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Biomedical subjects

Y Miyake

Publications and source records attributed to Y Miyake.

At least 631 records · Page 35Linked to original sources
PubMed1982

Syntheses of 15 alpha- and 15 beta-carboxymethyltestosterone bovine serum albumin conjugates: characteristics of the antisera to testosterone.

Syntheses of 15 alpha- and 15 beta-carboxymethyltestosterone (15 alpha- and 15 beta-CMT) were investigated in order to prepare testosterone-bovine serum albumin conjugates for radioimmunoassays of testosterone. A mixture of 15 alpha- and 15 beta-bis(ethoxycarbonyl)methyl-3 beta-hydroxy-5-androsten-17-one (IIa and IIb) obtained by a reaction of 3 beta-hydroxy-5,15-androstadien-17-one (I) and sodium diethyl malonate was oxidized to afford a mixture of 15 alpha- and 15 beta-bis(ethoxycarbonyl)methyl-4-androstene-3, 17-dione (Va and Vb). After the separation by silica gel chromatography, each epimer obtained was hydrolyzed by acid, followed by decarboxylation, and selective reduction of the 17-ketone to give 15 alpha- and 15 beta-CMT. The antisera, generated in rabbits by immunization with the bovine serum albumin (BSA) conjugates of 15 alpha- and 15 beta-CMT, respectively, exhibited high specificity for testosterone.

Animals↗
PubMed1982

A spin trap study on anaerobic dehalogenation of halothane by a reconstituted liver microsomal cytochrome P-450 enzyme system.

In the presence of a spin trapping reagent, N-tert-butyl-alpha-phenylnitrone, anaerobic incubation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) with a reconstituted cytochrome P-450 enzyme system of rabbit liver microsomes exhibited an electron spin resonance spectrum containing signals of a nitroxide radical, and all components of the reconstituted system were necessary to produce the nitroxide radical. Formation of 2-chloro-1,1,1-trifluoroethane, the product of anaerobic dehalogenation of halothane by the reconstituted system, was inhibited by N-tert-butyl-alpha-phenylnitrone. These results indicate that a radical intermediate of halothane is produced during the anaerobic dehalogenation reaction, and that the radical is trapped by N-tert-butyl-alpha-phenylnitrone to form a N-tert-butyl-alpha-phenylnitrone radical adduct.

Anaerobiosis↗
PubMed1982

Activation of kidney prorenin by kidney cathepsin B isozymes.

Kidney protein was converted to a form of active renin by kidney cathepsin B isozymes. The three isozymes showed similar catalytic behavior for prorenin. The optimal pH for the activation was in the range of 4.0-5.0 and the reaction was completely inhibited by leupeptin. The molecular weight and the isoelectric point of the activated prorenin were 40,000 and pH 4.9. As a minor product, an activated prorenin having an isoelectric point of 5.2 was also produced.

Animals↗
PubMed1982

A spin label study on human low density lipoprotein.

Selective labeling with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)-maleimide of human serum LDL has been performed. The spin-labeled LDL exhibited an ESR spectrum containing signals of a strongly immobilized component only. The signals were completely reversible between 4 degrees C and 37 degrees C and fairly stable at each temperature. The spin-labeled LDL which was prepared by the usual method exhibited an ESR spectrum containing signals of both strongly immobilized and weakly immobilized components (5, 6). The latter was unstable above 25 degrees C and changed irreversibly. The strongly binding site showed higher affinity for the nitroxide radical than the weakly binding site, and two kinds of the strongly binding site were demonstrated kinetically. The rate of binding of the nitroxide radical to the two kinds of strongly binding site were estimated to be 4.7 x 10(4) M-1 . day-1 and 0.16 x 10(4) M-1 . day-1 at pH 7.4 and 4 degrees C, respectively. Both the strongly immobilized and weakly immobilized radicals were reduced with ascorbate at the same rate. It was also shown on gel filtration of the SDS-treated LDL derivatives that the strongly immobilized component was on the apoprotein B moiety, whereas either noncovalent binding to LDL or binding to some small molecular species other than protein was suggested for the weakly immobilized component.

