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Biomedical subjects

M Negishi

Publications and source records attributed to M Negishi.

At least 397 records · Page 22Linked to original sources
PubMed1981

Distribution and induction of cytochrome P-450 in rat liver nuclear envelope.

Induction of cytochrome P-450s by 3-methylcholanthrene (MC) and phenobarbital (PB) and distribution of P-450s in the rat liver nuclear envelope were investigated by biochemical analyses and ferritin immunoelectron microscopy using specific antibodies against the major molecular species of MC- and PB-induced cytochrome P-450. It was found, in agreement with Kasper (J. Biol. Chem., 1971, 246: 577-581), that the total amount of cytochrome P-450s determined by biochemical analysis was markedly increased by MC, but not by PB, treatment. Immunoelectron microscopic analysis, however, showed marked and slight increases in ferritin labeling by MC and PB treatment, respectively. The latter finding was interpreted as resulting from the induction of a particular molecular species of PB-induced cytochrome P-450s. Ferritin immunoelectron microscopic analysis of intact isolated nuclei, naked nuclei from which the outer membrane of the nuclear envelope was partially detached (mechanically), and isolated nuclear envelopes have shown that the ferritin particles are found exclusively on the cytoplasmic face of the outer nuclear envelopes. Neither the nucleoplasmic face of the inner membrane of the nuclear envelope nor the cisternal face of both membranes of the nuclear envelope showed any labeling with ferritin. This indicates that cytochrome P-450 is located only on the outer membrane of the nuclear envelope and does not diffuse laterally into the domain of the inner membrane of the nuclear envelope across the nuclear pores. Our results suggest that a marked heterogeneity exists in the enzyme distribution between the outer and inner membrane of the nuclear envelope and that microsomal marker enzymes such as cytochrome P-450 exist exclusively in the outer membrane. In addition, it appears that cytochrome P-450 is probably not a transmembrane protein but an intrinsic protein located on the cytoplasmic face of the outer membrane of the nuclear envelope.

Animals↗
PubMed1981

Structural gene products of the murine Ah complex. Differences in ontogenesis and glucosamine incorporation between liver microsomal cytochromes P1-450 and P-448 induced by polycyclic aromatic compounds.

Antibodies against mouse-liver microsomal cytochromes P1-450 and P-448, two polycyclic aromatic inducible cytochromes, were previously developed [Negishi, M. and Nebert, D.W. (1979) J. Biol. Chem. 254, 11015-11023]. Liver microsomes from 3-methylcholanthrene-treated and phenobarbital-treated and control adult mice and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated adult and fetal mice were examined. Immunoprecipitable radioactivity was measured, following labeling with pyridoxal phosphate/NaB[3H]4 or with 125I-labeled p-aminosulfobenzoic acid/NaNO2 in vitro or with [3H]leucine, [14C]glucosamine, or [32P]O4 in vivo. (a) Induction of cytochrome P1-450 occurs developmentally earlier in gestation than induction of cytochrome P-448 when the mother is treated with polycyclic aromatic compounds. (b) There appears to be a basal form of cytochrome P-448 but no cytochrome P1-450 in control liver microsomes; inducibility of cytochrome P-448 thus ranges between 5--12-fold, whereas that of P1-450 is infinite. (c) Phenobarbital pretreatment induces no detectable P1-450 or P-448. (d) P-448 appears to be either greater in concentration than P1-450 in the membrane or more exposed than P1-450 on the microsomal membrane surface. (e) By the radioimmunoassay methods used, 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced P1-450 and P-448 in Ah-nonresponsive mice are indistinguishable from those in Ah-responsive mice; this is true in both the fetus and the adult. (f) Compared with P-448 expression, the expression of P1-450 is more closely associated with 3-methylcholanthrene-induced aryl hydrocarbon hydroxylase activity, and these two structural gene products are apparently regulated independently. (g) P-448 but not P1-450 appears to be a glycoprotein. These data illustrate further differences between two forms of polycyclic aromatic-inducible P-450 in mouse liver. Neither P1-450 nor P-448 appears to be a phosphoprotein. Neither anti-(P1-450) nor anti-(P-448) precipitates any forms of liver microsomal P-450 from beta-naphthoflavone-treated adult rabbits and, conversely, anti-LM4 (the antibody to rabbit liver microsomal P-450 form 4) does not precipitate any forms of liver microsomal P-450 from 3-methylcholanthrene-treated C57BL/6N mice.

Animals↗
PubMed1980

Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes.

Treatment of rats with phenobarbital leads to a substantial increase in levels of translatable liver cytochrome P-450 mRNA. This mRNA is primarily associated with ribosomes bound to endoplasmic reticulum membranes which in an in vitro system synthesized approximately 10 times more cytochrome P-450 than did free polysomes from the same animals. Cytochrome P-450 synthesized by rough microsomes in vitro appears to be directly inserted into the membranes because it was not released by a treatment with low detergent concentrations that released albumin and other microsomal content proteins. The amino-terminal amino acid sequence of cytochrome P-450 synthesized in an mRNA-dependent system resembles in hydrophobicity the signal segment of presecretory proteins and therefore may serve to insert the polypeptide into the membrane during synthesis. In contrast to the situation with secretory proteins and several other membrane proteins, however, the putative insertion signal of cytochrome P-450 is not removed by a membrane-associated peptidase and remains in the mature polypeptide.

Animals↗
PubMed1980

Effect of adenosine and adenosine 5'-monophosphate on cell division of cultured mastocytoma P-815 cells.

