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1.
The appearance of energy transfer factor B (Factor B) activity in discrete fractions derived from heart mitochondrial extracts has been demonstrated. Two subfractions (Fractions 50-2 and 50-10), besides Factor B (in Fraction 100-10) stimulated the activity of the ammonia-EDTA particle (AE-particle) in oxidative phosphorylation, ATP-P1 exchange and ATP-driven NAD+ reduction by succinate (Succ-NAD+ assay). Treatment of these factor preparations with p-Chloromercuriphenylsulfonate abolished their stimulatory activity in Succ-NAD+, indicating the involvement of thiol groups in their function. Based on sucrose density gradient centrifugation, the molecular weight of the active component in Fraction 50-10, which had the highest Factor B activity, was 47,000. In earlier work from this laboratory, the molecular weight of Factor B was found to be 29,000. Rabbit antiserum to Factor B showed precipitin bands with and completely inhibited the stimulation of the Succ-NAD+ activity produced by Fractions 50-2, 50-10 and 100-2 in the presence of the AE particle. 相似文献
2.
Reconstitution of energy-dependent transhydrogenase in ATPase-negative mutants of Escherichia coli 总被引:16,自引:0,他引:16
The aerobic-driven and ATP-driven energy-dependent transhydrogenase activities of membrane particles from two different Ca2+, Mg2+-activated ATPase-negative mutants of were examined. The activities were low or absent in one of the mutants (DL-54). Reconstitution of the aerobic-driven reaction could be obtained by addition to particles from this mutant of DCCD or of a coupling factor prepared from the parent strain. The coupling factor also restored the ATP-driven reaction. In the other mutant (N144) the aerobic-driven activity was unimpaired, and was not affected by DCCD or by the coupling factor. The difference between the two mutants could be rationalized if the coupling factor ATPase had both a stabilizing and an enzymic function. 相似文献
3.
Cathepsin BI1 was purified from rat liver lysosomal fraction by ammonium sulfate fractionation, followed by chromatography on Sephadex G-200 and DEAE-Sephadex. Formation of chemotactic factor for guinea pig polymorphonuclear (PMN) leukocytes was demonstrated when guinea pig serum was incubated with cathepsin BI. This factor formation was dependent on SH-reagent dithiothreitol (DTT) and was maximal at pH 6.0. ZnSO4, an inhibitor of cathepsin BI, inhibited the chemotactic factor formation likewise. 相似文献
4.
D L Stiggall Y M Galante R Kiehl Y Hatefi 《Archives of biochemistry and biophysics》1979,196(2):638-644
The coupling factor protein isolated previously in pure form with a molecular weight of 11–12 × 103 (K.-S. You and Y. Hatefi, 1976, Biochim. Biophys. Acta423, 398–412) has been shown to restore ATP-induced NAD reduction by succinate, transhydrogenation from NADH to NADP, and ATP-33Pi exchange to submitochondrial particles rendered deficient by extraction with 1 m NH4OH. The factor also stimulated the oxidative phosphorylation activity of the extracted particles 2.5- to >3-fold. The stimulatory effect of the factor was inhibited by mercurials, Cd2+, phenylarsine oxide, and diamide, indicating that it contains an essential dithiol. Dithiothreitol and dihydrolipoate did not replace the protein factor in stimulating the deficient particles. The purified dithiol-containing protein was precipitated and inhibited by antibody raised against coupling factor B. Since this antibody also inhibits coupling factor F2, it is concluded that the active principle of coupling factors B and F2 is the purified dithiol-containing protein of molecular weight 11–12 × 103 referred to above. 相似文献
5.
Belogrudov GI 《Archives of biochemistry and biophysics》2008,473(1):76-87
Treatment of the recombinant bovine factor B with trypsin yielded a fragment (amino acid residues 62-175) devoid of coupling activity. Removal of the N-terminal Trp2-Gly3-Trp4 peptide resulted in a significant loss of coupling activity in the FBΔW2−W4 deletion mutant. Sucrose density gradient centrifugation demonstrated co-sedimentation of recombinant factor B with the ADP/ATP carrier, which is present in preparations of H+-translocating F0F1-ATPase, but not in preparations of complex V. The N-terminally truncated factor B mutant FBΔW2−W4 did not co-sediment with the ADP/ATP carrier. Recombinant factor B co-sedimented with partially purified membrane sector F0, extracted from F1-stripped bovine submitochondrial particles with n-dodecyl-β-d-maltoside. Factor B inhibited the passive proton conductance catalyzed by F0 reconstituted into asolectin liposomes. A factor B mutant, bearing a photoreactive unnatural amino acid pbenzoyl-l-phenylalanine (pBpa) substituted for Trp2, cross-linked with F0 subunits e and g as well as the ADP/ATP carrier. These results suggest that the N-terminal domain and, in particular, the proximal N-terminal amino acids are important for the coupling activity and protein-protein interactions of bovine factor B. 相似文献
6.
