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Biomedical subjects

R Sato

Publications and source records attributed to R Sato.

At least 469 records · Page 26Linked to original sources
PubMed1975

Magnetic circular dichroism studies of hepatic microsomal cytochrome P-450.

Magnetic circular dichroism (MCD) spectra have been measured for cytochrome P-450 (P-450) purified from phenobarbital-induced rabbit liver microsomes. The temperature dependence of some of the MCD spectra has also been determined. The MCD spectrum of oxidized P-450 seems to suggest that it is in a state intermediate between the ferric low-spin states. Model experiments suggest that this anomaly arises from the coordination of a thiolate anion to the heme. Reduced P-450 shows a very peculiar MCD spectrum; the spectrum as well as its temperature dependence suggest that the heme in reduced P-450 is a "mixture" in terms of redox and/or spin states. The MCD spectrum of the CO complex of reduced P-450 exhibits an apparent Faraday A term around 450 nm which consists of about 50% C term and 50% the other terms, indicating that it is not in a purely ferrous low-spin state. The CO complex of reduced cytochrome P-420 (P-420), on the other hand, shows an MCD spectrum characteristic of a ferrous low-spin heme. It is suggested from model experiments that the thiolate anion coordinates to the heme trans to CO in the P-450-CO complex. The Soret region of the MCD spectrum of the EtNC complex of reduced P-450 is characterized by two apparent A terms around 430 and 455 nm, whereas that of the corresponding complex of P-420 has only one apparent A term around 434 nm.

Animals↗
PubMed1975

Ascorbate-synthesizing system in rat liver microsomes. II. A peptide-bound flavin as the prosthetic group of L-gulono-gamma-lactone oxidase.

L-Gulono-gamma-lactone oxidase [EC 1.1.3.8] was purified 80-fold from rat liver microsomes. In confirmation of our previous finding with a cruder preparation, the purified enzyme was shown to contain an L-gulono-gamma-lactone-reducible pigment as a prosthetic group. This pigment was not liberated from the protein by acid ammonium sulfate, 10% trichloroacetic acid or 2 M area, but was effectively released by proteolytic digestion. The pigment thus released showed a reduced-minus-oxidized difference spectrum characteristic of a flavin compound. The pigment was liberated from a trichloroacetic acid-treated preparation of the enzyme by pronase digestion and purified by Florisil column chromatography and paper chromatography. The absorption spectrum as well as the fluorescence emission and excitation spectra of the purified pigment indicated that it was actually a flavin peptide. It was, however, different not only from FMN but also from flavin peptides isolated from other sources such as succinate dehydrogenase [EC 1.3.99.1] and monoamine oxidase [EC 1.4.3.4] as regards the pH dependence of fluorescence intensity and the Rf value on thin-layer chromatography. A preliminary analysis showed that the purified flavin compound contained several amino acid residues. Alkaline photolysis of the purified flavin peptide suggested that the isoalloxazine ring of the flavin is involved in its binding to the peptide. The hypsochromic shift of the absorption peak in the near-ultraviolet region suggested further that the linkage between the flavin and the peptide may be mediated by the 8-methyl group of the isoalloxazine nucleus. It can be concluded that the prosthetic group of gulonolactone oxidase is a flavin which is covalently bound to the enzyme protein.

Alcohol Oxidoreductases↗
PubMed1975

Effect of metal ions in the culture medium on the stearoyl-coenzyme A desaturase activity of Mycobacterium phlei.

A particulate fraction prepared from Mycobacterium phlei grown in a metal-deficient medium exhibited a greatly reduced activity of stearoyl-CoA desaturase compared to that from normally grown cells. Metal deficiency, however, had no effect on the FAD-dependent NADPH-cytochrome C reductase activity, which has been suggested to participate in the desaturation process. When the cells were grown in the deficient medium supplemented with both Fe2+ and Mg2+, the desaturase activity was restored to the normal level. Supplementation with Mg2+ alone promoted growth but did not restore the desaturase activity, whereas Fe2+ alone did cause a significant restoration. Among the various metal ions tested, only Fe2+ and Fe3+ enhanced the formation of desaturase activity in the deficient medium. When added to the assay medium in vitro, Fe2+ and Fe3+ did not stimulate the desaturase activity of the particulate fraction from the deficient cells. Cultivation in the metal-deficient medium had essentially no effect on the levels of cytochromes in the particulate fraction, but dramatically decreased the non-heme iron content and the amount of a high-spin ferric species exhibiting an ESR signal at g=4.3. No labile sulfur could be detected in the normal or metal-deficient particulate fractions. It is concluded that the presence of iron ions in the culture medium is necessary for the synthesis and/or assembly of the terminal portion of the desaturase system.

Culture Media↗
PubMed1975

Purification and properties of the intact form of NADH-cytochrome b5 reductase from rabbit liver microsomes.

NADH-cytochrome b5 reductase [EC 1.6.2.2] has been solubilized with Triton X-100 and purified to homogeneity from rabbit liver microsomes. The purified enzyme is essentially free of the detergent and phospholipids and exists in aqueous media as an oligomeric aggregate of about 13 S. Its monomeric molecular weight is about 33,000 and 1 mole of FAD is associated with 1 mole of the monomeric unit. The enzyme catalyzes the reductions by NADH of ferricyanide and 2,6-dichlorophenol indophenol at an activity ratio of 1 : 0.09. Although the intact form of cytochrome b5 is a poorer electron acceptor than its hydrophilic fragment for the purified flavoprotein, electron transfer from the reductase to the intact cytochrome can be markedly stimulated by detergents or phospholipids, which also cause profound enhancement of the NADH-cytochrome c reductase activity reconstituted from the reducatse and cytochrome b5. Upon digestion with trypsin [EC 3.4.21.4], the ability of the reductase to form an active NADH-cytochrome c reductase system with the intact form of cytochrome b5 and Triton X-100 is rapidly lost. This loss of the reconstitution capability can be prevented by preincubation of the reductase with phosphatidylcholine liposomes. Trypsin digestion also results in the cleavage of the reductase molecule to a protein having a molecular weight of about 25,000 and a smaller fragment. The purified flavoprotein can bind to liver microsomes, liver mitochondria, sonicated human erythrocyte ghosts, and phosphatidylcholine liposomes. The reductase solubilized directly from liver microsomes by lysosomal digestion however, is devoid of membrane-binding capacity. It is concluded that the intact form of NADH-cytochrome b5 reductase is an amphipathic protein and its hydrophobic moiety, which is removable by lysosomal digestion, is responsible for the tight binding of the reductase to microsomes and for its normal functioning in the membrane.

Animals↗