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Y Funae

Publications and source records attributed to Y Funae.

At least 163 records · Page 9Linked to original sources
PubMed1989

Omega- and (omega-1)-hydroxylation of lauric acid and arachidonic acid by rat renal cytochrome P-450.

We resolved four cytochrome P-450s, designated as P450 K-2, K-3, K-4, and K-5, from the renal microsomes of untreated male rats by high-performance liquid chromatography (HPLC) and investigated the lauric acid and arachidonic acid hydroxylation activities of these fractions. P450 K-4 and K-5 had high omega- and (omega-1)-hydroxylation activities toward lauric acid. The ratio of the omega-/(omega-1)-hydroxylation activity of P450 K-4 and K-5 was 3 and 6, respectively. Also, P450 K-4 and K-5 effectively catalyzed the omega- and (omega-1)-hydroxylation of arachidonic acid. P450 K-3 was not efficient in the hydroxylation of either lauric acid or arachidonic acid. P450 K-2 had low omega- and (omega-1)-hydroxylation activities toward arachidonic acid, and efficiently catalyzed the hydroxylation of lauric acid at the (omega-1)-position only, not at the omega-position.

Animals↗
PubMed1989

Suppression of hepatic levels of an ethanol-inducible P-450DM/j by growth hormone: relationship between the increased level of P-450DM/j and depletion of growth hormone in diabetes.

The mechanism of the suppression of an ethanol-inducible cytochrome P-450 (P-450DM/j) by pituitary hormone has been studied in rats. The hepatic content of P-450DM/j protein quantitated by Western blots was low but was 2-fold higher in male than female untreated rats (75 and 34 pmol/mg of protein, respectively). The content was increased 2.6-fold (male) and 5.6-fold (female) by hypophysectomy and the sex-related difference was abolished. Treatment of hypophysectomized rats with human growth hormone (hGH), but not with prolactin, reversed the increased amounts of P-450DM/j protein. The hGH-induced suppression was more effective with the continuous infusion than intermittent injection. The hepatic level of P-450DM/j mRNA, determined by the use of a 23-mer oligonucleotide probe, was also changed by hypophysectomy and/or hGH-treatment, largely in parallel with the changes in the content of P-450DM/j protein and microsomal p-nitrophenol and aniline hydroxylations. These results suggest that growth hormone exerts the suppressive effect on P-450DM/j through a somatogenic receptor-mediated process. In another growth hormone-depleted condition, diabetes, the hepatic level of P-450DM/j mRNA was also increased to a level similar to that in hypophysectomized rats, but the protein content was 2- to 3-fold higher in diabetic than hypophysectomized rats. These results indicate, in addition to the reduction of serum growth hormone level, the presence of another stimulatory factor, which acts translationally or posttranslationally in livers of diabetic rats. On the other hand, coordinate changes in the level of P-450DM/j protein and the mRNA in hypophysectomized rats indicate that growth hormone acts rather directly and suppresses the level of P-450DM/j mainly at a pretranslational step in rat livers.

Animals↗
PubMed1989

Form-specific degradation of cytochrome P-450 by lipid peroxidation in rat liver microsomes.

Microsomal lipid peroxidation caused decreases in the activities of ethylmorphine N-demethylase and testosterone 6 beta-hydroxylase more markedly than aniline hydroxylase, p-phenetidine O-deethylase and testosterone 2 alpha- and 16 alpha-hydroxylases. Results of sodium dodecylsulfate-polyacrylamide gel electrophoresis of liver microsomes showed that proteins with molecular weights between 47kDa and 50kDa in liver microsomes were sensitively degraded by lipid peroxidation. Western-blot analysis of liver microsomes with antibodies to P-448-H, P-450 male and P-450 PB-1 which corresponded to P-450d, P-450h and P-450 PCN, respectively, showed that the amount of P-450 PB-1 was decreased more markedly by lipid peroxidation as compared to those of P-450 male and P-448-H. It seemed, therefore, likely that the stability of cytochrome P-450 to lipid peroxidation varies dependent on the forms of cytochrome P-450.

Animals↗
PubMed1988

Aminopyrine metabolism by multiple forms of cytochrome P-450 from rat liver microsomes: simultaneous quantitation of four aminopyrine metabolites by high-performance liquid chromatography.