Binding Sites↗
PubMed1982

A resonance Raman study on the reaction intermediates of D-amino acid oxidase.

Resonance Raman (RR) spectra of two reaction intermediates of D-amino acid oxidase with substrate analogs were obtained. The reaction intermediates studied were (1) the one in the aerobic oxidative reaction of the enzyme with beta-cyano-D-alanine and (2) the other in the reverse reductive reaction of the enzyme with chloropyruvate and ammonium. Both intermediates are characterized with the charge transfer absorption bands in the long wavelength region extending beyond 600 nm. The RR spectra of the two intermediates excited at 488.0 or 514.5 nm are those of oxidized flavin, which is consistent with our previous assumption that oxidized flavin is involved in these reaction intermediates. Relatively simple RR spectra were obtained for these intermediates with excitation at 632.8 nm which is within the region of the charge transfer bands. The resonance enhancement for the Raman lines around 1585 and 1350 cm-1 for either of the intermediates with excitation in the region of the charge transfer bands suggests that the charge transfer interaction involves the N(5)-C(4a) region extending to the C(10a)-N(1)-C(2) region of the isoalloxazine nucleus. The Raman line at 1657 cm-1 for the intermediate with chloropyruvate and ammonium was assigned to C = N of an imino acid from the isotopic frequency shift upon 15N-substitution. The assignment substantiates our previous conclusion that the intermediate involves an imino acid, alpha-imino-beta-chloropropionate.

Aerobiosis↗
PubMed1982

Purification and characterization of prorenin from porcine kidney.

Prorenin was purified extensively from porcine kidney by a procedure involving extraction at pH 6.5 in the presence of protease inhibitors, ammonium sulfate fractionation, affinity chromatography on pepstatin-aminohexyl-Sepharose, acetone fractionation, chromatography on DEAE-cellulose, gel filtration on Ultrogel AcA 44, chromatography on concanavalin A-Sepharose, and chromatography on CM-Sepharose. The resulting preparation was essentially homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide-gel electrophoresis. The molecular weight of the preparation was estimated to be 39,000 by SDS-gel electrophoresis and 44,000 by gel filtration on Ultrogel AcA 44, and the isoelectric point was at pH 5.1. The purified prorenin was stable for 60 min at 37 degrees C between pH 3.0 and 9.0 and on storage for 4 weeks at--80 degrees C. The prorenin did not show any renin activity which appeared after trypsinization. The molecular weight of trypsin-activated prorenin was estimated to be 40,000 by gel filtration on Ultrogel AcA 44. The specific renin activity was 2.06 mg angiotensin I/mg of prorenin per h, and the optimal pH for the renin activity was in the pH region from 6.0 to 6.5 with hog and rat plasma angiotensinogens as the substrates. Two distinct isoelectric points were identified at pH 4.9 and pH 5.0 with the trypsin-activated prorenin.

Animals↗
PubMed1982

Interaction and electron transfer between cytochrome b5 and cytochrome P-450 in the reconstituted p-nitroanisole O-demethylase system.

The interaction and electron transfer between cytochrome b5 and cytochrome P-450B1 were investigated using the reconstituted p-nitroanisole O-demethylase system. Apocytochrome b5 was prepared from detergent-solubilized cytochrome b5 by the acid-butanone method. The apocytochrome b5 thus obtained has been substituted with several metalloporphyrin derivatives. The reconstituted system containing cytochrome b5 substituted with heme derivatives such as proto-, meso-, and deuteroheme exhibited demethylation activity at the maximum turnover rates of 94, 58, 30%, respectively, compared to that containing the native cytochrome b5, while neither apocytochrome b5 nor cobaltic protoporphyrin-cytochrome b5 displayed the activity. Kinetic analysis showed the formation of a 1:1 complex between cytochrome P-450B1 and each of these substituted cytochrome b5's, except for cobaltic protoporphyrin-cytochrome b5; the affinities differed with the cytochrome b5 species used. The synergistic effect with the addition of the NADH-linked electron transport system was more remarkable at the lower reduction levels of cytochrome b5 in the steady state. Interaction between the components involved in NADH- and NADPH-linked electron transport systems was modulated by the existence of Triton X-100. The optimal concentration in the reconstituted system for the demethylation was observed at around 0.03% of Triton X-100, where the reduction rates for cytochrome b5 and cytochrome P-450B1 by the respective reductases were maximal. These results indicate that the two electron transport systems are closely coupled and exhibit the demethylase activity.

