1. Esterification of 32P1 by illuminated chloroplasts prepared on a sucrose gradient was examined to establish the optimal incubation conditions.
2. 2. The evidence is consistent with phosphorylation being closely coupled to the sum of noncyclic and pseudocyclic electron flow and with the rate of electron flow responding to the availability of electron acceptors.
3. 3. Apparent Km values for ADP and Mg2+ were found to be 40 and 250 μM, respectively. The Km value for Mg2+ was increased by the presence of Ca2+. Two apparent values were observed for P1 at 0.2 and 1.1 mM. Chloroplast damage resulted in increased apparent Km (P1) values.
4. 4. Acceleration of the esterification resulting from the addition of ADP and P1 to the medium indicated that these compounds were able to penetrate to the active site of esterification.
5. 5. Ribose 5-phosphate (Rib-5-P) was shown to inhibit P1 esterification without affecting the apparent Km for ADP or P1. The evidence suggests that Rib-5-P interferes with the uptake of P1, and possibly ADP.
2. Adenine nucleotide translocation in C. utilis mitochondria is an exchange-diffusion process. The whole pool of internal adenine nucleotides is exchangeable, ADP being the most readily exchangeable nucleotide. The rate of mitochondrial ADP exchange, but not the Km value, depends on growth conditions. At 0 °C, the rate is about 3 to 4 nmoles ADP/min per mg protein for mitochondria obtained from yeast grown in the presence of 1.5% glucose; it rises to 11.5 nmoles when glucose is replaced by 3% ethanol in the growth medium. The Km value for ADP is 2 μM. The Q10 is about 2 between 0 and 20 °C. Among other exchangeable adenine nucleotides are ATP, dADP and the methylene and the hypophosphate analogues of ADP. Unlike mammalian mitochondria, C. utilis mitochondria are able to transport external UDP by a carboxyatractyloside-sensitive process.
3. Under conditions of oxidative phosphorylation (phosphate and substrate present in an aerated medium), added ADP is exchanged with internal ATP. A higher ATP/ADP ratio was found in the extramitochondrial space than in the intramito-chondrial space. The difference between the calculated phosphate potentials in the two spaces was 0.9–1.7 kcal/mole.
4. Atractyloside, carboxyatractyloside, bongkrekic acid and palmityl-CoA inhibit mitochondrial adenine nucleotide translocation in C. utilis as they do in mammalian mitochondria, but 2 to 4 times less efficiently. The inhibition due to atractyloside or palmityl-CoA is competitive with respect to ADP whereas that due to bongkrekic acid and carboxyatractyloside is non-competitive. Carboxyatractyloside and atractyloside inhibitions are additive. The apparent Kd for the binding of [35S]-carboxyatractyloside and [14C]bongkrekic acid is 10–15 nM and the concentration of sites 0.4–0.6 nmole/mg protein in both cases. [35S]Carboxyatractyloside binding is competitively displaced by atractyloside and vice versa.
5. Binding of [14C]ADP has been carried out with mitochondria depleted of their endogenous adenine nucleotides by incubation with phosphate and Mg2+ at 20 °C. The amount of bound [14C]ADP which is atractyloside removable is 0.08–0.16 nmole/mg protein.
6. The rate of ADP transport is quite different in mitochondria isolated from C. utilis, according to whether it is grown on glucose, or on ethanol or in the presence of chloramphenicol; for instance, it decreases by 10 times when 3% ethanol in the growth medium is replaced by 10% glucose and by 5 times when chloramphenicol is added to the medium. These variations are accompanied by parallel variations in cytochrome aa3. The number of atractyloside-sensitive ADP binding sites is not modified by the above conditions of culture, nor the number of [35S]carboxyatractyloside binding sites. The affinity for ADP is apparently not significantly modified, nor the size of the endogenous adenine nucleotide pool. In contrast to glucose repression or chloramphenicol inhibition, semi-anaerobiosis in C. utilis lowers significantly the mitochondrial binding capacity for carboxyatractyloside. Strict anaerobiosis in S. cerevisiae results in a practical loss of the cytochrome oxidase activity, and also of the carboxyatractyloside and ADP binding capacity. Transition from anaerobiosis to aerobiosis restores the cytochrome oxidase activity and the ADP and carboxyatractyloside binding capacities. 相似文献
1. 1. The effect of Mg2+ on ATP-dependent processes catalysed by intact rat-liver mitochondria can be explained quantitatively by the formation of Mg-ATP complexes that cannot act as a substrate for the adenine nucleotide translocator.
