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M J Coon

Publications and source records attributed to M J Coon.

At least 163 records · Page 9Linked to original sources
PubMed1976

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.

Amino Acid Sequence↗
PubMed1976

Induction of multiple forms of mouse liver cytochrome P-450. Evidence for genetically controlled de novo protein synthesis in response to treatment with beta-naphthoflavone or phenobarbital.

The administration of polycyclic aromatic compounds such as beta-naphthoflavone or 3-methylcholanthrene is known to cause the induction of many liver microsomal monoxygenase activities and the appearance of a distinct cytochrome called P-448 in genetically responsive, but not in nonresponsive, inbred mouse strains. However, the administration of 2,3,7,8-tetrachlorodibenzo-p-dioxin induces these activities and cytochrome P-448 formation to the same extent in both responsive and nonresponsive inbred strains. In contrast, phenobarbital or pregnenolone-16 alpha-carbonitrile induces in both responsive and nonresponsive strains a different profile of enzyme activities and the appearance of cytochrome P-450 (rather than cytochrome P-448). In the present studies, electrophoresis of liver microsomal proteins from inbred C57BL/6N and DBA/2N and recombinant inbred AKXL-38 and AKXL-38A mouse strains revealed the presence of four polypeptides whose relative staining intensity could be correlated with the induction state of the microsomes as determined by enzymatic and spectral methods. Of these four bands, Band 4 (55,000 daltons) was increased whenever spectral measurements revealed an increase in the cytochrome P-448 content due to administration of beta-naphthoflavone or 2,3,7,8-tetrachlorodibenzo-p-dioxin. Administration of pregnenolone-16alpha-carbonitrile caused an increase in Band 3 (54,000 daltons), whereas administration of phenobarbital caused an increase primarily in Band 2 (51,000 daltons) but also smaller increases in Band 1 (49,000 daltons) and Band 4. The changes observed for phenobarbital and pregnenolone-16alpha-carbonitrile were the same for both responsive and nonresponsive strains. The same electrophoretic technique was used to measure the incorporation of radioactive leucine into microsomal proteins. Microsomes were prepared from liver combined from responsive mice (C57BL/6N) treated with beta-naphthoflavone and L-[14C]leucine and nonresponsive mice (DBA/2N) treated with beta-naphthoflavone and L-[3H-4,5]leucine. A significant increase in the 14C/3H ratio was observed for Band 4, and decreases were seen for Bands 1 and 2. In similar experiments with other mice and phenobarbital as the inducing agent with L-[14C]leucine and the vehicle alone with L-[3H-4,5]leucine, the 14C/3H ratio was markedly increased for Band 2, and smaller increases were observed for Bands 1 and 4. These results and other data presented indicate that the increased formation of cytochrome P-448 and P-450 by beta-naphthoflavone and phenobarbital, respectively, is primarily the result of an increased rate of de novo protein synthesis rather than a decreased degradation rate or a conversion of pre-existing polypeptides.

Animals↗
PubMed1975

Purified liver microsomal cytochrome P-450. Electron-accepting properties and oxidation-reduction potential.

The reduction of highly purified cytochrome P-450 from rabbit liver microsomes under anaerobic conditions requires 2 electrons per molecule. Similar results were obtained with dithionite, NADPH in the presence of NADPH-cytochrome P-450 reductase, or a photochemical system as the electron donor, with CO or other ligands, with substrate or phosphatidylcholine present, after denaturation to form cytochrome P-420, or with cytochrome P-450 partially purified from rat or mouse liver microsomes. The reduced cytochrome P-450 donates 2 electrons to dichlorophenolindophenol or to cytochrome c. Reoxidation of reduced cytochrome P-450 by molecular oxygen restores a state where 2 electrons from dithionite are required for re-reduction. Although these unexpected findings indicate the presence of an electron acceptor in addition to the heme iron atom, significant amounts of non-heme iron, other metals or cofactors, or disulfide bonds were not found, and free radicals were not detected by electron paramagnetic resonance spectrometry. Resolution of the cytochrome with acetone and acid yielded the apoenzyme, which did not accept electrons, and ferriprotoporphyrin IX, which accepted a single electron. A reconstituted hemoprotein preparation with the spectral characteristics of cytochrome P-420 accepted as much as 0.7 extra electron equivalent per heme. The midpoint oxidation-reduction potential of purified cytochrome P-450 from rabbit liver microsomes at pH 7.0 is -330 mv, and with CO present this value is changed to about -150 mv. The oxidation-reduction potential is unaffected by the presence of phosphatidylcholine or benzphetamine, a typical substrate. Laurate, aminopyrine, and benzphetamine undergo hydroxylation in the presence of chemically reduced cytochrome P-450 and molecular oxygen. Neither NADPH nor the reductase is required for substrate hydroxylation under these conditions.

Anaerobiosis↗
PubMed1975

Purified liver microsomal cytochrome P-450. Separation and characterization of multiple forms.

