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Biomedical subjects

M J Coon

Publications and source records attributed to M J Coon.

At least 145 records · Page 8Linked to original sources
PubMed1979

Mechanistic studies with purified components of the liver microsomal hydroxylation system: spectral intermediates in reaction of cytochrome P-450 with peroxy compounds.

Recent investigations in this laboratory on the mechanism of action of liver microsomal cytochrome P-450 (P-450 LM) and its interaction with other components of the hydroxylation system are presented. Two electrophoretically homogeneous forms of the cytochrome, phenobarbital-inducible P-450 LM2 and 5,6-benzoflavone-inducible P-450 LM4, so designated according to their relative electrophoretic mobilities, were used in these studies. Phosphatidylcholine is required in the reconstituted enzyme system for rapid electron transfer from NADPH to P-450 LM, catalyzed by NADPH-cytochrome P-450 reductase, as well as for maximal hydroxylation activity with either molecular oxygen or a peroxy compound serving as oxygen donor to the substrate. The phospholipid facilitates the binding of both substrate and reductase to P-450 LM and apparently causes a structural change in the cytochrome as shown by an increase in alpha-helical content, determined by circular dichroic spectrometry. P-450LM3 and LM4 are one-electron acceptors under anaerobic conditions, in accord with previous potentiometric titrations and product yield data, but in disagreement with previous titrations with reducing agents. The cause for the discrepancy between the present and earlier results is not yet fully understood. Stopped flow spectrophotometry was employed to detect intermediates in the reaction of peroxy compounds with P-450LM2. With m-chloroperbenzoic acid the intermediate formed has absorption maxima at 375, 425, and 540 nm in the absolute spectrum and at 370, 436, and 540 nm in the difference spectrum (intermediate minus oxidized form). A study of the magnitude of the spectral change at various peracid concentrations indicated that with this oxidant the reaction shows a dependence resembling a binding curve. These and other experiments with various oxidants, including cumente hydroperoxide, suggest a reversible two-step mechanism according to the reaction: P-450 LM + oxidant equilibrium C equilibrium D, where C may be an enzyme-oxidant complex and D is a spectral intermediate of unknown structure. A scheme is proposed for the mechanism of action of P-450 LM based on these and earlier studies, including evidence from deuterium isotope experiments for the formation of a substrate carbon radical prior to oxygen transfer.

Anaerobiosis↗
PubMed1979

Kinetics of reduction of purified liver microsomal cytochrome P-450 in the reconstituted enzyme system studied by stopped flow spectrophotometry.

Stopped flow studies were undertaken to examine the kinetics of reduction of 5,6-benzoflavone-inducible P-450 LM4 by NADPH in the presence of NADPH-cytochrome P-450 reductase and phospholipid under anaerobic CO at 25 degrees C. The reaction exhibited biphasic kinetics irrespective of NADPH concentration or of the molar ratio of reductase to P-450 LM4. The apparent first order rate constants for the fast and slow phases were determined to be 0.9 to 1.0 and 0.25 s-1, respectively. With the reductase and P-450 LM4 present in equimolar amounts, the total amount of P-450 LM4 reduced increased linearly with NADPH concentration; the titration gave a stoichiometry of 2 mol of NADPH per mol of reductase-cytochrome complex. The NADPH concentration had no appreciable effect on the magnitude of the first order rate constants for the fast and slow phases. The kinetics obtained in the presence of benzphetamine were essentially indistinguishable from those seen in the absence of this substrate, while the amount of P-450 LM4 reduced in the fast phase, but not the rate constant for this phase, decreased when phospholipid was omitted from the reaction mixture. Nearly maximal rates of NADPH oxidation by P-450 LM2 OR LM4 were obtained with a molar ratio of reductase to P-450 LM of 1.0. Benzphetamine enhanced the oxidation of NADPH by P-450 LM2 but had no effect on the activity of P-450 LM4. Rates of NADPH oxidation in the presence of P-450 LM2 and LM4 decreased by 80 and 40%, respectively, when phospholipid was omitted from the reconstituted enzyme system. These studies provide evidence for the formation of a catalytically functional 1:1 complex between the reductase and P-450 LM4, and indicate that P-450 LM2 and LM4 differ in their dependence on phospholipid.

Animals↗
PubMed1978

Purified liver microsomal NADPH-cytochrome P-450 reductase. Spectral characterization of oxidation-reduction states.