2. 2. The dinitrophenol-induced ATPase is characterized by two affinities of ATP: Km(1) = 6.7 μM and Km(2) = 63 μM, which contribute to the extent of 70% and 30%, respectively, to the total ATPase activity under the standard conditions employed.
3. 3. Km(1) of ATP is competitively increased by atractyloside, and is insensitive to changes in cation concentration or to oligomycin or aurovertin.
4. 4. Km(2) is as sensitive to atractyloside as the Km(1) and is also insensitive to oligomycin. However, it is increased by decreasing the cation concentration, and disappears in the presence of aurovertin.
5. 5. It is proposed that two conformations of the adenine nucleotide translocator exist, characterized by their different affinities for ATP. The distribution of the enzyme over these two conformations appears to be a function of the energy state of the mitochondria (coupled or uncoupled).
Abbreviations: PEP, phosphoenol pyruvate 相似文献
1. 1.|No obvious correlation was found between the temperature dependence of the apparent Km values for either mitochondrial succinoxidase or NADH oxidase systems prepared from rat or rainbow trout liver tissue and the previously observed pattern of susceptibility of these same enzymes to thermal inactivation.
2. 2.|Apparent activation energies (EA) have been calculated from the data and, although these are equivocal when considered in the above context, it is concluded that the possibility is not excluded that entropy may be a more important variable than enthalpy in the action of these enzymes in ectothermic animals.
Author Keywords: Mitochondria; Salmo gairdneri; rat; succinoxidase; NADH oxidase; temperature dependence; Km; activation energy; enzyme evolution 相似文献
1. 1. The effect of the Mg2+ concentration on the CO2 fixation activity in situ in isolated and intact spinach chloroplasts upon suspension in hypotonic medium was examined. CO2 fixation in the dark was activated 25–100 fold by 20 mM Mg2+ in the presence of added ATP plus either ribulose 5-phosphate or ribose 5-phosphate. 20 mM Mg2+-stimulated fixation only 2–3 fold in the presence of the substrate of fixation, ribulose 1,5-diphosphate. The highest Mg2+-stimulated rate of fixation in the dark observed with chloroplasts was 480 μmoles CO2 fixed per mg chlorophyll per h.
2. 2. The concentration of bicarbonate at half of the maximal velocity (apparent Km) during the Mg2+-stimulated fixation of CO2 was 0.4 mM in the presence of ATP plus ribose 5-phosphate and 0.6 mM with ribulose 1,5-diphosphate.
3. 3. Dithioerythritol or light enhanced Mg2+-stimulated CO2 fixation 1–3 fold in the presence of ATP plus ribose 5-phosphate but not ribulose 1,5-diphosphate.
4. 4. These results indicate that Mg2+ fluxes in the stroma of the chloroplast could control the activity of the phosphoribulokinase with a lesser effect on the ribulosediphosphate carboxylase. An increase in Mg2+ of 6–10 mM in the stroma region of the chloroplast would be enough to activate CO2 fixation during photosynthesis.
Abbreviations: Rib-5-P, ribose 5-phosphate; Ribul-5-P, ribulose 5-phosphate; Ribul-1,5-P2, ribulose 1,5-diphosphate; HEPES, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid 相似文献
The most highly purified nitrite reductase still exhibited cytochrome c oxidase activity with a Km of 27 μM for O2. This activity was also inhibited by KCN, NaN3 and NH2OH and by NO2−.