Animals↗
PubMed1982

Studies on the reaction mechanism of NADPH-adrenodoxin reductase with NADPH.

The existence of a multi-component equilibrium involving a reversible reaction was demonstrated in the anaerobic reaction of NADPH-adrenodoxin reductase or ferredoxin-NADPH reductase; EC 1.18.1.2 (AdR) with NADPH or in the photoreduction of AdR in the presence of NADP+ and EDTA. After anaerobic reduction of AdR with excess NADPH, not only the fully reduced state but also other states of the enzyme were demonstrated as a mixture of characteristic absorption spectra of a flavin semiquinone with a peak at 575 nm and a shoulder around 625 nm and a charge transfer complex between NADP+ and the fully reduced AdR with a broad absorbance in a longer wavelength region. The amount of oxidized form in the reaction mixture was only a trace judging from the absorbance at 450 nm. The oxidized form was observed in addition to the other spectral species when the concentration of NADPH was limited. The spectrum of the AdR reduced with excess NADPH changed reversibly between 2 degrees C and 29 degrees C with isosbestic points at 427 nm and 667 nm; the semiquinone form increased at the expense of the charge transfer complex at lower temperatures, and vice versa at higher temperatures. The anaerobic photoreduction of AdR in the presence of NADP+ and EDTA produced the same spectral species as seen in the NADPH-reduced AdR, and the spectrum of the photoreduced AdR also changed reversibly between 1 degree C and 33 degrees C in a similar way to that of the NADPH-reduced AdR; the oxidized form changed in parallel with the semiquinone form. It was also found that NADPH was produced continuously during the photoirradiation even after the spectral changes had ceased. The results show that anaerobic reduction of AdR with NADPH or the photoreduction of AdR in the presence of NADP+ and EDTA produces a multi-component equilibrium and the equilibrium is dependent upon the concentration of NADPH and the temperature. Attainment of the equilibrium involves not only the forward reaction of AdR with NADPH but also the reverse reaction of the fully reduced AdR with NADP+.

Anaerobiosis↗
PubMed1982

Purification and properties of glucosyltransferase responsible for water-insoluble glucan synthesis from Streptococcus mutans.

A glucosyltransferase responsible for water-insoluble glucan synthesis was purified from the culture fluids of Streptococcus mutans 6715-15 strain by column chromatography on Toyopearl HW-60 and subsequently on hydroxyapatite. The enzyme preparation gave a single band on analysis by polyacrylamide gel electrophoresis. The pH dependency of the activity showed two optimal peaks at 5.8 and 7.3 and the Km values for sucrose were 1.4 and 3.3 mM at the respective optimal pHs. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 180,000. Although the enzyme scarcely synthesized water-insoluble and water-soluble glucans from sucrose, water-insoluble glucan formed from sucrose in the presence of dextran T10 consisted of over 93% alpha-1, 3-glucosidic linkage. Analysis of the structure of water-insoluble glucan indicated that the enzyme catalyzed the formation of branch points in alpha-1,6-glucan (dextran) and transferred the glucosyl moiety of sucrose to the C-3 position of the branching glucose residue of dextran. Since this enzyme has not yet been registered, we named it mutansynthetase (EC 2.4.1.?).

Chemical Phenomena↗