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M J Coon

Publications and source records attributed to M J Coon.

At least 109 records · Page 6Linked to original sources
PubMed1984

On the amino acid sequence of cytochrome P-450 isozyme 4 from rabbit liver microsomes.

Isozyme 4 of rabbit liver microsomal cytochrome P-450 was shown earlier in this laboratory to contain multiple NH2-terminal residues, whereas isozymes 2, 3a, 3b, and 3c have single, unique NH2-terminal sequences. Similar results were obtained with isozyme 4 obtained from animals that were untreated, treated with phenobarbital (which does not induce this isozyme), or induced with beta-naphthoflavone or isosafrole. With the use of selective chemical blocking at seryl or at nonprolyl residues, the complexity of the NH2-terminal sequence has now been shown to be due to the presence of three forms of the cytochrome differing only in the absence of the first or the first two residues: NH2-Ala-Met-Ser-Pro-Ala-Ala-Pro-, NH2-Met-Ser-Pro-Ala-Ala-Pro-, and NH2-Ser-Pro-Ala-Ala-Pro-. These forms may result from variable biological processing. Peptides containing the seven cysteine residues were sequenced and compared with similar peptides reported for other P-450 cytochromes; homology was extensive with respect to two of the cysteine regions in isozyme 4, and a third cysteine region showed partial identity. The sequence of peptides representing about two-thirds of the amino acids in isosafrole-induced cytochrome P-450 isozyme 4 was determined. Comparison with phenobarbital-induced rabbit cytochrome P-450 isozyme 2 indicated about 25% homology. In contrast, comparison of isozyme 4 with rat cytochrome P-450d, which is also induced by isosafrole and for which the sequence has recently been deduced from cDNA [Kawajiri, K., Gotoh, O., Sogawa, K., Tagashira, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. USA 81, 1649-1653], showed about 70% homology.

Amino Acid Sequence↗
PubMed1984

Purification of liver microsomal cytochrome P-450 isozymes 3a and 6 from imidazole-treated rabbits. Evidence for the identity of isozyme 3a with the form obtained by ethanol treatment.

Two forms of cytochrome P-450 have been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated with imidazole. Several criteria indicate that the cytochrome of higher electrophoretic mobility is identical with ethanol-inducible isozyme 3a. "Imidazole-3a" and "ethanol-3a" exhibit the same chromatographic characteristics and have identical electrophoretic mobilities upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Furthermore, the two protein preparations have the same absorbance maxima and absorption coefficients in the oxidized, reduced, and reduced-CO states. A single immunoprecipitin band exhibiting complete identity was observed upon reaction of imidazole-3a and ethanol-3a with the immunoglobulin G fraction from sheep immunized with the latter protein. The amino acid composition and first 10 residues of the amino terminus of the two protein preparations are indistinguishable, as are the high-performance liquid chromatographic maps of the peptides obtained upon cleavage with trypsin, Staphylococcus aureus V8 protease, or Lys C endoproteinase . Furthermore, these preparations have very similar activities in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline. Evidence was obtained that the cytochrome of lower electrophoretic mobility isolated from imidazole-treated rabbits is probably identical with isozyme 6; the spectral characteristics, amino acid composition, and carboxyl-terminal sequence are described. As an inducer, imidazole has the advantage over ethanol of being less variable in its effects and requiring a shorter period of treatment. From the resulting liver microsomes, one can readily isolate, in addition to P-450 isozymes 3a and 6, isozymes 3c and 4 as well as epoxide hydrolase.

Amino Acids↗
PubMed1983

Properties of the oxygenated form of liver microsomal cytochrome P-450.