NADPH-cytochrome P-450 reductase was isolated from liver microsomes of phenobarbital-induced rats. The enzyme exhibits an apparent minimal molecular weight of 76,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contains 1 molecule each of FMN and FAD. Trypsin treatment of the reductase yields an enzyme with an apparent minimal molecular weight of 69,000 which retains the ability to reduce cytochrome c but has no activity toward cytochrome P-450. Various spectrophotometric titrations were performed to examine the electron-accepting properties of the purified NADPH-cytochrome P-450 reductase and, in particular, to determine the oxidation state of the stable semiquinone form produced by air oxidation of NADPH-reduced enzyme. Titration of the air-stable semiquinone form of the reductase with ferricyanide indicated that 1 mol/2 mol of flavin was required for complete oxidation. Furthermore, a spectrum corresponding to that of the air-stable semiquinone form was produced by the addition of approximately 0.5 mol of reductant/2 mol of flavin when the oxidized enzyme was titrate with NADPH or dithionite under anaerobic conditions. The spectral changes which accompanied the overall reduction of oxidized enzyme to the reduced form with dithionite produced four sets of isosbestic points, and the spectrophotometric titration curve consisted of four approximately equal phases. In the titration with NADPH, no significant further reduction was observed after the addition of approximately 1.5 mol/2 mol of flavin. However, the enzyme was fully reduced by NADPH when an NAPH-generating system was used to prevent the accumulation of NADP. Our results establish that the air-stable semiquinone form is a 1-electron-reduced form, rather than a half-reduced (2-electron-reduced) form as maintained by others and are in agreement with earlier studies (Iyanagi, T., Makino, N., and Mason, H.S. (1974) Biochemistry 13, 1701-1710) with the purified trypsin-solubilized reductase. Accordingly, the air-stable species represents a form of the NADPH-cytochrome P-450 reductase in which one of the two flavins exists in the semiquinone state and the other in the oxidized state.

Animals↗
PubMed1978

Regio- and stereoselectivity of various forms of purified cytochrome P-450 in the metabolism of benzo[a]pyrene and (-) trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene as shown by product formation and binding to DNA.

Highly purified cytochromes P-450(LM2) and P-450(LM4) and partially purified P-450(LM1), P-450(LM3b), and P-450(LM7) from rabbit liver microsomes exhibit different catalytic activities in the metabolism of benzo[a]pyrene (BzP) and (-)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(-)trans-7,8-diol] in a reconstituted enzyme system. The two highly purified cytochromes also exhibit differences in the activation of BzP and (-)trans-7,8-diol to intermediates that bind to DNA, as well as in the stereoselective conversion of (-)trans-7,8-diol to the highly mutagenic and carcinogenic diol-epoxides r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10- tetrahydrobenzo[a]pyrene (diol-epoxide I) and r - 7,t - 8 - dihydroxy - c - 9,10 - oxy - 7,8,9,10 - tetrahydrobenzo[a]pyrene (diol-epoxide II). P-450(LM2) is more active than P-450(LM4) in the metabolism of BzP and in its conversion to products that bind to DNA. In contrast, P-450(LM4) is more active than P-450(LM2) in the metabolism of (-)trans-7,8-diol and in its conversion to products that bind to DNA. The ratio of activity (percent substrate metabolized) with BzP relative to that with (-)trans-7,8-diol is 21 for P-450(LM2) and 0.3 for P-450(LM4); P-450(LM1), P-450(LM3b), and P-450(LM7) gave intermediate ratios. Marked stereoselectivity in the oxygenation of the (-)trans-7,8-diol to the highly mutagenic and putatively carcinogenic diol-epoxides I and II was observed with P-450(LM4), whereas the other preparations showed less selectivity. The ratio of diolepoxide I to diol-epoxide II ranges from 0.3 for P-450(LM7) to 11 for P-450(LM4). The substrate specificity and regio- and stereo-selectivity of the different forms of cytochrome P-450 may regulate the balance between activation and detoxification pathways of BzP and therefore determine the susceptibility of individual tissues, strains, and species to the carcinogenic action of BzP.

Animals↗
PubMed1977

Evidence that puriified liver microsomal cytochrome P-450 is a one-electron acceptor.

Two forms of highly purified liver microsomal cytochrome P-450, P-450LM2, and P-450LM4, have been titrated with standardized solutions of sodium dithionite under anaerobic conditions. Only 1 electron was consumed per hemin molecule, and reoxidation of the reduced heme is accompanied by the transfer of 1 electron to oxidizing agents such as cytochrome c, cytochrome b5, or potassium ferricyanide. The present results are in disagreement with earlier dithionite titrations and reoxidation experiments which indicate that liver microsomal cytochrome P-450 is a 2-electron acceptor, but are in accord with previous potentimetric titrations and product yield data which indicate that this hemeprotein is a 1-electron acceptor. The cause for the discrepancy between the present and previous results is not fully understood.

Animals↗