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M Nishikimi

Publications and source records attributed to M Nishikimi.

At least 91 records · Page 5Linked to original sources
PubMed1986

Interaction of anti-iron-sulfur protein and anti-ubiquinone binding protein antibodies with complex III of beef heart mitochondria.

Ubiquinol-cytochrome c reductase activity of Complex III was substantially inhibited by anti-iron-sulfur protein antibody, whereas it was not affected by anti-ubiquinone binding protein antibody. Enzyme-linked immunosorbent assay indicated that anti-ubiquinone binding protein antibody do not bind to the complex, but that it binds to Complex III of which iron-sulfur protein and phospholipids have been depleted. These results indicate that some of the antigenic sites of the iron-sulfur protein are located on the surface of Complex III, while the antigenic sites of the ubiquinone binding protein are inaccessible to antibody owing to the interaction with iron-sulfur protein and/or phospholipids in the complex.

Animals↗
PubMed1986

Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP-ribose) synthetase.

Calf thymus poly(ADP-ribose) synthetase (Mr = 120,000) is cleaved with papain into two fragments of M(r) = 74,000 and 46,000 and also split with chymotrypsin into two fragments of M(r) = 66,000 and 54,000. Each fragment purified to homogeneity is enzymatically inactive, but combined incubation of the 74,000 and 46,000 fragments in the presence of DNA restored 20% of the enzyme activity. In contrast, combined incubation of the 66,000 and 54,000 fragments does not restore any enzyme activity. In the former incubation, autopoly(ADP-ribosyl)ation reaction occurs exclusively on the 74,000 fragment. When each fragment is incubated with [adenine-U-14C]NAD in the presence of DNA and a catalytic amount of the native enzyme, poly(ADP-ribosyl)action occurs in the overlapped portion (22,000) of the 66,000 fragment and the 74,000 fragment. Nevertheless, the purified 22,000 fragment is a poor acceptor for poly(ADP-ribosyl)ation. The degree of poly(ADP-ribosyl)ation of the proteolytic fragments is significantly reduced by increasing NaCl concentration, probably due to the lack of the interaction between the enzyme fragments and DNA. These results, taken together, indicate that DNA is indispensable for the reconstitution of the catalytic activity as well as the poly(ADP-ribosyl)ation of the fragmented enzyme.

Amino Acids↗
PubMed1986

Purification of the iron-sulfur protein, ubiquinone-binding protein, and cytochrome c1 from a single source of mitochondrial complex III.

By detergent-exchange chromatography using a phenyl-Sepharose CL-4B column, Complex III of the respiratory chain of beef heart mitochondria was efficiently resolved into five fractions that were rich in the iron-sulfur protein, ubiquinone-binding protein, core proteins, cytochrome c1, and cytochrome b, respectively. Complex III was initially bound to the phenyl-Sepharose column equilibrated with buffer containing 0.25% deoxycholate and 0.2 M NaCl. An iron-sulfur protein fraction was first eluted from the column with buffer containing 1% deoxycholate and no salt after removal of phospholipids from the complex by washing with the buffer for the column equilibration, as reported previously (Y. Shimomura, M. Nishikimi, and T. Ozawa, 1984, J. Biol. Chem. 259, 14059-14063). Subsequently, a fraction containing the ubiquinone-binding protein and another containing two core proteins were eluted with buffers containing 1.5 and 3 M guanidine, respectively. A fraction containing cytochrome c1 was then eluted with buffer containing 1% dodecyl octaethylene glycol monoether. Finally, a cytochrome b-rich fraction was eluted with buffer containing 2% sodium dodecyl sulfate. The fractions of the iron-sulfur protein and ubiquinone-binding protein were further purified by gel chromatography on a Sephacryl S-200 superfine column, and the cytochrome c1 fraction was further purified by ion-exchange chromatography on a DEAE-Sepharose CL-6B column; each of the three purified proteins was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Animals↗
PubMed1986

Radioimmunoscintigraphy of human gastric carcinoma xenografts with 125I-labeled monoclonal antibody.

A monoclonal antibody (GC 302) established in our laboratory, which was reactive with gastric carcinoma and other epithelial carcinoma but not with normal gastric mucosa or other malignant tumors of mesenchymal origin, was used to investigate the radioimmunolocalization of tumors. Various kinds of target cells (5 X 10(5)) were incubated with 125I-labeled GC 302, and radioactivity was determined with a gamma counter. It was shown that there was a 500- to 1,000-fold increase in counts for gastric carcinoma (NUGC-2, NUGC-4, MKN-28 and MKN-45) as compared to those of normal lymphocytes and about 100-fold increase as compared to melanoma or leukemia. These findings were consistent with those obtained from the study of immunohistochemistry using GC 302. An in vitro assay was also carried out using nude mice bearing gastric cancer and inoculated with 125I-labeled GC 302. There was a 2- to 3-fold increase in radioactivity in the tumor and a 4- to 5-fold increase as compared with the visceral organs. Although the tumor:blood ratio was relatively low, radioimmunoscintigraphy could be done successfully with the aid of computed radiography. We thus conclude that further testing of GC 302 is worthwhile to establish whether or not it is useful for radioimmunoscintigraphy of metastatic lesions of gastric cancer for possible clinical application.