Four aminopyrine metabolites generated by hepatic microsomes were simultaneously assayed by high-performance liquid chromatography. The metabolites were 4-monomethylaminoantipyrine (MAA), 4-aminoantipyrine (AA), 3-hydroxymethyl-2-methyl-4-dimethylamino-1-phenyl-3-pyrazoline-5-one (AM-OH), and one unidentified metabolite. MAA was the major metabolite generated by the microsomes; its formation was induced by phenobarbital but not by 3-methylcholanthrene. Female rats had lower N-demethylation activity of aminopyrine than male rats. The production of AA by microsomes was low. The formation of AM-OH was strongly induced by phenobarbital, but treatment with 3-methylcholanthrene reduced its formation. These differences in the microsomal aminopyrine monooxygenase activity are dependent on the relative amounts of the individual cytochrome P-450 isozymes. Therefore, we examined aminopyrine metabolism in a reconstituted system with purified cytochrome P-450s. P-450 UT-2 (P-450h) had high aminopyrine N-demethylation and hydroxylation activities, but P-450 F-2 (P-450i) had low N-demethylation activity and no hydroxylation activities, but P-450 F-2 (P-450i) had low N-demethylation activity and no hydroxylation activity. P-450 PB-4 (P-450b) and P-450 PB-5 (P-450e) had high aminopyrine hydroxylation activity and their N-demethylation activity also was high. The 3-methylcholanthrene-inducible forms P-450 MC-1 (P-450d) and MC-5 (P-450c) had aminopyrine N-demethylation activity but no hydroxylation activity. P-450 UT-4 (RLM2) is a unique form that produced a large amount of the unknown metabolite. P-450 UT-7 had the highest N-demethylation activity. Addition of cytochrome b5 to the reconstituted system enhanced the aminopyrine hydroxylation activities of P-450s UT-1, UT-2, PB-2, and PB-5. Also, the N-demethylation activities of P-450s UT-1, PB-1, PB-2, and MC-1 were increased by cytochrome b5. Metyrapone inhibited the catalytic activities of P-450s PB-4, PB-5, MC-1, and MC-5, and especially those of P-450s UT-4, and UT-7. The kinetics of the four major cytochrome P-450s (P-450 UT-2, UT-4, PB-4, and MC-5) for aminopyrine N-demethylation and hydroxylation activities were studied. P-450s PB-4 and UT-2 had similar Km values (0.50 and 0.62 mM, respectively) in aminopyrine N-demethylation activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminopyrine↗
PubMed1988

Induction of renal cytochrome P-450 in hepatic microsomes of diabetic rats.

We purified two forms of cytochrome P-450 which was induced in hepatic microsomes of diabetic male rates treated with streptozotocin. One of these corresponded to P-450j. The other form, designated P450 DM-2, had a minimum molecular weight 53000 and a CO-reduced absorption maximum at 452 nm. The P450 DM-2 efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was not efficient in metabolizing aminopyrine, 7-ethoxycoumarin, aniline, N-nitrosodimethylamine, or testosterone. The NH2-terminal sequence of P450 DM-2 was identical to that of P450 K-5, the major renal cytochrome P-450. Both forms gave very similar electrophoretic patterns of proteolytic digests. P450 DM-2 and P450 K-5 are closely related forms.

Amino Acid Sequence↗
PubMed1988

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics.

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids.

Animals↗
PubMed1988

Constitutive testosterone 6 beta-hydroxylase in rat liver.

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

A sex-specific form of cytochrome P-450 catalyzing propoxycoumarin O-depropylation and its identity with testosterone 6 beta-hydroxylase in untreated rat livers: reconstitution of the activity with microsomal lipids.

