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M Negishi

Publications and source records attributed to M Negishi.

At least 361 records · Page 20Linked to original sources
PubMed1984

Mouse liver testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha). Purification, regioselectivity, stereospecificity, and immunochemical characterization.

Microsomal testosterone 16 alpha-hydroxylase (cytochrome P-450(16) alpha) was purified from the livers of male 129/J mice based on enzyme activity in the eluates from columns of DEAE Bio-Gel A, hydroxylapatite, and isobutyl-Sepharose 4B. The specific cytochrome P-450 content of the purified P-450(16) alpha fraction was 9.5 nmol/mg of protein. The specific testosterone 16 alpha-hydroxylation activity of the purified P-450(16) alpha fraction was 80 nmol/min/nmol of cytochrome P-450 or 764 nmol/min/mg of protein, and these values were about 40- and 400-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(16) alpha showed extremely high regioselectivity and stereospecificity for testosterone hydroxylation; more than 90% of the testosterone metabolites formed by the purified P-450(16) alpha fraction was 16 alpha-hydroxytestosterone. The purified anti-P-450(16) alpha antibody exhibited absolute specificity for inhibition of testosterone 16 alpha-hydroxytestosterone was inhibited by the anti-P-450(16) alpha. Anti-P-450(16) alpha inhibited the 16 alpha-hydroxylation activity of intact microsomes prepared from livers of male or female 129/J mice more than 90%, indicating that P-450(16) alpha is the major cytochrome P-450 isozyme catalyzing 16 alpha-hydroxylation activity of testosterone in these microsomal preparations. The purified P-450(16) alpha fraction also possessed high benzphetamine N-demethylation activity relative to the rates found with other xenobiotic substrates tested in this report.

Animals↗
PubMed1984

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction.

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.

Animals↗
PubMed1984

Mouse liver testosterone 15 alpha-hydroxylase (cytochrome P-450(15) alpha). Purification, regioselectivity, stereospecificity, and sex-dependent expression.

Testosterone 15 alpha-hydroxylase (cytochrome P-450(15) alpha) was purified from female 129/J mouse liver microsomes based on its specific activities in the eluates from the columns of octylamino-Sepharose 4B, hydroxylapatite, DEAE-Bio-Gel A, and CM52 chromatography. The 15 alpha-hydroxylation activity was five times higher in female than in male 129/J mouse liver microsomes. The specific cytochrome P-450 content of purified P-450(15) alpha fraction was 14.5 nmol/mg of protein. The Soret peak of the reduced cytochrome P-450-CO complex was 451 nm. The apparent subunit molecular weight of P-450(15) alpha was 48,000, and the protein appeared as only one major band on sodium dodecyl sulfate-polyacrylamide gels. The specific activity of testosterone 15 alpha-hydroxylation reconstituted with the purified P-450(15) alpha was 94 nmol/min/nmol of cytochrome P-450 and 1349 nmol/min/mg of protein, and these were about 65- and 1000-fold higher, respectively, than the activity of solubilized microsomes. The purified P-450(15) alpha exhibited high regioselectivity and stereospecificity for testosterone hydroxylation. More than 95% of the testosterone metabolites formed by the purified P-450(15) alpha was 15 alpha-hydroxytestosterone. Virtually 100% of mouse liver microsomal testosterone 15 alpha-hydroxylation activity can be accounted for by the purified P-450(15) alpha. The P-450(15) alpha fraction was able to catalyze benzphetamine N-demethylation, 7-ethoxycoumarin O-de-ethylation, aniline 4-hydroxylation, benzo(alpha)pyrene 3-hydroxylation, acetanilide 4-hydroxylation, and lauric acid (11 + 12)-hydroxylation at various turnover rates, indicating broad substrate specificity of the P-450(15) alpha for the oxidations of xenobiotics. This is in sharp contrast to high regioselectivity and stereospecificity for testosterone hydroxylation.

Animals↗
PubMed1983

Characterization of cytochrome P2-450 (20-S) mRNA. Association with the P1-450 genomic gene and differential response to the inducers 3-methylcholanthrene and isosafrole.