The growth of mouse mastocytoma P-815 cells in culture (37 degrees, 42 hr) was inhibited by exogenous adenosine (0.2 to 1.0 mM) and more effectively by AMP (0.01 to 0.1 mM), but not by adenine. The inhibited growth (a 25% inhibition by 0.5 mM adenosine and a 80% inhibition by 0.25 mM AMP) was restored to a near control level by the addition of uridine (0.5 mM) to the medium. The pretreatment (37 degrees, 3 hr) of the cells with adenosine or AMP caused a 60% inhibition of incorporation (37 degrees, 2 hr) of [U-14C]aspartate into uracil nucleotides, accumulating 14C-orotate and orotidine. Both dipyridamole, an inhibitor of adenosine uptake, and exogenous adenosine deaminase suppressed the growth inhibition induced by not only adenosine but also AMP. 2-Chloroadenosine, which is resistant to the action of adenosine deaminase, was a more potent growth inhibitor, while 3'AMP and 2'-AMP, which are not hydrolyzed to adenosine by membrane 5'-nucleotidase, were ineffective. Adenosine 5'-sulfate and other 5'-substituted adenosines were also ineffective. These observations indicate that AMP inhibits the growth of mastocytoma P-815 cells as a result of its continuous conversion to adenosine and a constant exposure of the cells to a low concentration of adenosine which readily permeates the cell membrane. In addition, adenosine, AMP and their agarose-linked forms rapidly (37 degrees, 20 min) elevated cellular levels of cAMP. This effect was not suppressed by dipyridamole. Apparently adenosine and AMP also act extracellularly for growth inhibition by regulating cAMP levels.

Adenine Nucleotides↗
PubMed1980

Effect of adenosine 3',5'-monophosphate on growth and several functions of cultured mastocytoma P-815 cells.

Growth-inhibited mouse mastocytoma P-815 cells at stationary phase contained more histamine, serotonin and adenosine 3',5'-monophosphate (cAMP), and higher activities of histidine decarboxylase and adenylate cyclase than the cells during exponential growth. The elevation of endogenous cAMP levels induced by several growth-inhibiting agents such as N6, O2'-dibutyryl cAMP (Bt2cAMP), prostaglandin E1, AMP and 2-chloroadenosine stimulated several functions characteristic of mastocytoma P-815 cells in culture, elevating the synthesis of histamine and serotonin, the activity of chymotrypsin-like protease, and the incorporation of [35S]sulfate into acidic glycosaminoglycans. 1-Methyl-3-isobutyl-xanthine (MIX), a potent inhibitor of cAMP phosphodiesterase, potentiated stimulatory effect of these agents. The results indicate that cAMP regulates the growth and functions of mastocytoma P-815 cells. [35S]-Sulfated acidic glycosaminoglycans synthesized in cells at stationary phase or in cells treated with Bt2cAMP plus MIX mainly localized in the 3000-10000 x g sedimentable fraction of cell homogenates, and had a molecular weight of 200000 to 400000 based on gel filtration. This acidic glycosaminoglycan was resistant to chondroitinase ABC and the heparin-degrading enzyme present in the 20000 x g sedimentable fraction of the cells, and was identified as a highly sulfated macromolecular heparin based on behaviors on DEAE-cellulose column and on acidic electrophoresis. Cycloheximide suppressed the stimulatory effect of Bt2cAMP on the synthesis of histamine and [35S]-sulfated acidic glycosaminoglycan.

Adenosine↗
PubMed1980

Aphidicolin: a specific inhibitor of DNA synthesis in synchronous mastocytoma P-815 cells.

The influence of aphidicolin on cell multiplication and DNA synthesis was examined using synchronous mouse mastocytoma P-815 cells. Aphidicolin was cytotoxic specifically to the cells of the S phase of the cell cycle. This cytotoxicity was reversed by appropriately washing the drug-treated cells, but not by the addition of deoxyribonucleosides. Aphidicolin, a potent inhibitor of DNA synthesis, selectively inhibited the activity of partially purified DNA polymerase alpha from the nucleus and the cytosol of mastocytoma cells, but did not affect the activity of DNA polymerase beta. Furthermore, aphidicolin had no effect on the synthesis of RNA and protein, and produced no changes in cell size at least for one generation.

Animals↗
PubMed1979

Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes.

Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.

Animals↗
PubMed1979

Purification and partial characterization of hepatic microsomal cytochrome P-450s from phenobarbital- and 3-methylcholanthrene-treated rats.

Hepatic microsomal cytochrome P-450 and P-448 have been purified from phenobarbital (PB)- and 3-methylcholanthrene (MC)-treated rats, by modifications of Imai and Sato's procedures )1974). The purified preparations of cytochrome P-450 and P-448 were homogeneous judging from their specific contents (17 and 16 nmol per mg protein, respectively) and the results of SDS-polyacrylamide gel electrophoresis and Ouchterlony immunodiffusion analyses. These two cytochromes are different in their physico-chemical and immunological properties, and their substrate specificities. In reconstituted systems containing the purified cytochrome and NADPH-cytochrome P-450 reductase, ethoxycoumarin deethylation and benzo(a)pyrene hydroxylation catalyzed by cytochrome P-450 and P-448 were completely inhibited by the homologous antibody, while essentially no effect was observed with heterologous conbinations of antigen and antibody. In contrast, the benzphetamine demethylation activities of cytochrome P-450 and P-448 were markedly inhibited by the heterologous antibody as well as by the homologous one. These results suggest that the two cytochromes are immunologically different but have some antigenic determinants in common. Drug metabolizing activities of microsomes from PB- and MC-treated rats were inhibited by the antibodies, essentially as expected from the results with the reconstituted systems. The remaining activities in the presence of excess concentrations of the antibody, however, were higher in MC-microsomes treated with anti P-448 antibody than in PB microsomes treated with anti P-450 antibody. These results suggest that cytochrome P-448 molecules may be so localized in the microsomal membrane that the membrane structure may hinder the access of the antibody to the antigenic determinant.

Amino Acids↗