The formation and autoxidative decomposition of the oxygenated forms of two isozymes of rabbit liver microsomal cytochrome P-450 were studied: 5,6-benzoflavone- or isosafrole-inducible P-450LM4, isolated in the high spin state, and P-450LM3b, isolated in the low spin state. When an anaerobic solution of photochemically reduced isozyme 4 was mixed with aerobic buffer in a stopped flow spectrophotometer, the dioxygen complex with absorption maxima at 555 and 418 nm was rapidly formed. The monophasic reaction had a pseudo-first order rate constant of about 58 s-1. Autoxidation of the complex, which was complete in about 20 s, exhibited biphasic first order kinetics at 580 nm with rate constants of about 0.92 and 0.22 s-1. The results obtained with isozyme 3b were similar, except that the decomposition of the ferrous oxy intermediate appeared to be triphasic. Superoxide could not be detected as a product of the autoxidative decomposition of the oxy form of P-450 isozyme 4. Hydrogen peroxide was produced in about 70% yield when oxygen was in excess, whereas in a titration in which small increments of oxygen were added to an excess of the ferrous cytochrome the reduction apparently led to the formation of water. The biphasic kinetics of some of the reactions involving purified mammalian cytochrome P-450 has in some instances been attributed to the formation of aggregates by these hydrophobic proteins. This was ruled out as an explanation of the kinetics observed for the autoxidative decay of ferrous dioxygen P-450LM4, since two phases were also observed with a preparation converted from the usual aggregated state to the monomeric state by exposure to a zwitterionic detergent.

Anaerobiosis↗
PubMed1983

Comparison of six rabbit liver cytochrome P-450 isozymes in formation of a reactive metabolite of acetaminophen.

This laboratory has recently reported the isolation of an ethanol-inducible form of rabbit liver microsomal cytochrome P-450, designated isozyme 3a. In view of the reports of others that the hepatotoxicity of acetaminophen is increased in ethanol-treated animals and the human alcoholic, we have determined the activity of the six available P-450 isozymes in the activation of the drug to give an intermediate which forms a conjugate with reduced glutathione. Isozymes 3a, 4, and 6, all of which are present in significant amounts in the liver microsomes from rabbits chronically administered ethanol, exhibited the highest activities in the reconstituted enzyme system, whereas isozymes 3b and 3c were 10- to 20-fold less effective, and phenobarbital-inducible isozyme 2 was essentially inactive, even in the presence of cytochrome b5. The results obtained thus indicate that induction by ethanol of P-450 isozyme 3a (or a homologous enzyme in other species) may contribute to the toxicity of acetaminophen but that other cytochromes also play a significant role.

Acetaminophen↗
PubMed1983

Alcohol oxidation by isozyme 3a of liver microsomal cytochrome P-450.

Liver microsomes from rabbits treated chronically with ethanol were solubilized and fractionated to yield a new isozyme of cytochrome P-450 in a homogeneous state. This cytochrome, designated as isozyme 3a on the basis of its relative electrophoretic mobility, is distinct from the known terminal amino acid sequences. In addition, peptide mapping by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome has unusually high activity in the oxidation of ethanol and other alcohols to aldehydes and in the rho-hydroxylation of aniline as compared with the other isozymes of P-450. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH-cytochrome P-450 reductase and is stimulated by the presence of phosphatidylcholine, is not due to contamination by catalase or an NAD+-or NADP+-dependent alcohol dehydrogenase.

Acetaldehyde↗
PubMed1983

Complete amino acid sequence and predicted membrane topology of phenobarbital-induced cytochrome P-450 (isozyme 2) from rabbit liver microsomes.

The complete amino acid sequence of phenobarbital-induced isozyme 2 of rabbit liver microsomal cytochrome P-450 (P-450LM2) is presented. The polypeptide consists of 491 residues with a calculated Mr of 55,755. The rabbit isozyme is 77% identical to the corresponding rat cytochrome, P-450b, as deduced from cDNA, with 96% of the hydrophobic, 88% of the anionic, and 83% of the cationic positions conserved. The secondary structure of isozyme 2 was predicted and a model was developed for the membrane topology of this cytochrome. Of the two highly conserved cysteinyl peptides in P-450LM2, P-450b, and bacterial P-450cam, we favor, on the basis of our model, the one nearer the NH2 terminus (Cys-152 in P-450LM2) as the source of the thiolate ligand to the heme iron atom. The recently reported sequence of the apparently identical protein [Heinemann, F. S. & Ozols, J. (1983) J. Biol. Chem. 258, 4195-4201] has two fewer residues and differs in 14 other amino acid assignments.