During the purification of rabbit liver microsomal cytochrome P-450 (P-450LM), evidence was obtained for the occurrence of at least four distinct forms. These were distinguished by polyacrylamide gel electrophoresis after treatment with sodium dodecyl sulfate in the presence or absence of mercaptoethanol and were shown to have characteristic spectra as the reduced carbon monoxide complexes. They are designated by their relative electrophoretic mobilities. P-450LM2, which was purified to apparent homogeneity, is induced by phenobarbital and has a subunit molecular weight of 50,000. P-450LM4, which was also extensively purified, is induced by beta-naphthoflavone and has a molecular weight of 54,000. P-450LM1,7, which is induced neither by phenobarbital nor beta-naphthoflavone, is a mixtureMIXTURE OF ABOUT EQUAL AMOUNTS OF TWO FORMS WITH MOLECULAR WEIGHTS OF 47,000 AND 60,000 RESPECTIVELY. Some preparations were obtained containing primarily P-450LM1 or P-450LM7. Benzphetamine, ethylmorphine, and p-nitroanisole are hydroxylated preferentially by P-450LM2, and benzpyrene by P-450LM1,7. Biphenyl is hydroxylated in both positions 2 and 4 by all of the preparations, but the latter position is strongly favored by the action of P-450LM2. Testosterone is hydroxylated primarily in position 16alpha by P-450LM2 and in position 6beta by P-450LM1,7. Although the occurrence of additional forms of the cytochrome with highly similar electrophoretic behavior is not ruled out, it appears that the presence of these forms differing in subunit molecular weight may account for the variety of catalytic activities attributed to this pigment of liver microsomes.

Animals↗
PubMed1975

Biochemical characterization of highly purified cytochrome P-450 and other components of the mixed function oxidase system of liver microsomal membranes.

Cytochrome P-450 has been purified from liver microsomes of phenobarbital-induced rabbits in the presence of ionic and nonionic detergents to concentrations over 17 nmoles per mg of protein. The purified cytochrome P-450 LM gives a single major band on SDS-polyacrylamide gel electrophoresis representing about 90 per cent of the total protein. The polypeptide chain has a molecular weight of about 49,000 daltons. NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-induced rats in the presence of ionic and nonionic detergents to a stage where it catalyzes the reduction of 33,000 nmoles of cytochrome c per min per mg of protein. The ratio of activities toward cytochrome P-450 and cytochrome c is constant throughout purification. The purified reductase contains equimolar amounts of FMN and FAD and gives a single major band on SDA-polyacrylamide gel electrophoresis accounting for about 70 per cent of the total protein; the molecular weight is about 80,000 daltons. The purified cytochrome P-450 is free of cytochrome b5 but contains another electron acceptor, provisionally called Factor C, which is equivalent in amount to the heme present. Two electrons are taken up per molecule of cytochrome P-450 from dithionite or from NADPH in the presence of catalytic amounts of the reductase, and both electrons are readily transferred from the reduced cytochrome P-450 to molecular oxygen or artificial electron acceptors. The reconstituted enzyme system containing purified cytochrome P-450, purified NADPH-cytochrome P-450 reductase, and phosphatidylcholine retains the ability to catalyze the hydroxylation of drugs, fatty acids, hydrocarbons, and aniline in the presence of NADPH and molecular oxygen.

Anaerobiosis↗
PubMed1975

Position-specific oxygenation of benzo(a)pyrene by different forms of purified cytochrome P-450 from rabbit liver.

High-pressure liquid chromatography was used to detect oxygenated products of benzo[a]pyrene formed in a reconstituted microsomal mixed-function oxidase system containing cytochrome P-450 (P-450LM), phospholipid, and NADPH-cytochrome P-450 reductase (NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4). Three cytochrome fractions purified from a single source, hepatic microsomes from phenobarbital-treated rabbits, were studied; the various forms of the cytochrome are designated by their relative electrophoretic mobilities. The total benzo[a]pyrene oxygenation rate was greatest for P-450LM1,7, intermediate for P-450LM2, and least for P-450LM4. The phenolic products were eluted in two peaks, A and B, that contained primarily 9-hydroxy- and 3-hydroxybenzo[a]pyrene, respectively. The ratio of peak A to peak B phenols was 0.11 for P-450LM2 and 0.45 for P-450LM4. Thus, the relative amounts of the various phenols formed by these two cytochrome fractions differ markedly. The positional specificity of the hydroxylation is also indicated by large differences in the fluorescence spectra of the phenolic products formed by the two cytochromes. P-450LM2 and P-450LM4 did not form benzo[a]pyrene dihydrodiols, thereby showing that benzo[a]pyrene oxide hydratase activity was absent from these purified preparations. Ninety percent of the phenols formed by P-450LM1,7 were eluted in peak B; the metabolites produced by this preparation also included dihydrodiols, thus indicating the presence of hydratase activity. The positional specificities of different forms of cytochrome P-450 may channel polycyclic aromatic hydrocarbon metabolism into the various activation and detoxification pathways and thereby help determine the cytotoxic and carcinogenic activity of these compounds.

Animals↗