The B to A transition of DNA in solution 总被引:31,自引:0,他引:31
V I Ivanov L E Minchenkova E E Minyat M D Frank-Kamenetskii A K Schyolkina 《Journal of molecular biology》1974,87(4):817-833
7.
C L Hsin-Hsiung-TaiTai C S Hollander 《Biochemical and biophysical research communications》1974,57(2):457-462
15-Hydroxyprostaglandin dehydrogenase has been purified from swine kidney to a specific activity of near 100 miliunits per mg of protein. The purified enzyme was found to be inhibited by thyroid hormone analogues of which triiodothyroacetic acid was the most potent inhibitor. The concentration required for 50% inhibition was 5 μM for triiodothyroacetic acid. The inhibition by thyroid hormones was uncompetitive and non-competitive with regard to NAD+ and prostaglandin E1, respectively. The sensitivity of this enzyme to thyroid hormones suggests that these hormones may regulate the metabolism of prostaglandins . 相似文献
8.
Exposure of rat liver mitochondria to inorganic arsenate followed by reisolation and washing to remove the added arsenate results in uncoupled respiration with succinate and ascorbate (), but and values of 1.3 to 1.6 with 3-hydroxybutyrate or glutamate. and values greater than 1.0 with NAD+-linked substrates arise as a result of partial reactivation of coupling at sites 2 and 3 by these substrates. In the presence of rotenone, NAD+-linked substrates can still reactivate coupling with succinate or ascorbate at these sites. The extent of reactivation in the presence of rotenone by 3-hydroxybutyrate is decreased by simultaneous addition of acetoacetate. The results suggest that the coupling at sites 2 and 3 is amenable to control through changes in the reduction state of some specific components of the respiratory chain located remotely from these sites. 相似文献
9.
Complexes of the formula cis-[Pt(H~N)(L)Cl2], where (H~N) are the protonated diamines including 3-aminoquinuclidine, N-aminopiperidine, piperazine, N-methylpiperazine, 1,1,4-trimethylpiperazine, and N-methyl-1,4-diazabicyclo [2,2,2] octane (N-methyl-dabco) and L = SCN?, NO2?, Br?, and F?, were synthesized from the protonated diamine complexes, [Pt(H~N)Cl3]. The antitumor activities of the complexes were evaluated in vitro against L1210 murine leukemia cells, and ID50 values for the L-substituted complexes were compared to values of the parent complexes. In each case it was found that replacement of a chloride ion by SCN?, NO2?, Br?, or F?, either reduced or completely eliminated antitumor activity. This effect is explained in terms of the trans-directing ability of the ligand, L, compared to chloride. The NO2-substituted complex of 3- aminoquinuclidine was tested in vivo and found to exhibit little or no antitumor activity. 相似文献
10.
The stimulation of protein synthesis by NAD+ in rabbit reticulocyte lysates has been reported. [Lennon M. B., Wu, J., and Suhadolnik, R. J., (1976) Biochem. Biophys. Res. Commun. 72, 530–538]. NAD+ can replace the creatine phosphate-creatine phosphokinase () energy regenerating system normally used in in vitro protein synthesizing systems. The replacement of by NAD+ is optimal at 37 °C. A significant lag in the rate of protein synthesis with NAD+ is observed with decreasing temperatures. Analysis of the adenylate energy charge with NAD+ shows an initial rapid decrease. This decrease in the energy charge recovers with increasing NAD+ concentrations. The energy level correlates with the rates of incorporation of d,l-[4,5-3H(N)]leucine into protein. ATP production via NAD+ pyrophosphorylase or oxidative phosphorylation does not explain the stimulation by NAD+. Rather, the stimulation is correlated with the activation of glycolysis. Glycolysis is not active in lysate preparations because NAD+ is absent. Additional possible roles of NAD+ in protein synthesis are discussed. 相似文献
11.