Amino Acid Sequence↗
PubMed1983

Inactivation of key metabolic enzymes by mixed-function oxidation reactions: possible implication in protein turnover and ageing.

Several mixed-function oxidation systems catalyze the inactivation of Escherichia coli glutamine synthetase. Inactivation involves modification of a single histidine residue in each enzyme subunit and makes the enzyme susceptible to proteolytic degradation. We show here that 10 key enzymes in metabolism are inactivated by a bacterial NADH oxidase and by an oxidase system comprised of NADPH, cytochrome P-450 reductase, and cytochrome P-450 isozyme 2 from rabbit liver microsomes. Most of the inactivatable enzymes require a divalent cation for activity and all but one (enolase) possess a nucleotide binding site. Glutamine synthetase, pyruvate kinase, and phosphoglycerate kinase are protected from inactivation by their substrates; substrate protection of other enzymes was not tested. We propose that inactivation involves mixed-function oxidization system-catalyzed synthesis of H(2)O(2) and reduction of Fe(III) to Fe(II) followed by oxidation of enzyme-bound Fe(II) by H(2)O(2) to generate oxygen radicals that attack a histidine (or other oxidizable amino acid) at the metal binding site of the enzyme. This is supported by the following: (i) most of the inactivation reactions are inhibited by EDTA and by catalase, (ii) both mixed-function oxidation systems reduce Fe(III), and (iii) H(2)O(2) together with Fe(II) catalyzes nonenzymic inactivation of glutamine synthetase. In view of the fact that inactivation of glutamine synthetase makes it susceptible to proteolytic degradation, it is possible that mixed-function oxidation system-catalyzed inactivation of enzymes is a regulatory step in enzyme turn-over. In addition, the implication of oxidative inactivation reactions in ageing is suggested by the fact that many of the enzymes inactivated by mixed-function oxidation systems are known to accumulate as inactive forms during ageing.

Aging↗
PubMed1982

Structural features of isozyme 2 of liver microsomal cytochrome P-450. Identification of a highly conserved cysteine-containing peptide.

Earlier findings in several laboratories have suggested that the various forms of cytochrome P-450 within a given species or from different species are not closely related. However, our present studies on the primary structure of the phenobarbital-inducible cytochrome from rabbit liver microsomes (P-450LM2) show that this protein is about 80% identical to the corresponding rat protein (P-450b), the sequence of which was recently deduced by others. For example, of the first 70 residues of the NH2-terminal sequences, 52 are identical, with most differences structurally conservative and attributable to point mutations. No evidence was found for heterogeneity in the region of the rabbit cytochrome corresponding to the variable region proposed for the rat cytochrome. A comparison of the known primary structure of P-450LM2 with that of P-450b and that of the bacterial camphor-hydroxylating cytochrome (P-450cam) as recently reported by others reveals only one highly conserved cysteine-containing peptide present in approximately the same position in each cytochrome. This peptide in the rabbit enzyme, -Arg-Ile-Gln-Glu-Glu-Ala-Arg-Cys147-Leu-Val-Glu-Glu-Leu-Arg-, and the corresponding peptides containing Cys152 in P-450b and Cys134 in P-450cam may serve an essential function such as providing the axial thiolate ligand to the heme iron atom.

Amino Acid Sequence↗
PubMed1982

Catalytic activity of cytochrome P-450 isozyme 3a isolated from liver microsomes of ethanol-treated rabbits. Oxidation of alcohols.

