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Biomedical subjects

T Yamano

Publications and source records attributed to T Yamano.

At least 199 records · Page 11Linked to original sources
PubMed1985

Light and electron microscopic immunohistochemistry on the localization of cytochrome P-450 of the side chain cleavage system and of cytochrome P-450 of 11 beta-hydroxylase in the bovine adrenal cortical cells.

In order to know the mechanism of steroid hormone biosynthesis, the localization of cytochromes P-450 of cholesterol side-chain cleavage enzymes (P-450SCC) and P-450 of 11 beta-hydroxylase (P-450(11)beta) were studied in bovine adrenal glands by light as well as electron microscopic immunohistochemistry, using monoclonal antibodies. With light microscopy the cytoplasm of the glomerulosa cells was faintly immunostained, while that of the fasciculata-reticularis cells was intensely immunostained by both monoclonal antibodies for cytochrome P-450SCC and cytochrome P-450(11)beta, though the capsular connective tissue cells and the adrenal medullary cells were entirely negative for these reactions. Electron microscopic immunohistochemistry revealed that the positive reaction products for cytochromes P-450 were present on the matrix side of the inner mitochondrial membrane including the tubulovesicular cristae of the cortical cells, and especially of the fasciculata and reticularis cells. The present results indicate that both cholesterol side-chain cleaving enzymes and 11 beta-hydroxylase are present in the inner mitochondrial membrane of bovine adrenal cortical cells.

Adrenal Cortex↗
PubMed1985

Monoclonal antibodies against bovine adrenal cytochrome P-450(11 beta) and cytochrome P-450SCC. Their isolation, characterization and application to immunohistochemical analysis of adrenal cortex.

Seven monoclonal antibodies directed against bovine adrenocortical cytochrome P-45011 beta were isolated. They were found to be immunoglobulins each having distinct affinity to the antigen. Five clones had IgG1 heavy chains, whereas the other two had IgG2b heavy chains. All the clones had kappa light chains. All the monoclonal antibodies recognized a protein among mitochondrial proteins of bovine adrenal cortex, whose mol. wt, 50,000, was the same as that of cytochrome P-45011 beta. Among the monoclonal antibodies isolated, monoclonal antibody 258 recognized a protein of Mr 50,000 in rat adrenal mitochondria, a protein of Mr 47,000 in pig adrenocortical mitochondria, a protein of Mr 50,000 in guinea pig adrenal mitochondria, a protein of Mr 55,000 in rabbit adrenal mitochondria and a protein of Mr 50,000 in human adrenocortical mitochondria. These results suggest that each of these proteins, having a mol. wt around 50,000, recognized by monoclonal antibody 258 is very likely to be the cytochrome P-45011 beta of adrenocortical mitochondria of each animal species. Monoclonal antibody 42718 inhibited by 50% the steroid hydroxylation activity of cytochrome P-45011 beta. In addition to the monoclonal antibodies against cytochrome P-45011 beta, three monoclonal antibodies directed against bovine adrenocortical cytochrome P-450SCC were also isolated and characterized. Immunohistochemical staining of bovine adrenal cortex with the use of these two kinds of monoclonal antibodies revealed that the contents of both cytochrome P-45011 beta and cytochrome P-450SCC were greater in the zona fasciculata and zona reticularis than in the zona glomerulosa. Electron microscopical observation of immunoperoxidase-stained preparations confirmed the presence of these cytochromes in the mitochondria.

Adrenal Cortex↗
PubMed1984

18,19-Dihydroxydeoxycorticosterone, a new metabolite produced from 18-hydroxydeoxycorticosterone by cytochrome P-450(11) beta. Chemical synthesis and structural analysis by 1H NMR.

A new metabolite was produced from 18-hydroxydeoxycorticosterone by the cytochrome P-450(11) beta linked hydroxylase system purified from bovine adrenocortical mitochondria. It was identified as 18,19-dihydroxydeoxycorticosterone by chemical synthesis on the basis of high-performance liquid chromatography, gas chromatography-mass spectrometry, and proton nuclear magnetic resonance (1H NMR) spectroscopy, and detailed structural analysis of it was performed by 1H NMR spectroscopy. The methylene protons at the C-19 position of the steroid were nonequivalent and coupled with each other, having a coupling constant of 10.6 Hz. These protons had different coupling constants, 6.7 and 3.4 Hz, for the hydroxy proton at the C-19 position. Due to these couplings, the signals of the methylene protons were observed around 3.9 ppm as two double doublets. The methylene protons at the C-21 position were also nonequivalent, having a coupling constant of 11.1 Hz. Coupling constants between these methylene protons and the hydroxy proton at the C-21 position were 8.2 and 4.2 Hz, respectively. These results indicate that both hydroxymethyl groups at the C-19 and C-21 positions do not freely rotate in chloroform solution. The signals of hydroxy protons at the C-19 and C-21 positions were found at 1.25 and 1.87 ppm, respectively, by means of decoupling of the corresponding methylene protons. The hydroxy proton at the C-18 position was found to scarcely couple with any proton. This fact suggests that this hydroxy group is linked to the C-20 position, making a hemiketal bridge between the C-18 and the C-20.

