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1.
The role of cytochrome 5 in the -nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome 5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome 5 reductase, and Triton X-100 in addition to cytochrome 5. The omission of cytochrome 5 from the complete system entirely abolished the activity. These results clearly show that cytochrome 5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome 5 in such a way that the second electron is transferred from cytochrome 5 and thus exhibits the demethylase activity. 相似文献
2.
S Kominami K Shinzawa S Takemori 《Biochemical and biophysical research communications》1982,109(3):916-921
The energy requirements for mitochondrial protein synthesis were investigated in isolated rat liver mitochondria. Controlled changes in coupling efficiency were obtained by titration with FCCP in the presence of various substrates. No relationship was observed between the efficiency of oxidative phosphorylation and the inhibition of protein synthesis. With succinate-ADP as the substrate the ADP:O ratio was decreased by 70–80% with no effect on protein synthesis. In contrast, with acetate-ADP as substrate, a 10–20% reduction in the ADP:O ratio gave complete inhibition of protein synthesis. The data suggest that the rate of ATP production is more important for maintenance of protein synthesis than the efficiency of coupling . Thus, certain substrates can support maximal rates of protein synthesis even in relatively poorly coupled mitochondria. Analysis of mitochondrial translation products formed in the presence of increasing FCCP concentrations also showed that decreased efficiency of oxidative phosphorylation had no influence on the nature of the products. 相似文献
3.
Julián Gómez-Cambronero María L. Nieto JoséM. Mato Mariano Sánchez-Crespo 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,845(3):511-515
The enzyme lyso-platelet-activating factor:acetyl-CoA acetyltransferase (EC 2.3.1.67) was assayed in microsomal fractions from rat spleens. The addition of micromolar Ca2+ rapidly enhanced acetyltransferase activity and this activation was reversed by the addition of EGTA in excess of Ca2+. The effect of Ca2+ was on the apparent of the enzyme for the substrate acetyl-CoA without showing any significant effect on the of the acetylation reaction. When microsomes were isolated in the presence of 5 mM EGTA, to remove endogenous calmodulin, the same enhancing effect of Ca2+ on the acetylation reaction was observed. The addition of exogenous calmodulin to this preparation had no effect on the enzyme activity. Preincubation of spleen microsomes with the calmodulin inhibitor trifluoperazine decreased acetyltransferase in both the presence and the absence of Ca2+, indicating an effect of this drug independently of calmodulin. The addition of Mg-ATP to the assay mixture also had no effect on the acetylation reaction. These data suggest that Ca2+ modulates acetyltransferase activity from rat spleen microsomes by a mechanism that seems to be independent of calmodulin or protein phosphorylation. 相似文献
4.
The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca2+, Mg2+-activated ATPase of . In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an mutant of was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked. 相似文献
5.
C H Seiter R Margalit R A Perreault 《Biochemical and biophysical research communications》1979,86(3):473-477
Cytochrome was chemically coupled to cytochrome oxidase using the reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) which couples amine groups to carboxyl residues. The products of this reaction were analyzed on 2.5–27% polyacrylamide gradient gels electrophoretically. Since cytochrome binds to cytochrome oxidase electrostatically in an attraction between certain of its lysine residues and carboxyl residues on the oxidase surface, EDC is an especially appropriate reagent probe for binding-subunit studies. Coupling of polylysine to cytochrome oxidase using EDC was also performed, and the products of this reaction indicate that polylysine, an inhibitor of the cytochrome reaction with oxidase, binds to the same oxidase subunit as does cytochrome , subunit IV in the gel system used. 相似文献
6.
Hideaki Yamada Toru Nagasawa Haruyuki Ohkishi Bunsei Kawakami Yoshiki Tani 《Biochemical and biophysical research communications》1981,100(3):1104-1110
Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide is catalyzed by highly purified 3-chloro-D-alanine hydrogen chloride-lyase from . The synthetic reaction proceeds optimally at pH 8.5, as a function of enzyme concentration and incubation time. The enzymatically synthesized D-cysteine was isolated from the large scale reaction mixture and identified by physicochemical means. 相似文献
7.