M Tanabe 《Experimental parasitology》1979,48(1):135-143
NADH oxidase activity was detected in the 105,000g supernatant (“soluble”) fraction of Trichomonas vaginalis and the enzyme was purified 50-fold by centrifugation, ammonium sulfate precipitation, Sephadex G-200, and DEAE-Sephadex A-25 chromatography. The ratio of oxygen uptake to NADH oxidation was approximately one-half. Addition of catalase did not affect the rate of oxygen uptake elicited by NADH. Since the purified fraction was free from interfering enzymes, the postulated reaction is as follows: . Among numerous substances tested, only NADH was a functional substrate, whereas NADPH was not oxidized. The purified enzyme had a Vmax of 16.5 μmole of oxygen consumed/min/mg protein, and the apparent Km for NADH was 7.4 μM. Substrate inhibition was observed at 3.7 mM NADH. The purified NADH oxidase was competitively inhibited by NAD+ as well as by NADP+ with 50% inhibition at 1 and 5 mM, respectively. The enzyme was also markedly inhibited by p-chloromercuribenzoate, hydrogen peroxide, and transient metal-chelators such as bathophenanthroline or o-phenanthroline. A flavoprotein antagonist, atebrin was slightly less inhibitory. Various quinones, flavin nucleotides and artificial dyes, except for p-benzoquinone, ferricyanide and cytochrome c, did not function in accepting electrons from NADH oxidase. These three compounds, however, were still poor electron acceptors in the enzymatic reaction suggesting that the trichomonad NADH oxidase has little diaphorase activity. All of these findings indicate that T. vaginalis has an unique NADH oxidizing enzyme in that H2O seems to be the prdouct of oxygen reduction. This NADH oxidase appears important in the aerobic metabolism of this parasite. 相似文献
12.
Exogenous NAD Effects on Plant Mitochondria: A Reinvestigation of the Transhydrogenase Hypothesis 总被引:4,自引:4,他引:0
Addition of NAD+ to purified potato (Solanum tuberosum L.) mitochondria respiring α-ketoglutarate and malate in the presence of the electron transport inhibitor rotenone, stimulated O2 uptake. This stimulation was prevented by incubating mitochondria with N-4-azido-2-nitrophenyl-aminobutyryl-NAD+ (NAP4-NAD+), an inhibitor of NAD+ uptake, but not by 1 mm EGTA, an inhibitor of external NADH oxidation. NAD+-stimulated malate-cytochrome c reductase activity, and reduction of added NAD+ by intact mitochondria, could be duplicated by rupturing the mitochondria and adding a small quantity to the cuvette. The extent of external NAD+ reduction was correlated with the amount of extra mitochondrial malate dehydrogenase present. Malate oxidation by potato mitochondria depleted of endogenous NAD+ by storing on ice for 72 hours, was completely dependent on added NAD+, and the effect of NAD+ on these mitochondria was prevented by incubating them with NAP4-NAD+. External NAD+ reduction by these mitochondria was not affected by NAP4-NAD+. We conclude that all effects of exogenous NAD+ on plant mitochondrial respiration can be attributed to net uptake of the NAD+ into the matrix space. 相似文献
13.
contains both NAD+- and NADP+-linked malic enzyme activities when grown on malate/nitrate. The enzyme is inactive in the absence of NH4+. AcetylCoA inhibits both activities competitively with respect to L-malate. Glyoxylate (0.5 mM) causes 60% inhibition of the NADP+-linked activity but has little effect on the NAD+-linked activity. Citrate, aspartate, AMP, ADP, and ATP, at 0.5mM, have little effect on either of the two activities. The results are discussed with regards to the control of malic enzyme activity within the cell. 相似文献
14.
Two proteins (A and B) from are required for synthesis of the NAD+ precursor, quinolinate, from L-aspartate and dihydroxyacetone phosphate. The requirement for B protein and L-aspartate in this system can be replaced by millimolar concentrations of oxaloacetate and ammonia if they are added together. This finding supports the concept that the B protein (L-aspartate oxidase) functions to form iminoaspartate which is condensed with dihydroxyacetone phosphate by the A protein to form quinolinate. 相似文献
15.
The surface activity and enzymic properties of the factor F1, the catalytic moiety of Streptococcus faecalis H+-ATPase, has been studied at the air-water and phospholipid-water interfaces. F1 does not interact with the monolayer phospholipids, hence its adsorption on a biological membrane must be due mainly to its recognition of proteins of the hydrophobic complex. The dimensions of the F1 molecule at the air-water interface have been estimated. In the presence of Mg2+, base area is , height . Bearing in mind the size of a globular subunit, it follows from the measurements that the major F1 subunits should all lie in the same plane. The ATPase activity of F1 at the interface is inversely proportional to the monolayer density. With low density monolayer, the specific ATPase activity is higher at the interface than in the bulk of the solution.Adsorption of F1 at the interface shifts the isoelectric point of the protein, apparently due to changes in its conformation. The findings are discussed relative to the proton-active transport mechanism. 相似文献
16.
