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Biomedical subjects

Y Miyake

Publications and source records attributed to Y Miyake.

At least 685 records · Page 38Linked to original sources
PubMed1979

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.

3-Hydroxysteroid Dehydrogenases↗
PubMed1979

Pigmentary paravenous chorioretinal degeneration: fundus appearance and retinal functions.

A case of pigmentary paravenous chorioretinal degeneration initially diagnosed as retinitis pigmentosa is presented. The focal macular visual evoked response (VER) was normal. Both rod and cone components in the electroretinogram showed reduced amplitude with normal peak time. The psychophysical threshold in the dark adaptation was nearly normal in some parts of the retina and extremely elevated in other parts of the retina. Fundus appearance and retinal function studies suggest that the impaired area in the retina and choroid is localized in this disease.

Adult↗
PubMed1978

Identification of organic sulfur compounds transferred to fish from petroleum suspension.

The authors attempted to determine if the organic sulfur compounds usually contained in a crude oil could serve as a marker of oil pollution in fish. Eels (Anguilla Japonica Temminck et Schlegel) were maintained in a controlled laboratory environment of water with a suspension of crude oil. Gas chromatography-mass spectrometry and mass chromatography of eel flesh extract showed the presence of organic sulfur compounds of alkyl-(from mono- to pentametyl) benzothiophenes, dibenzothiophene and alkyl-(from mono- to trimethyl) dibenzothiophenes, and other organic sulfur compounds of alkyl-(from mono- to pentamethyl) naphthalenes.

Animals↗
PubMed1977

Simultaneous recording of electroretinogram and visual evoked response. Focal stimulation under direct observation.

A system has been tested that allows simultaneous recording of the retinal response (electroretinogram [ERG]) and the occipital response (visual evoked response [VER]) with focal photic stimulation of the retina under direct observation of the fundus. A helium-neon gas laser is used as a stimulus source. The laser is chopped either by a pen motor or a rotating disc. The laser is attached to a biomicroscope through which the examiner can observe the fundus of the subject during the entire recording session. The optically clear contact lens is made with a flat surface that neutralizes refraction due to the cornea, thereby allowing fundus observation by microscope. Two metal wires mounted inside and outside of the lens serve as the electrode for the ERG. Graticules consisting of concentric circles and radial lines are projected onto the subject's fundus, providing a pattern that the examiner can use to determine the exact location to be stimulated in the fundus. With proper adjustment of stimulus and background illumination, local ERG and VER can be recorded simultaneously by stimulating the macula.

Electroretinography↗
PubMed1977

Analysis of the reactivity of microsomal cytochrome P-450 toward drugs and exogenous ligands.

Three forms of cytochrome P-450 in rat liver microsomes (Comai, K. and Gaylor, J.L. (1973) J. Biol. Chem. 284, 4947-4955) were characterized as two types of P-450 in the membrane-bound state. One was reactive not only toward cyanide but also aniline, aminopyrine, and hexobarbital, and the other was less reactive toward cyanide and rather unreactive toward the drugs. Cyanide titrations of microsomes in the presence and absence of the drugs were performed spectrophotometrically for analysis of the interactions. The affinity of cyanide for the less reactive P-450 was about twenty times less than that for the reactive P-450. About 40% of P-450 in the microsomal membrane was reactive and the rest was the less reactive form. The reactive P-450 involved two forms having different affinities for cyanide. The ratio of their amounts was 1:2. Triton WR-1339 interfered with cyanide binding to the reactive P-450 mainly. The relative amount of each form of P-450 as well as the dissociation constant of cyanide could be estimated by the present method, and the modes of interaction of the membrane-bound P-450 with the drugs and exogenous ligands were deduced.

Aminopyrine↗
PubMed1977

Effect of halide anions on the binding of FAD to D-amino acid oxidase and the tryptophanyl fluorescence of the apoenzyme.

The quenching of tryptophanyl fluorescence of native and denatured D-amino acid oxidase from hog kidney was measured. About 60% of the tryptophanyl fluorescence of the native apoenzyme was quenched by iodide at pH 8.3, and 25 degrees C. All of the tryptophanyl fluorescence of the apoenzyme in 6 M guanidine hydrochloride was quenched. The tryptophanyl fluorescence quenching of the holoenzyme by 1-methyl nicotinamide chloride was low in comparison with that of the apoenzyme. These results of the quenching experiments are discussed based on the intermolecular collision quenching mechanism. By measuring the fluorescence intensities of the tryptophanyl residues and FAD of the holoenzyme solution, and the fluorescence polarization of the holoenzyme solution containing halide anions such as iodide, bromide, chloride, or fluoride, we found that FAD dissociates from the holoenzyme in the presence of iodide, bromide, or chloride, and the ability to dissociate FAD from the holoenzyme decreases in order iodide, bromide, and chloride. However, fluoride seems to enhance the association reaction of FAD with the apoenzyme. These results were consistent with the visible absorption spectra and derivative spectra of free FAD and the holoenzyme in the presence and absence of halide anions.

Animals↗
PubMed1976

An oxygraphic method for the determination of cholesterol and measurement of the slow oxygen-consuming reactions of hepatic microsomes.