18-Hydroxydesoxycorticosterone↗
PubMed1984

Aldosterone biosynthesis by a reconstituted cytochrome P-45011 beta system.

[3H]Corticosterone was incubated with cytochrome P-45011 beta purified to electrophoretic homogeneity from bovine adrenocortical mitochondria, and the reaction products were analyzed by high performance liquid chromatography. The production of aldosterone (21.2 pmol/nmol P-450/min) and 18-hydroxycorticosterone (1.17 nmol/nmol P-450/min) was observed. When lipidic extracts from mitochondria of bovine adrenocortical zona glomerulosa were added to the reaction mixture, the rate of production of aldosterone was increased 28-fold. When [3H]18-hydroxycorticosterone was incubated with cytochrome P-45011 beta, the amount of aldosterone produced was 55.7 pmol/nmol P-450/min in the absence of the lipidic extracts and the enhancing effect of the lipidic extracts was 4-fold.

18-Hydroxycorticosterone↗
PubMed1984

Purification of intracellular phospholipase A2 from rat spleen supernatant by reverse-phase high-performance liquid chromatography.

Intracellular phospholipase A2 was purified to homogeneity from rat spleen supernatant by reverse-phase high-performance liquid chromatography with a trifluoroacetic acid-acetonitrile solvent system. The method simplified the purification procedure, which includes three consecutive chromatographic steps. The recovery of the enzyme activity was greater than 70% with an about 23,000-fold purification. The solvent system did not affect the catalytic properties of the enzyme. Phospholipases A2 from rat spleen, human pancreatic juice, and porcine pancreas were eluted in that order from a column of octadecasilyl silica gel in a similar concentration range of acetonitrile. This result suggests that the phospholipases A2 examined have similar hydrophobicities. This method may be applicable to the purification of phospholipases A2 from other sources.

Animals↗
PubMed1984

Gel chromatographic evidence for the participation of the higher polymers in the self-association system of a flavoenzyme D-amino acid oxidase.

The self-association of subunits of D-amino acid oxidase holoenzyme was studied by high-speed gel filtration with a short column of TSK-GEL G3000 SW in 0.1 M sodium pyrophosphate (pH 8.3) at 25 degrees C. Over the range of the peak concentrations of 0.009-4.45 mg/ml in the presence of FAD the apparent Stokes radii increased with an increase of the concentrations and did not level off. The largest value obtained in this study was 61.5 A. This would correspond to that calculated for the hexamer with linear subunit arrangement which has the largest Stokes radius among the various arrangements. These results provide the first gel chromatographic evidence that the higher polymers greater than the dimer participate in the self-associating system of the enzyme.

Animals↗
PubMed1984

Chemical modification of proteins: plotting of the remaining activity against the number of residues modified.

In the study of chemical modification of proteins, it has been a common practice to plot the fractional remaining activity against the number of residues modified per protein molecule. Extrapolation of the initial, nearly linear portion of the curve to the axis giving numbers of residues has often been presumed to specify the actual number of critical groups modified, i.e., the stoichiometry of the modification and the inactivation reactions. However, this extrapolation method is not generally applicable (Horiike, K. & McCormick, D.B. (1979) J. Theor. Biol. 79, 403-414). This paper describes further examination of the underlying theoretical framework of the extrapolation method. The properties and features of the extrapolated values are considered and presented with numerical and graphical examples. And the theoretical conditions with which the extrapolated value gives the number of essential residues are derived.

Amino Acids↗
PubMed1984

A 13C-NMR study on the interaction of riboflavin with egg white riboflavin binding protein.

The interaction between riboflavin and riboflavin binding protein (RBP) was studied by 13C-NMR spectroscopy. The 13C-NMR spectra of riboflavin selectively enriched at the 2-, 4-, 4a-, and 10a-positions and of (3-[13C]methyl)riboflavin were measured both in the free and RBP-bound forms. The 13C signals of 13C-enriched riboflavin or 3-methylriboflavin bound to RBP are broader than those of the free form, reflecting the restriction of flavin mobility. The 2-, 4-, and 10a-13C signals of riboflavin show no pH-dependent shift in the neutral to acidic pH region either in the bound or free form but the 4a-13C signal of bound riboflavin shifts to lower field in the acidic pH region while that of the free form remains unshifted. The 2-, 4-, 4a-, and 10a-13C signals of free riboflavin exhibited pH-dependent change in the alkaline pH region with a pK value of about 10, in association with the N(3)-H deprotonation. The pH titration profile of the 2-, 4-, and 4a-13C signals of bound riboflavin indicates that the pK of N(3)-H is shifted substantially to the alkaline side when riboflavin is bound to RBP. The 3-methyl-13C signal of 3-methylriboflavin shows no pH-dependent shift whether the compound is free or bound to RBP. The binding of riboflavin and 3-methylriboflavin was also studied spectrofluorometrically. The analysis of the pH dependence of the association constant revealed that one ionizable group in RBP with pK of about 5 and N(3)-H of riboflavin play important roles in the binding. We conclude that RBP preferentially binds the neutral, i.e., N(3)-protonated, form of riboflavin and that the neutral form in turn is stabilized by the hydrophobic environment of RBP surrounding the N(3) region of the bound riboflavin molecule.