A constitutive cytochrome oxidase associated with membranes was also isolated from cells grown anaerobically with NO2−. It was inhibited by smaller amounts of KCN, NaN3 and NH2OH than the cytochrome oxidase activity of the nitrite reductase enzyme and also differed in having a pH optimum of about 8 and a Km for O2 of less than 0.1 μM. Spectroscopically, cytochromes b and c were found to be associated with the constitutive oxidase in the particulate preparation. Its activity was also inhibited by NO2−.
The physiological role of the cytochrome oxidase activity associated with the purified nitrite reductase is likely to be of secondary importance for the following reasons: (a) it accounts for less than 10% of total cytochrome c oxidase activity of cell extracts; (b) the constitutive cytochrome c oxidase has a smaller Km for O2 and would therefore be expected to function more efficiently especially at low concentrations of O2. 相似文献
The Km value of ester hydrolysis increased with an increase in the solvent content. Dioxane was the most effective and dimethyl sulfoxide (DMSO) the least, whilst Kcat showed a tendency to increase slightly in N, N-dimethylformamide (DMF) and DMSO. For dioxane and acetonitrile (MeCN) a maximum was observed.
In peptide formation from Fua-Phe-OEt and Gly-NH2, dioxane and MeCN supported high product yield at molar fractions smaller than ca. 0.05 but the yield decreased significantly at higher fractions, although a relatively constant selectivity (ratio of the peptide bond formed to the ester consumed) was maintained. DMSO gave rather low peptide yields and selectivity even at lower molar fractions. DMF showed an intermediate tendency.
An apparent saturation parameter of the amine component was evaluated and the dissociation constant of a complex between acyl-enzyme and amino acid amide (Kn), as well as the rate constant of aminolysis exerted by the amino acid amide bound correctly on the enzyme (Kn), was calculated by initial rate analysis of peptide formation. In contrast to Km values, Kn decreased with increasing concentrations of organic cosolvent. while a suppressive effect was observed (except for DMSO) on the Kn parameter.
Effects of the solvent practically immiscible in water was also studied by use of the enzyme physically “immobilized” on glass beads. 相似文献
One of these ATPases, Mg2+-dependent and with a low pH optimum, has been solubilized with the aid of detergents and purified approx. 20-fold. The optimal activity of the enzyme is at pH 5.5, and the optimal Mg2+ concentration is 5·10−3 M. The enzyme hydrolyzes dATP more rapidly than ATP, although the Km for both substrates is the same. Other nucleotide triphosphates are hydrolyzed very slowly while ADP or pyrophosphate are not hydrolyzed at all. Oligomycin, carbonylcyanide m-chlorophenylhydrazone, and ADP inhibit the enzyme, the latter competitively.
The level of the enzyme is highest in chloroplasts from rapidly dividing cells, and reaches a minimum in chloroplasts from maturing cultures which have attained their maximal photosynthetic activity. It is postulated that the enzyme has a role in controlling chloroplast development. 相似文献
The possibility of “spill-over” of light absorbed by System II to System I was studied. There is no probability that this spill-over, if any, exceeds 25% in Chlorella.
The apparent equilibrium constant is decreased by 3(3,4-dichlorophenyl)-1,1-dimethylurea. This is not in favor of the hypothesis of fully independent electron-transfer chains in photosynthesis; it is therefore likely that some communication between those chains exists. 相似文献
2. A particulate, heat-labile nitrite oxidase having an absolute requirement for O2 was prepared from N. agilis cells using sonic oscillation and differential centrifugation. The particles also possessed NADH oxidase, succinoxidase, formate oxidase and traces of NADPH oxidase activity. The stoichiometry of the nitrite oxidase reaction approached the theoretical value of 2 moles of NO2− consumed per mole of O2 consumed. The pH optimum of the nitrite oxidase system shifted to progressively more alkaline values as the NO2− concentration was increased, changing from a pH value of 6.8 at 0.6 mM KNO2 to pH 8.0 at 0.01 M KNO2 with apparent Km's of 0.2 and 1.2 mM NO2−, respectively. Computations of the HNO2 concentrations present under the above conditions showed an approx. 500-fold greater affinity for HNO2 which was independent of pH, suggesting the involvement of HNO2 as both a substrate and an inhibitor (at higher concentrations) of the nitrite oxidase system. The marked inhibition by NaN3, NaCN and Na2S, as well the light-reversible inhibition by CO, indicated the presence of cytochrome oxidase which was subsequently characterized. NO2− proved to be a competitive inhibitor of the nitrite oxidase system.