Characteristics of a typical male-dominant reaction, dealkylation of n-propoxycoumarin, in rat livers were studied in relation to microsomal testosterone 6 beta-hydroxylase. The depropylation was more than 10-fold higher in the liver of male than female adult rats, but the sex-related difference was eliminated by neonatal castration. Hypophysectomy of adult male rats, which decreased the rates of male-specific P-450-male-dependent reactions, increased the depropylation of propoxycoumarin, while the rate was decreased by either intermittent injection or continuous infusion of human growth hormone to hypophysectomized rats. With regard to age-related difference, microsomal depropylation was detectable at neonate and reached a maximal level at 14 to 20 d of age, but was abruptly diminished only in female rats at puberty. These changes are in good agreement with those of testosterone 6 beta-hydroxylation and the content of a male-specific P-450(6)beta-1/PB-1. In reconstituted systems using extracted microsomal lipids, P-450(6)beta-1/PB-1 and P-450-male catalyzed the depropylation of propoxycoumarin. However, the microsomal depropylation was inhibited by antibodies which recognize P-450(6)beta-1/PB-1, but not P-450-male. These results indicate that microsomal depropylation of propoxycoumarin is catalyzed mainly by a male-specific P-450(6)beta-1/PB-1 in livers of untreated rats.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1988

Purification and characterization of diabetes-inducible cytochrome P-450.

A diabetes-inducible form of cytochrome P-450, termed P-450DM, was purified to electrophoretical homogeneity (MW 51,000) by high-performance liquid chromatography from liver microsomes of diabetic rats induced with streptozotocin. The CO-reduced absorption maximum of P-450DM was at 452 nm and the oxidized heme iron appeared to be predominately in the high-spin state as deduced from the Soret maximum at 395 nm. P-450DM was active in aniline hydroxylation and N-nitrosodimethylamine demethylation. The dealkylation activity toward 7-ethoxycoumarin by P-450DM was much enhanced by the addition of cytochrome b5.

Animals↗
PubMed1987

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver.

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.

Amino Acid Sequence↗
PubMed1987

Purification and characterization of liver microsomal cytochrome P-450 from untreated male rats.

Different forms of cytochrome P-450 from untreated male rats were simultaneously purified to homogeneity using the HPLC technique. The absorption maximum, molecular weight, NH2-terminal sequence and catalytic activity of them were determined. The NH2-terminal sequences of six forms of cytochrome P-450 (designated P450 UT-1, UT-2, UT-4, UT-5, UT-7 and UT-8) indicate that these cytochrome P-450 isozymes are of different molecular species. The hydrophobicity values of the NH2-terminal sequences of P450 UT-1 and P450 UT-8 were lower than that of other forms. P450 UT-8 has the highest molecular weight, 54,000, of the six forms of P-450. P450 UT-2 was active in demethylation of benzphetamine, P450 UT-4 was active in the metabolism of 7-ethoxycoumarin and p-nitroanisole. P450 UT-1 and P450 UT-2 were active in the 2 alpha- and 16 alpha-hydroxylation of testosterone, whereas P450 UT-4 was active in the 6 beta-, 7 alpha- and 15 alpha-hydroxylation of the same steroid. We believe that P450 UT-1, P450 UT-7 and P450 UT-8 are as yet unrecognized forms of cytochrome P-450.

Amino Acid Sequence↗
PubMed1987

Genotoxic and mutagenic activation of aflatoxin B1 by constitutive forms of cytochrome P-450 in rat liver microsomes.

Cytochrome P-450-mediated activation of aflatoxin B1 (AFB1) to genotoxic and mutagenic products which subsequently cause induction of an umu gene expression in Salmonella typhimurium TA1535/pSK1002 has been studied in a rat liver microsomal or reconstituted monooxygenase system. Liver microsomes from male Sprague-Dawley rats had a 1.5-fold higher activity to catalyze AFB1 than did those from female rats. In addition, the activation was not increased in liver microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, a polychlorinated biphenyl mixture, or dexamethasone, suggesting that the constitutive forms of cytochrome P-450 have important roles for the activation of AFB1 in rat liver microsomes. Using 15 forms of cytochrome P-450 purified from liver microsomes of untreated and phenobarbital- and 3-methylcholanthrene-treated rats, three isozymes from untreated male rats and one isozyme from untreated female rats were found to have high reactivities in metabolizing AFB1 to genotoxic products. Cytochrome P-450 forms isolated from inducer-treated rats were relatively less active. The close correlation between induction of umu gene expression and mutagenicity with Ames/S. typhimurium TA98 system by activated metabolites of AFB1 in the reconstituted monooxygenase system suggested that the constitutive forms of cytochrome P-450 had major roles for genotoxic and mutagenic activation of AFB1 in rat liver microsomes.