Animals↗
PubMed1985

Novel purification of cytochrome c1 from mitochondrial Complex III. Reconstitution of antimycin-insensitive electron transfer with the iron-sulfur protein and cytochrome c1.

Complex III of beef heart mitochondria was separated into the iron-sulfur protein and the complex devoid of it as described previously (Shimomura, Y., Nishikimi, M., and Ozawa, T. (1984) J. Biol. Chem. 259, 14059-14063). From the latter preparation, cytochrome c1 was subsequently purified by detergent-exchange chromatography on a phenyl-Sepharose column and DEAE-Sepharose column chromatography. In the former chromatography, the resolution of the iron-sulfur protein-depleted complex was achieved by changes of detergents on the surface of the complex; nearly homogeneous cytochrome c1 was eluted from the column with dodecyl octaethylene glycol mono-ether after dissociation of core proteins and subunit VI with guanidine and cholate. The purified cytochrome c1 consists of a single polypeptide as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 39 nmol of heme/mg of protein. The isolated iron-sulfur protein catalyzes reduction of cytochrome c by ubiquinol, which is insensitive to antimycin, at a rate of 0.03 mumol of cytochrome c reduced/min/nmol of protein, while the purified cytochrome c1 has no such catalytic activity. When cytochrome c1 and the iron-sulfur protein form a complex, the rate of cytochrome c reduction increases to 0.12 mumol/min/nmol of the iron-sulfur protein. In this reaction, cytochrome c1 mediates antimycin-insensitive electron transfer from the iron-sulfur protein to cytochrome c, thereby constituting a pathway of electrons: ubiquinol----iron-sulfur protein----cytochrome c1----cytochrome c. The complex formation between the iron-sulfur protein and cytochrome c1 was verified by binding of cytochrome c1 to a column of protein A-Sepharose to which the iron-sulfur protein was linked with immobilized anti-iron-sulfur protein antibody. The electron-transfer activity of the mixture is at a comparable level to that of antimycin-inhibited Complex III, and both activities are partially sensitive to superoxide dismutase. Thus, the above-described coupling of the iron-sulfur protein and cytochrome c1 is considered as reconstitution of the antimycin-insensitive pathway of electrons in Complex III.

Animals↗
PubMed1985

The iron-sulfur protein of cytochrome bc1 complex. Its occurrence in the mitochondrial inner membrane in excess of the amount constituting the complex.

Radioimmunoassay and quantitative immunoblot analysis have been developed for quantitation of the iron-sulfur protein of cytochrome bc1 complex in order to compare its content in isolated cytochrome bc1 complex with that in electron transport particles. The result by radioimmunoassay indicated that the content of the iron-sulfur protein/mol of cytochrome b is higher by approximately 30%, on the average, in electron transport particles than in cytochrome bc1 complex. This observation was supported by the data of immunoblot analysis. Since approximately 1/3 of cytochrome b in electron transport particles is not attributed to cytochrome bc1 complex, but to succinate-ubiquinone oxidoreductase complex (Davis, K.A., Hatefi, Y., Poff, K. L., and Butler, W. L. (1973) Biochim. Biophys. Acta 325, 341-356), the ratio of the iron-sulfur protein detectable by radioimmunoassay in electron transport particles to that in cytochrome bc1 complex is calculated to be approximately 2 on the basis of the content of 2 mol of b-type heme/mol of the complex. Therefore, it appears that the mitochondrial inner membrane contains approximately two times as much of the immunoreactive iron-sulfur protein as what is expected from the stoichiometry of one iron-sulfur center and two b-type hemes for cytochrome bc1 complex. This finding affords an interesting aspect in the study of biogenesis of cytochrome bc1 complex.

Animals↗
PubMed1984

Isolation and reconstitution of the iron-sulfur protein in ubiquinol-cytochrome c oxidoreductase complex. Phospholipids are essential for the integration of the iron-sulfur protein in the complex.