3. The particulate preparation also possessed a heat-labile nitrite-cytochrome c reductase activity which was energy independent and routinely measured under anaerobic conditions. As in the case of nitrite oxidase, the affinity of the enzyme for NO3− increased as the pH was lowered, but the pH optimum remained unaffected. In terms of calculated HNO2 concentration an approximately constant Km of about 0.2 μM was estimated at the several pH's examined. The inhibition by NO3− was shown to be competitive. The marked sensitivity of the reductase to several metal-binding agents implicated a metal component in the electron transport chain at the site prior to cytochrome c.
4. The membrane-like composition of the nitrite oxidase system is indicated. 相似文献
1. 1. Fuscin, a mould metabolite, is a colored quinonoid compound which reacts readily with −SH groups to give colorless addition derivatives.
2. 2. Binding of fuscin to mitochondria has been monitored spectrophotometrically. Fuscin binding is prevented by −SH reagents such as N-ehylmaleimide, N-Methylmaleimide, mersalyl or p-chloromercuribenzoate. Conversely, fuscin prevents the binding of −SH reagents as shown with N-[14C]ethylmaleimide. Once bound to mitochondria, fuscin is not removable by washing of mitochondria.
3. 3. High affinity-fuscin binding sites (Kd = 1 μM, N = 4–8 nmoles/mg protein) are present in whole mitochondria obtained from rat heart, rat liver, pigeon heart or yeast (Candida utilis). They are lost upon sonication but are still present in digitonin inner membrane + matrix vesicles. On the other hand, lysis of mitochondria by Triton X-100 does not increase the number of high affinity binding sites indicating that all these sites are accessible to fuscin in whole mitochondria. The number of fuscin high affinity sites appears to correlate with the glutathione content of mitochondrial preparations.
4. 4. Fuscin as well as N-ethylmaleimide and avenaciolide are penetrant SH-reagents;
5. 5. Fuscin interferes with the ADP-stimulated respiration of mitochondria on NAD-linked substrates, several functions of the mitochondrial respiratory apparatus being inhibited by fuscin in a non-competitive manner, but to various extents: (a) The electron transfer chain (Ki in the range of 0.1 mM); (b) the lipoamide dehydrogenase system (Ki = 5–10 μM); (c) the transport systems of phosphate (Ki ≈ 20 μM) and of glutamate (Ki = 3–5 μM); (d) the ADP transport, indirectly (Ki ≈ 10 μM).
6. 6. Like N-ethylmaleimide, fuscin inhibits the glutamate-OH− carrier, the inhibition of that carrier bringing about an apparent increase of aspartate entry in glutamate-loaded mitochondria by the glutamate-aspartate carrier.
7. 7. The inhibition of phosphate transport by fuscin probably accounts for the inhibition of the reduction of endogenous NAD by succinate in intact pigeon heart mitochondria.
8. 8. By binding the −SH groups of mitochondrial membrane specifically unmasked by addition of micromolar amounts of ADP, fuscin, like N-ethylmaleimide, prevents the functioning of ADP translocation.
9. 9. Because of their specific and analogous effects on some well defined mitochondrial functions such as glutamate transport and ADP transport, fuscin and N-ethylmaleimide can be distinguished from other −SH reagents. The lipophilic nature of fuscin and N-ethylmaleimide which accounts for the accessbility of these compounds to hydrophobic sites in the mitochondrial membrane or on the matrix side of this membrane may be partly responsible for their characteristic inhibitory effects on mitochondrial functions.
Abbreviations: DTNB, 5,5′-dithio-bis-(2-nitrobenzoic acid); PCMB, p-chloromercuribenzoate 相似文献