Aflatoxin B1↗
PubMed1987

Purification and characterization of six cytochromes P-450 from hepatic microsomes of immature female rats.

Six rat hepatic cytochromes P-450, named P-450IF-1-6, were purified from hepatic microsomes of immature female rats by high-performance liquid chromatography with anion-exchange, cation-exchange, and hydroxylapatite columns. The purified forms, except for P-450IF-4, gave a single protein-staining band on SDS-polyacrylamide gel electrophoresis, with a minimum molecular weight of 50,000 for P-450IF-1, 49,000 for P-450IF-2, 47,000 for P-450IF-3, 53,500 for P-450IF-5, and 54,000 for P-450IF-6. The CO-reduced spectral maximum of these forms was at 450 nm for P-450IF-1, 448 nm for P-450IF-2, 451 nm for P-450IF-3, 449 nm for P-450IF-4, 449 nm for P-450IF-5, and 450 nm for P-450IF-6. All of these cytochromes had the low-spin state of heme in the oxidized form. P-450IF-4 had high metabolic activity for both benzphetamine and 7-ethoxycoumarin. P-450IF-5 had moderate activity toward 7-ethoxycoumarin. P-450IF-3 catalyzed the hydroxylation of testosterone at the 7 alpha-position effectively, but the other forms did not hydroxylate testosterone. Analysis of the NH2-terminal sequence showed that P-450IF-1, 2, 3, 5, and 6 differed structurally from each other. The sequences of P-450IF-1 and IF-2 were somewhat homologous, but the NH2-terminal sequences of the other forms were all different. Based on these results, we concluded that P-450IF-1 corresponded to one of the phenobarbital-inducible forms in male rat liver. P-450IF-2 was a female-specific form and its concentration was low.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Purification and NH2-terminal sequence of cytochrome P-450 from kidney microsomes of untreated male rats.

The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes.

Amino Acid Sequence↗
PubMed1986

Two-step purification of cytochrome P-450 from rat liver microsomes using high-performance liquid chromatography.

Cytochrome P-450 from rat liver microsomes treated with phenobarbital (PB) was separated into six fractions, as was cytochrome P-450 treated with 3-methylcholanthrene (MC), by high-performance liquid chromatography (HPLC) with an anion-exchange column. PB and MC induced three forms and one form of cytochrome P-450, respectively. The major forms induced by PB and by MC were further purified to apparent homogeneity based on sodium dodecyl(lauryl)sulphate--polyacrylamide gel electrophoresis by HPLC using a hydroxyapatite column. These new HPLC techniques are simple, rapid and useful for the purification of major forms of cytochrome P-450 from solubilized microsomes.

Animals↗
PubMed1985

Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography.

14 microsomal cytochromes P-450 were purified from the liver of untreated and phenobarbital- or 3-methylcholanthrene-treated male rats. Following solubilization of microsomes with sodium cholate, poly(ethylene glycol) fractionation and aminohexyl-Sepharose 4B chromatography, cytochromes P-450 were purified by high-performance liquid chromatography (HPLC), using a preparative DEAE-anion-exchange column. The pass-through fraction was further purified by HPLC using a cation-exchange column. Other fractions eluted on preparative DEAE-HPLC were further applied onto an HPLC using a DEAE-column. Five kinds (P-450UT-2-6), four kinds (P-450PB-1,2,4 and 5) and five kinds (P-450MC-1-5) of cytochromes P-450 were purified from untreated rats or rats treated with phenobarbital or 3-methylcholanthrene, respectively. HPLC profiles of tryptic peptides of cytochromes P-450UT-2 and P-450MC-2 were identical and the other profiles obtained from seven purified cytochromes P-450 were distinct from each other. Amino-terminal sequences of eight forms of cytochrome P-450 (UT-2, UT-5, PB-1, PB-2, PB-4, PB-5, MC-1 and MC-5) were distinct except for cytochromes P-450PB-4 and P-450PB-5.

Amino Acid Sequence↗