An iron-sulfur protein has been purified from beef heart ubiquinol-cytochrome c oxidoreductase (Complex III) of the mitochondrial respiratory chain by phenyl-Sepharose column chromatography and Sephacryl S-200 gel chromatography. Depletion of most of the endogenous phospholipids in the complex was a prerequisite to the dissociation of the protein from the complex in the former chromatography. The iron-sulfur protein was nearly homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 76 ng atoms of nonheme iron and 66 nmol of acid-labile sulfide/mg of protein. When this preparation was incubated with an iron-sulfur protein-depleted complex in the presence of soybean phospholipids, the enzymic activity was restored up to 90% of that of the parent Complex III, whereas the recovery of the activity was marginal in the absence of the phospholipids. Thus it is clear that the iron-sulfur protein is integrated into the complex with the aid of phospholipids.

Animals↗
PubMed1984

Detection of iron-sulfur center-containing subunits of mitochondrial NADH dehydrogenase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by high-performance gel permeation chromatography.

Soluble NADH dehydrogenase resolved from Complex I of the mitochondrial electron-transfer chain was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate at 4 degrees C, and then the gel was stained for iron with bathophenanthroline disulfonate and thioglycolic acid. The 23,000-dalton subunit was markedly stained, and the 51,000-dalton subunit was also stained, but only slightly. High-performance gel permeation chromatography using an eluant containing 0.1% sodium dodecyl sulfate also demonstrated that these subunits contain an iron-sulfur center: the elution pattern recorded by light absorption at 400 nm gave two peaks corresponding to the positions of the subunits.

Animals↗
PubMed1984

Novel isolation and reconstitution of the iron-sulfur protein of mitochondrial Complex III.

An iron-sulfur protein was isolated from mitochondrial Complex III as a single band on SDS-polyacrylamide gel electrophoresis by hydrophobic interaction chromatography, in which the complex was dissociated into the iron-sulfur protein and the rest of the complex with a loss of enzymic activity. The critical condition for the dissociation was depletion of boundary phospholipids to 0.6% (w/w). After gel filtration of the isolated iron-sulfur protein, 76 ng atom of non-heme iron and 66 nmol of acid-labile sulfide were found per mg of the protein. By incubating the protein with the iron-sulfur protein depleted-complex in the presence of a soybean phospholipid mixture, the enzymic activity was fully recovered. The electrophoretic pattern of the reconstituted complex showed a polypeptide composition similar to that of the original Complex III. These results indicate that the iron-sulfur protein is attached in the complex with the aid of boundary phospholipids.

Animals↗
PubMed1983

Immunochemical demonstration of the iron-sulfur protein in Complex III of mitochondrial electron-transfer chain.

An iron-sulfur protein of Complex III was purified by phenyl-Sepharose column chromatography and DEAE-Sepharose column chromatography. The purified preparation was homogeneous as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and a specific antibody directed against this protein was raised in a rabbit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electrophoretic blotting and immunoperoxidase reaction indicated that Complex III possesses a single polypeptide which reacts with the antibody. It was also found that the iron-sulfur center-containing subunits identified so far in Complex I did not cross-react with the antibody, indicating that they are antigenically unrelated to the iron-sulfur protein of Complex III.

Animals↗
PubMed1982

Poly(ADP-ribose) synthetase. The DNA binding domain and the automodification domain.

Poly(ADP-ribose) synthetase is known to require double-stranded DNA for activity and to be susceptible to automodification with poly(ADP-ribose). This enzyme is cleaved into two fragments by limited proteolysis with papain. Their molecular weights are 74,000 and 46,000, respectively, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of Mr 46,000 binds to a nicked DNA-cellulose column with the same affinity as that of native enzyme, while the fragment of Mr 74,000 has little affinity for the DNA ligand. When the enzyme previously automodified with [adenine-U-14C]NAD (2 ADP-ribose units incorporated per molecule of enzyme) is digested with papain, the label is detected only in the fragment of Mr 74,000. These results indicate that poly(ADP-ribose) synthetase contains two domains, one acting as the site for binding of DNA and the other acting as the site(s) for accepting poly(ADP-ribose).

Animals↗
PubMed1982

Participation of poly(ADP-ribosyl)ation in the depression of RNA synthesis caused by treatment of mouse lymphoma cells with methylnitrosourea.

When mouse lymphoma cells (L-1210) are treated with methylnitrosourea, a DNA-damaging agent, polyadenosine diphosphoribose (poly(ADP-ribose)) synthetase activity increases 5-8-fold in 2-3 h, while RNA polymerase activity remains constant for an initial 2 h and then gradually decreases to 25-30% of the control level in 5 h. Both alpha-amanitin-sensitive and -resistant RNA polymerase activities are depressed to the same degree by the treatment with methylnitrosourea. The depression in RNA synthesis is virtually prevented when the treated cells are cultured in the presence of 3-aminobenzamide, a specific inhibitor of poly(ADP-ribose) synthetase. Analyses of the RNA extracted from the cells labeled with [3H]uridine by agarose gel electrophoresis and by poly(U)-Sepharose column chromatography show that the contents of ribosomal precursor RNA and poly(A)-containing RNA are both low in the methylnitrosourea-treated cells as compared with those in the untreated cells and that the reduction in the contents of these kinds of RNA is almost completely prevented by the addition of 3-aminobenzamide to the culture medium. These results suggest that the enhancement of poly(ADP-ribosyl)ation causes the decrease in both synthesis of ribosomal RNA and messenger RNA.