Oxygen-consuming reactions of cholesterol oxidase [EC 1.1.3.6] and microsomes were measured with a galvanic oxygen electrode which was attached to an offset amplifier for sensitive measurement of the reaction processes. The sensitivity of this oxygraphic method for detection of oxygen consumption was ten times greater than that of the usual method. The minimum rate of slow oxygen-consuming reactions which could be estimated was about 5 nmoles of oxygen per min, and the minimum amount of oxygen consumption which could be determined was also about 5 nmoles. An oxygraphic method for direct and rapid determination of cholesterol was demonstrated using one-twentieth the amount of cholesterol oxidase which is used for the colorimetric method. The processes of cyanide-suppressed beta-NADH-dependent oxygen consumption and cyanide-insensitive alpha-NADH-dependent oxygen consumption, which were difficult to follow by the usual method, were followed using a small amount of microsomes (less than 1mg protein/ml). Furthermore, the temporary cessation of alpha-NADH-dependent oxygen consumption caused by ferricyanide and the corresponding oxidation-reduction of reduced cytochrome b5 were followed in the presence of ADP. ADP did not inhibit the oxygen consumption. The results indicate that the oxygen consumption with alpha-NADH is due to electron transfer from alpha-NADH via NADH-cytochrome b5 reductase and cytochrome b5, in which the rate-determining step lies at some reaction after the reduction of cytochrome b5.

Adenosine Diphosphate↗
PubMed1976

Stereospecificity of conversion of uric acid into allantoic acid by enzymes of Canadida utilis.

1. Allantoinase [EC 3.5.2.5] was isolated from cells of Candida utilis and unpurified by chromatography on columns of DEAE-cellulose and Sephadex G-200 after treatment with urea to remove urate oxidase [EC 1.7.3.3.]. 2. The purified allantoinase catalyzed the hydrolysis of allantoin into allantoic acid. However, only half of the allantoin produced from uric acid by urate oxidase was converted. The rest of the allantoin was unchanged, and showed a negative optical rotation. 3. On the other hand, the combined action of crude urate oxidase and allantoinase resulted in nearly complete conversion of uric acid into allantoic acid. Furthermore, the unpurified allantoinase preparation hydrolyzed racemic allantoin to allantoic acid completely. 4. These results indicate that the urate oxidase produces racemic allantoin from uric acid and that the allantoinase attacks only allantoin of positive optical rotation. The results also suggest that allantoin racemase is present in the yeast cells.

Amidohydrolases↗
PubMed1976

Spectral intermediates during the reduction of hepatic microsomal cytochrome P-450.

The slow reduction of microsomal cytochrome P-450 by dithionite consists of an initial fast and then a slow phase. During reduction of aniline and azide complexes with cytochrome P-450, an intermediate spectrum developed in the fast phase and changed to that of the reduced form in the slow phase. Only the spectra in the slow phase had an isosbestic point. No intermediate spectrum detectable during reduction of the cyanide complex and native-cytochrome P-450. Carbon monoxide accelerated the reaction, causing complete reduction in the initial phase. The electron spin resonance spectrum of cytochrome P450 was greatly reduced in the initial phase of reduction with dithionite. These results indicate that reduction of the aniline and azide complexes of cytochrome P-450 involves two steps: first reduction of cytochrome P-450 and then some changes in reduced state. The aniline and cyanide difference spectra of reduced cytochrome P-450 showed peaks at 423 nm and 429 nm, respectively, while that of azide had a peak at 445 nm and a trough at 404 nm. An essay method to obtain the difference spectrum of reduced minus oxidized cytochrome P-450 using a spectral data processor is reported. The effects of other NADH-nonreducible pigments on the spectrum is eliminated by this procedure, provided these pigments are rapidly reduced by dithionite. Therefore, the spectrum obtained was slightly differed from that measured by the usual method, especially in the region of 425 nm.

Anilides↗
PubMed1975

Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes.

The stoichiometry of 4-methyl sterol oxidase has been investigated by concurrent assays of rates of oxygen consumption, oxidation of reduced pyridine nucleotide, and formation of steroid 4alpha-oic acid, which is the oxidized product of attack of 4-methyl sterol precursors of cholesterol. The basal, steroid-independent rates of oxidation of alpha-NADH and alpha-NADH-dependent oxygen consumption by rat liver microsomes are about 10 to 15% of the rates observed with beta-NADH. Thus, alpha-NADH is substituted for beta-NADH; alpha-NADH oxidation is observed spectrophotometrically. The slow rate of oxygen consumption is measured accurately with a galvanic oxygen electrode that is attached to an offset amplifier. For maximal velocity, 4alpha-hydroxymethyl-5alpha-cholest-7-en-3beta-ol is the steroid substrate, and oxidase activity is induced 2-fold with a dietary bile acid sequestrant. Under these conditions, accurate measurements are obtained for substrate-dependent increments, which are equal to or greater than basal, substrate-independent rates. For each equivalent of hydroxymethyl group oxidized to carboxylic acid, 2 eq each of oxygen and alpha-NADH are consumed. Thus, the stoichiometry is consistent with that expected for two sequential attacks of the 4alpha-hydroxymethyl group by an external mixed function oxidase. In addition to establishing the stoichiometry of the 4-methyl sterol oxidase, the results further demonstrate that the steroidal 4alpha-carboxylic acid is formed from the hydroxymethyl intermediate by catalysis of a mixed function oxidase rather than dehydrogenases.

Animals↗