Carbon Isotopes↗
PubMed1984

Rat pancreatic phospholipase A2: purification, characterization, and N-terminal amino acid sequence.

Phospholipase A2 was purified from rat pancreas by heat treatment of the homogenate and the sequential use of DEAE-Sepharose chromatography, CM-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (HPLC). Prophospholipase A2 was not separated from the phospholipase A2 by CM-Sepharose chromatography under the conditions used, but it was well resolved by the reverse-phase HPLC. The enzyme was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and on analytical HPLC, and its molecular weight was estimated to be 14,000. The enzyme specifically hydrolyzed an acylester bond at the sn-2-position of the phospholipid examined. The purified enzyme has a pH optimum in the range of pH 9.5 to 10.5 and requires the presence of Ca2+ (3 mM) and sodium deoxycholate (0.1%) for optimum activity. The amino acid sequence of the first 32 residues in the N-terminal region of the enzyme was determined. The sequence revealed a marked homology with those of pancreatic phospholipases A2 of man, pig, ox, and horse, and porcine intestinal phospholipase A2 reported previously.

Amino Acid Sequence↗
PubMed1984

Cytochrome P-450-dependent oxidative cleavage of 1-(tetrahydro-2-furanyl)-5-fluorouracil to 5-fluorouracil.

Cytochrome P-450-dependent oxidative cleavage of 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT) was investigated in a reconstituted system containing purified phenobarbital-inducible cytochrome P-450 (P-450(1)) or 3-methylcholanthrene-inducible cytochrome P-450 (P-448(1)). FT was converted into 5-fluorouracil (5-FU) in the reconstituted system, and its rate was 71 pmol 5-FU formed/min/nmol P-4501 and 45 pmol 5-FU/min/nmol P-448(1). Cytochrome P-450, NADPH-cytochrome P-450 reductase and NADPH were required for 5-FU production. Inhibitors of cytochrome P-450 such as carbon monoxide and metyrapone markedly decreased the rate. FT was found to interact with the purified cytochrome P-450, causing a reverse type I spectral change. From these observations, we concluded that the hepatic microsomal cytochrome P-450-dependent mixed function oxidase system participates in the oxidative cleavage of FT.

Animals↗
PubMed1984

Cholesterol accumulation in adrenocortical mitochondria after ACTH-stimulation.

Rat adrenocortical cell suspensions (10(6) cells) were incubated with ACTH (40 nM) in 2 ml of Krebs-Ringer bicarbonate buffer for 90 min. About 42 nmol of corticosterone and 14 nmol of 18-hydroxydeoxycorticosterone were generated and released into the medium. Aminoglutethimide at 50 microM inhibited the steroidogenesis to 16%. Mitochondrial pellets were prepared from the cells incubated in the absence, or in the presence, of ACTH and aminoglutethimide, and cholesterol content was determined. The mitochondria of the cells incubated without the drugs contained 25.2 micrograms cholesterol/mg protein. Cholesterol content increased by 10% in the mitochondria of the ACTH-stimulated cells. The mitochondria of the cells incubated in the presence of both ACTH and aminoglutethimide contained 143% of cholesterol compared to those of the nontreated cells. When rats were subjected to ether stress after aminoglutethimide pretreatment, cholesterol content of the mitochondrial fraction increased to about 200% compared to that of the control rats. These results suggest that a cholesterol pool exists in the adrenocortical mitochondria and that the amount increases during the steroidogenic stimulation of the cells. The mitochondria were fixed with filipin-containing fixative and examined by freeze-fracture electron microscopy. Accumulations of filipin-cholesterol complexes were observed in the inner membrane of the mitochondria as protuberances or pits 25 nm in diameter.

Adrenal Cortex↗
PubMed1984

Effect of phospholipid on aldosterone biosynthesis by a cytochrome P-450(11) beta-reconstituted system.

