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M Mizugaki

Publications and source records attributed to M Mizugaki.

At least 163 records · Page 9Linked to original sources
PubMed1985

Studies on the metabolism of unsaturated fatty acids. XVI. Purification and general properties of 2,4-dienoyl-CoA reductase from Candida lipolytica.

2,4-Dienoyl-CoA reductase has been purified to homogeneity from Candida lipolytica cultivated in the presence of linoleic acid. The native enzyme had a molecular weight close to 360,000 as estimated by gel filtration on Sepharose CL-4B, whereas the subunit molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 33,000. The purified 2,4-dienoyl-CoA reductase from C. lipolytica gave a single precipitin line with antibodies raised against the purified enzyme from C. lipolytica. The general properties of the 2,4-dienyl-CoA reductase from C. lipolytica were examined. The enzyme had optimal pH at 6.5 and was inactivated by heat treatment at 50 degrees C for 10 min. trans-2,trans-4-Octadienoyl-CoA was the most active substrate of the dienoyl-CoA esters examined.

Candida↗
PubMed1984

Studies on the metabolism of unsaturated fatty acids. XIII. Properties of 2,4-dienoyl-CoA reductase from beef liver.

It is demonstrated that 3-alkenoyl-CoA is the product of the enzymic reduction catalyzed by 2,4-dienoyl-CoA reductase with the highly purified enzyme preparation from beef liver mitochondria. Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver was investigated. When trans-2, trans-4-decadienoyl-CoA was incubated with the reductase in the presence of 4R-[4-2H1]NADPH and H2O, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to pyrrolidine amides of fatty acids. When the substrate was incubated with the enzyme in the presence of 4S-[4-2H1]NADPH and H2O, one deuterium atom was incorporated into the product, 3-decenoate, at the C-5 position. In the case of 3-decenoate enzymatically prepared in the presence of NADPH and 2H2O, two deuterium atoms were incorporated into the product at the C-2 position. These results indicate that the reaction catalyzed by 2,4-dienoyl-CoA reductase from beef liver is a unique example of 1,4-addition of hydrogen to a 1,3-diene system conjugated with a carbonyl group of a thioester in biochemical fields. Chain length specificity of the reductase, isotope effects on reaction rates and competitive inhibitors are also discussed.

Animals↗
PubMed1984

Studies on the metabolism of unsaturated fatty acids. XIV. Purification and properties of NADPH-dependent trans-2-enoyl-CoA reductase of Escherichia coli K-12.

NADPH-dependent trans-2-enoyl-CoA reductase was purified to homogeneity from Escherichia coli. The molecular weight of the native enzyme was estimated to be 80,000 by gel filtration and that of the subunit was estimated to be 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was most active toward trans-2-hexenoyl-CoA and trans-2-octenoyl-CoA but was less active toward longer chain substrates, whereas the Km values decreased progressively with increasing carbon chain length of substrates. The reductase appears to have a functional thiol group. The enzyme activity was rapidly decreased by p-hydroxymercuribenzoate and 5,5'-dithiobis(2-nitrobenzoic acid), and slowly by N-ethylmaleimide. The enzyme was protected from inhibition by these SH-reagents by the addition of NADPH. The enzyme was also inhibited by saturated acyl-CoA esters.

Acyl Coenzyme A↗
PubMed1984

Studies on the metabolism of unsaturated fatty acids. XV. Purification and properties of 2,4-dienoyl-CoA reductase from rat liver peroxisomes.

Peroxisomal 2,4-dienoyl-CoA reductase was purified from rat liver to homogeneity. The subunit molecular weight of 33,000 was determined by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. The native molecular weight close to 120,000 was estimated by gel filtration on Sephacryl S-300 Superfine. trans-2, trans-4-Decadienoyl-CoA was the most active substrate among the dienoyl-CoA's of various chain lengths. The total activity of peroxisomal 2,4-dienoyl-CoA reductase exceeded that of the mitochondrial one even in the livers of rats fed with a standard diet. Furthermore both reductases were remarkably and coordinately induced in the livers of clofibrate-treated rats.

Animals↗
PubMed1983

The architecture of the animal fatty acid synthetase. II. Separation of the core and thioesterase functions and determination of the N-C orientation of the subunit.

Chicken fatty acid synthetase is cleaved by alpha-chymotrypsin into two fragments of molecular weight 230,000 and 33,000. These fragments may be easily separated by ammonium sulfate fractionation and gel filtration to yield pure preparations. The large 230,000-Da fragment contains all of the core activities of the fatty acid synthetic sequence i.e. acetyl and malonyl transacylases, condensing enzyme, beta-ketoacyl and enoyl reductases, the dehydratase, and the acyl carrier protein. The smaller 33,000-Da fragment retains the thioesterase activity which catalyzes the release of the completed acyl chains from the complex. Antibodies against the purified thioesterase fragment cross-react with analogous (Mr 33,000) peptides released from the complex by other proteases, as well as with all proteolytic intermediates that were predicted by peptide mapping to contain the thioesterase segment (Mattick, J. S., Tsukamoto, Y., Nickless, J., and Wakil, S. J. (1983) J. Biol. Chem. 258, 15291-15299). Amino acid sequence analyses demonstrate that the thioesterase domain is located at the carboxyl terminus of the synthetase monomer, thereby orienting the proteolytic (and functional) sites within the complex with respect to the direction of transcription and translation.