James D. Luketich Michael H. Melner F.Peter Guengerich David Puett 《Biochemical and biophysical research communications》1983,111(2):424-429
The mitochondrial and microsomal cytochrome P-450 contents of mouse testis have been measured using difference spectroscopy on stable enzyme preparations containing the ferrous-carbon monoxide complex. Results were obtained on control animals (52 ± 3 days of age) and on animals injected subcutaneously with human choriogonadotropin (0.017 μg/g body weight 24 h prior to sacrifice). The high ratio of testicular mitochondrial cytochrome oxidase to P-450, which has previously precluded measurements of basal P-450 levels, was overcome by using N,N,N′,N′-tetramethyl-p-phenylene diamine to bypass site II, in combination with antimycin A to prevent reverse electron flow. The basal levels of mitochondrial and microsomal P-450 in mouse testis were 37.9 ± 3.5 and 28.9 ± 1.6 pmol/mg protein, respectively. Following administration of a desensitizing dose of gonadotropin, the respective values were lowered to 19.9 ± 1.4 and 19.6 ± 2.1 pmol/mg protein in 24 h. This is the first report of a gonadotropin-mediated decrease in mitochondrial P-450 and thus demonstrates that desensitization leads to alterations in both microsomal and mitochondrial P-450 in mouse testis. 相似文献
8.
Addition of -nitroanisole to a reaction mixture containing phenobarbital-pretreated rabbit liver microsomes brings about an increase the reoxidation rate of NADH-reduced cytochrome b5. Addition of partially purified cytochrome b5 to a solution containing microsomes results in a marked increase in both NADH- and NADPH-dependent O-demethylation of -nitroanisole. -Nitroanisole also increases the rate of NADH mediated cytochrome P-450 reduction. From these and other results described in the Discussion section, we confirm that electrons required for NADH-dependent O-demethylation of -nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b5 and that cytochrome P-450 is the enzyme which catalyzes -nitroanisole O-demethylation. 相似文献
9.
Isolation and properties of the cytochrome B-C1 complex from Rhodopseudomonas sphaeroides 总被引:2,自引:0,他引:2
A highly purified and active cytochrome -1 complex has been isolated from the chromatophores of the photosynthetic bacteria R-26, through steps of Triton X-100 solubilization, salt fractionation and calcium phosphate column chromatography. The isolated enzyme complex catalyzes fully antimycin A sensitive oxidation of ubiquinol by cytochrome with a turnover number of 1500 per minute at 23° based on cytochrome 1. It contains 8.3 nmoles of cytochromes and 1 per mg protein and shows four polypeptides in the sodium dodecylsulfate polyacrylamide gel electrophoresis. 相似文献
10.
F. Peter Guengerich David P. Ballou Minor J. Coon 《Biochemical and biophysical research communications》1976,70(3):951-956
Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450LMredComplex I→Complex II→P-450LMox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome is added. The latter protein does not appear to function as an electron carrier in this process. 相似文献
11.
J Wiegel 《Biochemical and biophysical research communications》1978,82(3):907-912
The α-isopropylmalate synthase (EC 4.1.3.12) from was inactivated by EDTA in a time-dependent reaction. Only the addition of Mn++ plus dithiothreitol could restore the activity. The substrate, α-ketoisovalerate, prevented the inactivation; the feedback inhibitor, leucine, and it's antagonist, valine, increased the rate of inactivation. Except for α,α′-bipyridyl, chelating reagents, other than EDTA had no effect on the enzyme stability. It is suggested that the α-isopropylmalate synthase is a metallo enzyme - the evidence points to Mn++ as the metal ion - and that this enzyme uses a mechanism of catalysis which differs from that of the analogous malate synthase (EC 4.1.3.2) and citrate synthase (EC 4.1.3.4). 相似文献
12.
M Erecińska R Oshino D F Wilson 《Biochemical and biophysical research communications》1980,92(3):743-748
[3H]-p-Azidophenacylbromide-(methyl-4-mercaptobutyrimidate)-cytochrome from was prepared and its properties determined. The radioactive photoaffinity-labeled cytochrome was linked by irradiation into a covalent complex with cytochrome oxidase. Analysis of the complex on SDS-polyacrylamide gels showed that cytochrome bound to one of the smaller subunits of cytochrome oxidase with an apparent molecular weight of 15,000. 相似文献
13.
[Porphyrin-14C] cytochrome (isolated from tissues of dogs injected with [14C] ALA) was given intravenously to one normal and one bile fistula dog. Essentially all of the injected 14C was excreted in the urine during the first six days. No (unchanged) cytochrome was detectable in the urine. Analysis of 14C and of light absorption at 400 nm in the successive eluates from Florisil columns showed that all 14C peaks coincided with pigment peaks, but some pigment peaks has no increase in 14C. The relative distribution of 14C in these pigment peaks changed markedly between the first and third days. Delayed excretion of some 14C suggested cellular uptake of cytochrome prior to the urinary excretion of its endogenous metabolites. 相似文献
14.