Animals↗
PubMed1981

Homology of L-gulonolactone oxidase of species belonging to Mammalia, Aves, and Amphibia.

Immunological cross-reactivity of L-gulonolactone oxidase of different species (rat, chicken, and bullfrog) was tested by the Ouchterlony technique. Antiserum directed against the enzyme from chicken kidney reacted with rat liver enzyme as well as with bullfrog kidney enzyme. This finding suggests that there is, at least partly, sequence homology among the enzyme from species belonging to the three classes, Mammalian, Aves, and Amphibia.

Animals↗
PubMed1981

Studies on the sulfhydryl group of L-galactonolactone oxidase.

L-Galactonolactone oxidase was found to be inactivated by various sulfhydryl reagents: the order of inactivation rate was HgCl2 greater than p-chloromercuribenzoate greater than 4,4'-dipyridyldisulfide greater than 2,2'-dipyridyldisulfide greater than 5,5'-dithio-bis-(2-nitrobenzoate) greater than N-ethylmaleimide greater than iodoacetamide. The inactivation by 4,4'dipyridyldisulfide was studied in detail, and it was found that the maximum degree of inactivation attained with increasing reagent concentration was 93%, and that the kinetics of inactivation was first order with respect to the reagent concentration. The pH dependence of the second-order rate constant of the inactivation revealed that sulfhydryl group with a pKa of approximately 9.8 was involved in the inactivation process. The value of pKa is high compared with that of low-molecular weight thiols, indicating that the ionization of the sulfhydryl group is affected by the electric field of a negatively charged group located in its vicinity. The values of Km and Vmax for the L-galactonolactone oxidase reaction did not change greatly around pH 9.8. This finding is consistent with the view that the sulfhydryl group does not participate in the catalytic process. The velocity of inactivation in the presence of substrate was considerably greater than that observed in its absence. It appears that the sulfhydryl group becomes more reactive during the catalytic cycle of the enzyme. It was also noted that the absorption spectrum of the flavin prosthetic group (the oxidized form) was modified by adding p-chloromercuribenzoate.

Chloromercuribenzoates↗
PubMed1981

Correlation of T4 to T3 converting activity with T3 concentration in rat tissues.

The correlation between T4 5'-deiodinase activity and thyroid hormone concentration in rat tissues was sought. Studies with normal and thyroidectomized rats indicated a significant correlation of the hepatic T3 concentration with the enzyme activity in liver homogenate (r = 0.80, p less than 0.01) or in liver microsomes (r = 0.08, p less than 0.01). The correlation between the hepatic T4 concentration and activity of the enzyme was less significant (r = 0.55, p less than 0.05 for homogenate, and r = 0.66, p less than 0.01 for microsomes). The degree of T4 5'-deiodinase activity in various organs was found to increase in line with tissue concentration of T3. It seems likely that the level of T4 5'-deiodinase activity is controlled by the tissue concentration of T3.

Animals↗
PubMed1980

L-gulonolactone oxidase activity and vitamin C status in riboflavin-deficient rats.

The effect of riboflavin deficiency on the activity of L-gulonolactone oxidase [L-gulono-gamma-lactone: oxygen 2-oxidoreductase, EC 1.1.3.8] and on vitamin C status was studied. A marked decrease in the specific activity of L-gulonolactone oxidase was observed in the liver microsomes isolated from riboflavin-deficient rats: the specific activity was approx. one-third of that in the microsomes isolated from control rats. The L-ascorbic acid content in the liver of the riboflavin-deficient rats was approx. one-half of that in the liver of the control rats. It seems that the rate of production of L-ascorbic acid in the riboflavin-deficient rats is limited by the decreased level of L-gulonolactone oxidase activity. Immunotitration using rabbit antiserum directed to L-gulonolactone oxidase revealed that a substantial amount of an inactive form of this enzyme is present in the liver microsomes of the riboflavin-deficient rats. L-Gulonolactone oxidase activity in the microsomes of these rats increased by approx. 35% upon addition of FAD, but it was slightly decreased by the addition of FMN or riboflavin. These results indicate that the liver microsomes of the riboflavin-deficient rats contain a protein which exhibits L-gulonolactone oxidase activity upon addition of Fad.

Animals↗