Cytochrome P-450 isozyme 3a, isolated from hepatic microsomes of rabbits treated chronically with ethanol, was found to have a unique substrate specificity when compared with isozymes 2, 3b, 3c, and 4. Form 3a has unusually high activity in the p-hydroxylation of aniline and in the oxidation of alcohols to aldehydes. These properties are reflected in the increased activities of these substrates in microsomes from ethanol-treated rabbits as compared to microsomes from untreated animals or those administered phenobarbital or 5,6-benzoflavone. The ethanol-oxidizing activity of isozyme 3a, which requires the presence of NADPH and NADPH cytochrome P-450 reductase and is stimulated by the presence of phospholipid, was shown not to be due to contaminating catalase or an NAD- or NADP-dependent alcohol dehydrogenase. Isozyme 3a catalyzes the oxidation of methanol, 1-propanol, and 1-butanol as well as ethanol; the relationships between the apparent Km values for these alcohols and their octanol-water partition coefficients is in accord with the known hydrophobic nature of the P-450 binding site. Whereas typical substrates of isozyme 2 are known to be metabolized with the stoichiometry predicted of a monooxygenase reaction, with isozyme 3a the sum of acetaldehyde formed from ethanol and of hydrogen peroxide generated is inadequate to account for the NADPH and oxygen consumed. Free hydroxyl radicals appear to mediate the slow oxidation of ethanol in the presence of the reductase alone but not the faster rate catalyzed by P-450 isozyme 3a. The results obtained, however, do not rule out the involvement of hydroxyl radical equivalent generated and bound at the active site of the cytochrome.

Animals↗
PubMed1982

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5.

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.

Alprostadil↗
PubMed1982

Oxidation-reduction states of FMN and FAD in NADPH-cytochrome P-450 reductase during reduction by NADPH.

NADPH-cytochrome P-450 reductase, a component of the multisubstrate monooxygenase system of liver microsomes, is an unusual flavoprotein in that it contains both FMN and FAD. In recent studies in this laboratory, a procedure was devised for selective removal of FMN from the purified enzyme, thus leading to the identification of FMN and FAD as the prosthetic groups of high and low reduction potential, respectively, and to the assignment of known reduction potentials to the individual flavin half-reactions. In the present study, the reaction of NADPH with the reductase was examined under anaerobic conditions by stopped flow spectrophotometry. The results were shown to correspond to those predicted on the basis of a model for the rapid exchange of reducing equivalents between the two flavins, the distribution being governed at any time by the reduction potentials for the individual flavin half-reactions. The reaction is divided into three steps, as follows (a) In a rapid first phase with a first order rate constant of 28 s-1, a mixture of about 70% (FMNH2, FAD) and 30% disemiquinone (FMNH ., FADH .) is generated; (FMN, FADH2), the presumed transient intermediate in the reduction of the oxidized flavoprotein by NADPH, does not accumulate under these conditions. (b) In a second phase characterized by a first order rate constant of 5.4 s-1, a mixture of 65% (FMNH2, FADH2), 24% (FMNH2, FAD), and 11% (FMNH ., FADH .) is produced. (c) Regardless of the NADPH concentration employed, a third phase occurs with very slow changes leading to an equilibrium mixture of the nine oxidation-reduction states of the reductase. The absorption spectra for all possible oxidation-reduction states of the FMN moiety of the reductase as well as of the native reductase are presented.

Cytochromes↗
PubMed1982

Purification and characterization of a unique isozyme of cytochrome P-450 from liver microsomes of ethanol-treated rabbits.

A new isozyme of cytochrome P-450 has been purified to electrophoretic homogeneity from hepatic microsomes of rabbits treated chronically with ethanol. Several criteria indicate that the ethanol-inducible cytochrome, which has a minimal molecular weight of 51,000 and is designated form 3a on the basis of its relative electrophoretic mobility, is distinct from the known isozymes of P-450. As judged spectrally, the new isozyme is high spin in the oxidized state, as is form 4, but differs in that the spin state is unperturbed by nonionic detergents. The absolute spectrum of the ferrous carbonyl complex of form 3a is red shifted as compared to that of forms 2, 3b, 3c, 4, and 6 and exhibits a maximum at 452 nm. The amino acid composition of form 3a is different from that of the other isozymes, and both the NH2- and COOH-terminal sequences are distinct; form 3a has an NH2-terminal alanine and a carboxyl-terminal leucine residue. Peptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis following treatment with papain, chymotrypsin, or Staphylococcus aureus V8 protease and by high performance liquid chromatography following trypsinolysis indicates that form 3a is a unique gene product. This cytochrome displays the highest activity of all of the rabbit isozymes in the oxidation of ethanol to acetaldehyde and the p-hydroxylation of aniline when reconstituted with NADPH-cytochrome P-450 reductase and phospholipid in the presence of NADPH and oxygen.