C R Geren L M Geren K E Ebner 《Biochemical and biophysical research communications》1975,66(1):139-143
A thermostable protein that strongly inhibits the soluble D-alanine carboxypeptidase was isolated from a cell-free extract of . The inhibitor was purified 140-fold by heat treatment, selective precipitation at pH 4.5, ion exchange chromatography on DEAE-cellulose and gel chromatography on Sephadex G-100. Inhibition of soluble D-alanine carboxypeptidase by this inhibitor is reversed by cations such as Mg++ or Na+ and abolished by digestion of the inhibitor with proteolytic enzymes. The inhibitor does not affect either the particulate D-alanine carboxypeptidase of or the growth of the bacteria. 相似文献
15.
Haruko Meyer Justus Mueller Franz Meyer 《Biochemical and biophysical research communications》1978,82(3):834-839
An acyl-CoA carboxylase, which catalyzes the carboxylation of acetylpropionyl-, and butyryl-CoA, has been isolated from the tapeworm . The enzyme has an absolute requirement for ATP, Mg2+, and HCO3 and, in addition, requires K+ for full catalytic activity. The enzyme has been purified 50-fold by a combination of calcium phosphate gel adsorption, ion-exchange column chromatography, and gel filtration. In its substrate specificity, K+ requirement, molecular size, and antigenic behavior, the tapeworm enzyme is similar to the acyl-CoA carboxylase of another helminth— the free-living nematode . 相似文献
16.
W. Maier D. Erge B. Schumann D. Gröger 《Biochemical and biophysical research communications》1981,99(1):155-162
A cell-free system of has been described which incorporates [14C] leucine into the peptide-type ergot alkaloid ergosine. Cell-free extracts may be prepared either from protoplasts or lyophilized mycelium. Production was markedly stimulated by addition of agroclavine compared with d-lysergic acid and by agitation of the incubation mixture. The synthesis of ergosine is strongly dependent on the presence of ATP in the reaction mixture. 相似文献
17.
Identification of the structural gene for yeast cytochrome c oxidase subunit I on mitochondrial DNA.
E Keyhani 《Biochemical and biophysical research communications》1979,89(4):1212-1216
Mitochondrial protein synthesis was analyzed in the yeast mit? mutants of which specifically lack cytochrome oxidase. [3H]leucine labeled polypeptides synthesized in yeast OXI 3 mutant were analyzed by means of immunoprecipitation and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When compared to control, subunit I was not detectable. This result was substantiated by growing OXI 3 mutant in the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis. Under such conditions SDS-PAGE analysis of [3H]leucine labeled immunoprecipitate shows the absence of subunit I. These data show that the OXI 3 locus contains the structural gene for cytochrome oxidase subunit I. 相似文献
18.
Yoichi Taya Susumu Nishimura 《Biochemical and biophysical research communications》1973,51(4):1062-1068
A new enzyme, which catalyzes the transfer of a methyl group to tRNA to form 5-methylaminomethyl-2-thiouridylate, was isolated from by a procedure including affinity chromatography. The purified enzyme was nearly homogeneous upon disc electrophoresis. Using methyl-deficient tRNAGlu of as substrate, the 5-methylaminomethyl-2-thiouridylate residue synthesized was mostly found in the anticodon loop, showing a coincidence of the modification site with that . 相似文献
19.
Kazuyuki Morihara Tatsushi Oka Hiroshige Tsuzuki Yoshiharu Tochino Takashi Kanaya 《Biochemical and biophysical research communications》1980,92(2):396-402
We have established a procedure for converting porcine insulin into human insulin using a serine protease from M497-1 which shows unique specificity against lysine residues on the carboxyl side of the splitting point. Desalanine-(B30)-insulin (DAI) was prepared by digestion of porcine insulin with protease. The coupling between DAI and Thr-OBut was performed by the same enzyme at pH 6.5 with a large excess of the amine component (Thr-OBut) in the presence of high concentrations of organic co-solvents. The highest yield was 85% by 20 h reaction at 37°C. The synthesized [Thr-OBut-B30]-insulin was isolated, then deprotected with trifluoroacetic acid in the presence of anisole to obtain semisynthetic human insulin. 相似文献
20.
Steven J. Scheinman Gerard N. Burrow Theoharis C. Theoharides Zoe N. Canellakis 《Life sciences》1977,21(8):1143-1147
High titer antiserum to hepatic ornithine decarboxylase was prepared by employing enzyme·monospecific antibody complex as the immunizing antigen. This new antiserum preparation was successfully labeled with 125I and was found to retain its specific immune properties. Iodinated antiserum was used to precipitate thyroid ornithine decarboxylase induced by a mixture of thyroid stimulating hormone and methyl xanthine in rat thyroids . 125I-labeled antibody incorporation into the enzyme antibody complex after induction showed an increase which paralleled the increase in enzymatic activity and thus suggested synthesis of thyroid enzyme protein. 相似文献