Amino Acid Sequence↗
PubMed1983

Studies on the metabolism of unsaturated fatty acids. XII. Reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli.

Incorporation of deuterium atoms from deuterium-labeled NADPH and 2H2O during the reaction catalyzed by 2,4-dienoyl-CoA reductase of Escherichia coli (E. coli) was investigated. When trans-2,cis-4-decadienoyl-CoA was incubated with 4R- or 4S-[4-2H1]NADPH in the presence of purified 2,4-dienoyl-CoA reductase, no deuterium was detected in the reaction product by gas chromatography-mass spectrometry after derivatization to its pyrrolidine amide. On the other hand, when the dienoyl-CoA was incubated in the presence of NADPH and the reductase in 2H2O, two deuterium atoms were incorporated: One deuterium atom was located at the C-4 position of trans-2-decenoate, and the other at the C-5 position. The UV and shorter wavelengths of the visible spectrum of the reductase solution revealed that the reductase contained flavin as a prosthetic group. Therefore it is considered that a hydrogen atom of NADPH was first transferred to the flavin moiety of the reductase, and then the hydrogen atom was rapidly exchanged for one in the medium before its direct transfer to the substrate.

Catalysis↗
PubMed1982

Studies on the metabolism of unsaturated fatty acids. VIII. Induction of 2,4-dienoyl-CoA reductase in Escherichia coli on the addition of unsaturated fatty acids.

2,4-Dienoyl-CoA reductase has been detected in crude extracts of E. coli. The reductase was shown to be induced many fold when the cells were grown in the presence of linoleic or oleic acid. The activity profile of the reductase on gel filtration was different from those of other enoyl reductases. These results suggest that there are two pathways even in E. coli for the degradation of cis-4-decenoyl-CoA, which is an intermediate in the beta-oxidation of linoleic acid, as recently proposed in rat liver.

Chemical Phenomena↗
PubMed1982

Studies on the metabolism of unsaturated fatty acids. IX. Stereochemical studies of the reaction catalyzed by trans-2-enoyl-coenzyme A reductase of Escherichia coli.

The steric course of the reaction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase from Escherichia coli was investigated. trans-2-[6,7,8-2H7]Octenoyl-CoA was synthesized as a substrate and incubated with partially purified trans-2-enoyl-CoA reductase in the presence of 4R- or 4S-[4-2H1]NADPH. The deuterium-labeled octenoyl-CoA was also incubated with the reductase in the presence of NADPH in 2H2O. Aliquots of octanoic acids formed were analyzed, after esterification, by gas chromatography-mass spectrometry (GC-MS) to demonstrate that the pro-4R hydrogen of NADPH was incorporated into the C-3 position of octenoyl-CoA and that hydrogen from water was introduced into the C-2 position of octenoyl-CoA. The remaining portions of octanoic acids isolated from the incubation mixtures were converted to their CoA esters by the action of acyl-CoA synthetase, and they were dehydrogenated by treatment with acyl-CoA oxidase, which had previously been shown to catalyze the anti-elimination of the pro-2R and pro-3R hydrogen atoms of acyl-CoA. The deuterium contents of the products were also analyzed by GC-MS, and the results indicated that the reduction catalyzed by NADPH-dependent trans-2-enoyl-CoA reductase occurred by an anti-addition of hydrogen via a 2-Re, 3-Re attack on the trans-double bond of the substrate.

Deuterium↗
PubMed1982

Studies on the metabolism of unsaturated fatty acids. X. Purification and some properties of 2,4-dienoyl-CoA reductase from Escherichia coli.

2,4-Dienoyl-CoA reductase has been separated from Escherichia coli grown in the presence of linoleic acid and purified to homogeneity. The enzyme has a molecular weight close to 50,000 as determined by gel filtration on Sephacryl S-200 Super-fine. The reductase was rather stable in a buffer containing citric acid and kept its full activity on heating at 55 degrees C for 10 min in the pH range of 5.5 to 6.5, but was completely inactivated on heating at 58 degrees C for 10 min. Phosphocellulose column chromatography revealed that the reductase was not involved in the multi-enzyme complex (molecular weight of 260,000) of fatty acid oxidation.

Escherichia coli↗
PubMed1982

Studies on the metabolism of unsaturated fatty acids. XI. Alterations in the activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase and 2,4-dienyl-CoA reductase in rat liver mitochondria and peroxisomes by clofibrate.

Alterations in the activities of enoyl-CoA hydratase, 3-hydroxyacyl-CoA epimerase, and 2,4-dienoyl-CoA reductase in rat liver mitochondria and peroxisomes caused by clofibrate were compared in order to clarify the metabolic pathways for unsaturated fatty acids. Our results suggest that there are two pathways for fatty acids having (a) double bond(s) at the even-numbered carbon atom(s) both in mitochondria and in peroxisomes. One is the hydratase-epimerase participating pathways, and the other is the reductase participating pathway. It is considered that the former in mitochondria occurs mainly under normal conditions, and the latter becomes significant when the degradation of fatty acids is stimulated.

Animals↗