Mouse liver cytochrome P2-450 is defined as the major isosafrole-inducible form of P-450 which is most specific for isosafrole metabolism. lambda AhP-1 represents a 15.5 X 10(3)-base-pair segment of mouse genomic DNA having the cytochrome P1-450 gene (approximately equal to 4600 base pairs) located in the middle portion. Using various subclones as probes, we investigated the differential expression of P1-450 mRNA and P2-450 mRNA induction as a function of association with the Ah locus, 3-methylcholanthrene or isosafrole dosage, tissue specificity, and developmental age. Both P1-450 (23-S) mRNA and P2-450 (20-S) mRNA induction processes are regulated by the Ah receptor. P2-450 mRNA is about 10-fold more sensitive than P1-450 mRNA to induction by either 3-methylcholanthrene or isosafrole. Phenobarbital pretreatment has no effect at all on either P1-450 mRNA or P2-450 mRNA. Whereas both P1-450 mRNA and P2-450 mRNA are induced by 3-methylcholanthrene in C57BL/6N liver, P1-450 (23-S) mRNA but not P2-450 (20-S) mRNA is induced by 3-methylcholanthrene in C57BL/6N kidney. P1-450 mRNA induction by 3-methylcholanthrene is measurable in C57BL/6N liver at day 15 of gestation, and the expression becomes enhanced with increasing age. P2-450 mRNA induction by 3-methylcholanthrene in C57BL/6N liver appears about 7 days later during development than 3-methylcholanthrene-inducible P1-450 mRNA. Both 3-methylcholanthrene-induced P1-450 mRNA and P2-450 mRNA are detectable in DBA/2N liver; their appearance is later in development, however, and at lower concentrations than that seen with C57BL/6N liver. P1-450 (23-S) mRNA and P2-450 (20-S) mRNA appear to hybridize to a common 5' fragment of the P1-450 gene.

Animals↗
PubMed1983

Structure of the mouse cytochrome P1-450 genomic gene.

Clone 46 was previously shown to represent mouse cytochrome P1-450 cDNA by both translation arrest experiments and segregation of induced P1-450 mRNA with induced aryl hydrocarbon hydroxylase activity among individual 3-methylcholanthrene-treated offspring of the (C57BL/6N)(DBA/2N)F1 X DBA/2N backcross. With clone 46 as a probe, a MOPC 41 mouse genomic-DNA library was screened. lambda 3NT12, a 16 X 10(3)-base-pair insert of genomic DNA grown in a recombinant Charon 4A lambda vector phage, was isolated and characterized. It was determined that clone 46 hybridizes to the extreme 5' end of lambda 3NT12. pMJE12, a 3.0 X 10(3)-base-pair fragment in the 5' region of lambda 3NT12, was subcloned in plasmid pBR322 and used as a probe to screen again the same mouse-DNA library; recombinant phages lambda 3NT13, lambda 3NT14, and lambda AhP-1 were isolated and characterized. The relative orientation of each of the four genomic clones on the mouse chromosome was determined. Only lambda AhP-1 contains the entire P1-450 genomic gene, which by R-loop analysis spans about 46 X 10(2) base pairs and contains at least five exons. Clone 46 is shown to be a 3' unique sequence of the genomic P1-450 gene. The lambda AhP-1 genomic-DNA clone from the MOPC 41 plasmocytoma is shown by a series of restriction enzymes to be the same as genomic DNA from normal mouse liver. With a subclone in the 5' portion of the P1-450 gene, two and three hybridizable fragments are found with mouse genomic DNA that has been digested with EcoRI and BamHI, respectively.

Animals↗
PubMed1983

Inhibition of cellular growth and nutrient transport induced by calcium ionophore A23187 in mastocytoma P-815 cells.

Kinetic analysis of Ca2+ metabolism in mastocytoma P-815 cells showed that at non-cytotoxic and growth-inhibitory concentrations (less than 10 micrograms/ml), the calcium ionophore A23187 did not affect the rate constant (36.2 min-1) or the compartment size (8.32 nmol/10(6) cells) of the fast phase of calcium exchange at the cell surface. In contrast, A23187 (1 to 10 micrograms/ml) dose-dependently increased the compartment size of slow phase I of the intracellular calcium uptake from 0.54 to 2.39 nmol/10(6) cells without affecting the rate constant (2.38 min-1). In addition, A23187 (5 to 10 micrograms/ml) produced an additional compartment [slow phase II] with a rate constant of 0.25 min-1 and increased its compartment size from 0.42 to 1.26 nmol/10(6) cells. A23187 (10 micrograms/ml) in the presence of Ca2+ (0.9 mM) non-competitively inhibited the membrane transport of amino acids, glucose and nucleosides; and it also reduced the cellular ATP level and the membrane fluidity. Apparently, A23187 inhibits the growth of mastocytoma cells by elevating the intracellular concentration of Ca2+, which in turn reduces the membrane fluidity and the carrier-dependent as well as the active membrane transport of various nutrients required for cellular growth.

Adenosine Triphosphate↗