Effect of phospholipid on aldosterone synthesis catalyzed by a cytochrome P-450(11)beta-reconstituted system was examined. Corticosterone was incubated with P-450(11)beta in the presence of phosphatidylcholine, phosphatidylethanolamine, cardiolipin, phosphatidylinositol, or phosphatidylserine. These phospholipids stimulated the rate of aldosterone- and 18-hydroxycorticosterone-synthesis, although the mode of stimulation by neutral phospholipids was different from that by acidic phospholipids. In the presence of a phospholipid mixture containing the lipids in the same molar ratios as found in adrenocortical mitochondria, the rate of production of aldosterone increased 10-fold over the rate without the lipids, and that of 18-hydroxycorticosterone increased 3-fold. The maximal synthetic rates of aldosterone and 18-hydroxycorticosterone were 2 and 5 nmol/nmol P-450/min, respectively.

18-Hydroxycorticosterone↗
PubMed1983

Hepatic microsomal cytochrome p-450-dependent N-demethylation of methylguanidine.

Cytochrome P-450-dependent N-demethylation of methylguanidine, a uremia toxin, was investigated. Methylguanidine was stoichiometrically converted into equal amounts of guanidine and formaldehyde by aerobic incubation with phenobarbital-induced microsomes and NADPH. The guanidine formation in the incubation mixture followed Michaelis-Menten kinetics and required the presence of molecular oxygen and NADPH. Methimazole, a non-formaldehyde-producing substrate specific for FAD-containing monooxygenase, did not inhibit significantly formaldehyde formation, suggesting that microsomal FAD-containing monooxygenase does not play a significant role in N-demethylation of methylguanidine. The direct involvement of cytochrome P-450 in the N-demethylation is supported by the observations that addition of methylguanidine to purified cytochrome P-450 preparation caused a type I spectral change and that inhibitors of cytochrome P-450, such as carbon monoxide and metyrapone, markedly decreased the rate of demethylation. Neither superoxide anion nor hydrogen peroxide was directly involved in the demethylation reaction. In addition, guanidine formation was observed in the reconstituted system containing purified cytochrome P-450. Thus, these findings indicate that the hepatic microsomal mixed function oxidase system catalyzes N-demethylation of methylguanidine to guanidine.

Animals↗
PubMed1983

A prominent feature of the conversion of P-450 to P-420 of cytochrome P-450B1 among the cytochrome P-450 isozymes.

Conversion of cytochrome P-450 to P-420 was observed with the use of three isozymes of P-450, P-450PB, P-450MC and P-450B1. The last one, which was isolated and characterized in our laboratories, is the cytochrome P-450 with high affinity for cytochrome b5. Of these isozymes, cytochrome P-450B1 is predominantly fast in the rate of conversion from P-450 to P-420 in the reduced state under carbon monoxide. p-Nitroanisole, which is the substrate of P-450B1 for demethylation, accelerated the conversion, whereas the effects of the compound on the rate of conversion of the other P-450S were small. The effect of cholate on the conversion was distinct and rapid but not very selective among the isozymes. Stabilization with glycerol for prevention of the conversion was found to be effective, for any of these isozymes. No remarkable difference was observed in the stability of the oxidized state among these isozymes when detected in the CO-reduced form. The rates of the reaction from the oxidized to the CO-reduced form were measured with these isozymes. The rate of P-450B1 was the highest in both the medium with glycerol and that without glycerol. Circular dichroism was measured with respect to conversion of P-450 to P-420. The absorption at 450 nm was related to the significant circular dichroism, while the increased absorption at 420 nm due to the conversion was not accompanied by distinct circular dichroism. These data support the concept that the heme vicinity of cytochrome P-450B1 is more labile in the structure of the reduced form under carbon monoxide than those of the other isozymes.

Animals↗
PubMed1983

19-Hydroxylation of 18-hydroxy-11-deoxycorticosterone catalyzed by cytochrome P-45011 beta of bovine adrenocortex.

When 18-hydroxy-11-deoxycorticosterone was incubated with adrenocortical mitochondria fortified by adding exogenously adrenodoxin reductase and adrenodoxin in the presence of an NADPH-generating system, the steroid was converted to two products which were separated by high performance liquid chromatography. The retention time of one product was identical with that of 18-hydroxycorticosterone, an expected product of 11 beta-hydroxylation, whereas the retention time of the other was not coincident with those of any other corticosteroids examined. Production of the unidentified substance was also shown when 18-hydroxy-11-deoxycorticosterone was incubated with purified cytochrome P-45011 beta and its electron transport system. The ratio of the two products was always constant, even when various incubation conditions were employed with regard to reaction time, the substrate concentration, and the cytochrome concentration. Structural determination of the unidentified product was conducted by mass spectrometry and 1H NMR spectrometry. The results of these analyses indicated that the substance was 18,19-dihydroxy-11-deoxycorticosterone, a hitherto unreported corticosteroid.

18-Hydroxydesoxycorticosterone↗