D D Hackney 《Biochemical and biophysical research communications》1979,91(1):233-238
The soluble mitochondrial ATPase, F1, can be slowly inactivated by incubation with Mg+2 in a manner consistent with the observations of Moyle and Mitchell , 55 (1975)). This inhibition results in a low initial rate of ATP hydrolysis upon addition to an ATPase assay medium of F1 which has been incubated with Mg+2. This inhibition, however, is completely reversible by Mg·ATP in a time dependent process and results in the rate of ATP hydrolysis increasing during the ATPase assay to reach control levels after 30 sec. The length of the lag is independent of the F1 concentration in the ATPase assay and the lag is also completely reversed by subsequent incubation with excess EDTA before assay.F1 is unstable if incubated with EDTA in the absence of free nucleotides or Mg+2. The rate of inactivation increases with decreasing protein concentration until a limiting rate is reached at high dilution. Mg+2 in excess of the EDTA or 50 μM ADP stabilize the F1 against the inactivation but cannot reverse prior denaturation. 相似文献
17.
Raymond L. Houghton Robert J. Fisher D. Rao Sanadi 《Biochemical and biophysical research communications》1976,73(3):751-757
A partially purified preparation of pyridine nucleotide transhydrogenase (E.C. 1.6.1.1.) (energy-independent) has been obtained from membranes of by means of deoxycholate extraction and DEAE-cellulose chromatography in the presence of Triton X-100. The enzyme was lipid-depleted by treating with cholate and ammonium sulfate. The preparation was reactivated by various phospholipids, in particular, bacterial cardiolipin and phosphatidyl glycerol. Phosphatidyl ethanolamine, the major phospholipid in the outer membrane of , was relatively ineffective in stimulating activity. The membrane-bound pyridine nucleotide transhydrogenase is slowly inhibited by N-ethylmaleimide. Protection against inhibition was achieved with NAD+ and NADP+, but NADPH served to accelerate the rate of inhibition. 相似文献
18.
Hybrids were constructed between K12 ? mutants uncoupled in oxidative phosphorylation, and thus defective in ATP biosynthesis, and an F′ plasmid carrying nitrogen fixation genes from . Examination of these hybrids showed that expression of +Kp genes in K12 does not require coupling of oxidative phosphorylation but needs the contribution of an anaerobic electron transport system involving fumarate reduction. The Kp cluster of genes does not contain functions able to complement a defective Mg2+-ATPase aggregate but does contain a function(s) which appears to interact with the B? mutant over the formation of a redox system. 相似文献
19.
An alkali metal ion-sensitive NAD+-specific glyceraldehyde 3-phosphate dehydrogenase has been purified 250-fold from germinating green gram (Phaseolus aurieus). The purified enzyme shows a single protein band on gel electrophoresis. It has been shown to be a tetrameric protein (molecular weight 160,000) made up of apparently identical monomers (subunit molecular weight 40,000). It shows an ratio equal to 1.38, which is not changed on treatment with animal charcoal or cellulosic ion exchangers. Direct estimation shows less than 0.07 mol bound NAD+/mol enzyme. Green gram glyceraldehyde 3-phosphate dehydrogenase is inhibited fairly strongly at physiological concentrations of Na+ ions. The inhibition is stronger at higher pH and lower protein concentration. Deproteinated extract, cysteine, and reduced glutathione reverse the Na+ ion inhibition. The effect of deproteinated extract is attributable to the presence of some SH-containing compounds. Potassium and rubidium ions have a mild activating effect at lower concentration (below 100 mm) and are inhibitory at higher, nonphysiological, concentrations. Ammonium and lithium ions have no effect. The inhibition due to Na+ ions is noncompetitive with respect to NAD+ and phosphate ions but competitive with respect to glyceraldehyde 3-phosphate, with Ki about 60 mm. Sodium ions protect the enzyme against proteolysis with trypsin. It is suggested that Na+ ions and the small molecular weight SH-compounds may possibly be involved in regulation of the overall rate of glycolysis via modulation of glyceraldehyde 3-phosphate dehydrogenase activity. 相似文献
20.
Incubation of spinach thylakoids with HgCl2 selectively destroys Fe–S center B (FB). The function of electron acceptors in FB-less PS I particles was studied by following the decay kinetics of P700+ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t
1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t
1/20.25 ms). In FB-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in FB-less particles, electron transfer can proceed normally at room temperature from FX to FA and that the charge recombination between P700+ and FX
-/A1
- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of FB. Even in the presence of 60% glycerol, FB-less particles can transfer electrons to FA at room temperature as efficiently as untreated particles.Abbreviations DCIP
2, 6-dichlorophenol indophenol
- FA, FB, FX
iron-sulfur center A, B and X, respectively
- PMS
phenazine methosulfate 相似文献