Amino Acid Sequence↗
PubMed1982

Structural features of liver microsomal NADPH-cytochrome P-450 reductase. Hydrophobic domain, hydrophilic domain, and connecting region.

Detergent-solubilized liver microsomal NADPH-cytochrome P-450 reductase is known to retain the ability to transfer electrons to cytochrome P-450, whereas the trypsin-solubilized reductase transfers electrons only to artificial acceptors. Due to the loss of a hydrophobic fragment by the action of trypsin, the altered reductase is no longer capable of binding cytochrome P-450. In the present study the primary tryptic attack on the rabbit reductase was shown to be at the Lys 44-Ile 45 bond to liberate the hydrophilic domain (molecular weight, 71,000) from the intact enzyme (molecular weight, 77,000). The other fragment (molecular weight, 4,800) undergoes tryptic attack at the Lys 34-Lys 35-Lys 36 sequence to yield a polypeptide representing the hydrophobic domain of the reductase and a nona- or decapeptide (Lys 35 or Lys 36 through Lys 44) which serves as the connecting region. The hydrophobic peptide, which is derived from the NH2-terminal end of the reductase, has an acetylated NH2 terminus and a region (Val 16 through Phe 32) which is exceptionally hydrophobic, with a predicted beta-sheet structure, and is believed to be involved in the binding of cytochrome P-450 and phospholipid. The site of attack on the reductase by various proteases is different, but the cleavage points are localized within a short segment of the polypeptide chain. A comparison of the tryptic forms (representing the hydrophilic domains) of the rabbit and rat reductases by terminal sequence analysis showed a high degree of similarity, with about 80% of the residues in exact correspondence and only a short variable region near the Ile NH2 terminus.

Amino Acid Sequence↗
PubMed1982

Heme ligand replacement reactions of cytochrome P-450. Characterization of the bonding atom of the axial ligand trans to thiolate as oxygen.

Evidence of several types has accumulated that cytochrome P-450 has a thiolate anion as one of the axial ligands to heme (the fifth ligand). On the other hand, there is as yet no general agreement on the nature of the axial ligand trans to thiolate (the sixth ligand), although nitrogen and oxygen have been proposed. To resolve the controversy, the ligand exchange reactions of cytochrome P-450 were investigated by the use of optical spectroscopy. Two isozymes of rabbit liver microsomal cytochrome P-450 were examined: the isozyme induced by phenobarbital (P-450LM2), which has a sixth ligand and is low spin and the isozyme induced by 5,6-benzoflavone (P-450LM4), which is without a sixth ligand and is high spin. A series of artificial ligands was chosen to model the coordination of each of the potential native ligands, including water and certain amino acid residues. When the artificial ligand coordinated through an oxygen atom, the spectrum of pentacoordinate P-450LM4 changed to one closely resembling that of native, hexacoordinate P-450LM2. The spectrum of P-450LM2 was unchanged in the presence of oxygen-coordinating ligands. However, when artificial ligands which coordinate through nitrogen or sulfur were added to either P-450LM2 or P-450LM4, the induced spectra did not resemble the native spectrum and, in fact, were distinctive and characteristic of the particular ligand type. With three of the artificial ligands, 1-butanol, 1-benzylimidazole, and diethylphenylphosphine, the binding was found to be reversible by dilution, ultrafiltration, or gel filtration. The binding of 1-pentanol and 1-benzylimidazole was competitive, as expected for heme ligands, and the number of 1-benzylimidazole binding sites per molecule of P-450LM2 was estimated as 1.1. These results provide strong evidence that the native sixth ligand in P-450LM2 is oxygen rather than nitrogen.

Animals↗
PubMed1982

Mobility and clusterlike organization of liposomal cytochrome P-450 LM2: saturation transfer EPR studies.

Rotational diffusion of the electrophoretically homogenenous isozyme cytochrome P-450 LM2 from rabbit liver microsomes has been studied in buffer solution and in phospholipid vesicles by means of saturation transfer EPR spectroscopy. Sulfhydryl groups of the enzyme were selectively modified using a maleimide spin label. The effective rotational correlation time of 220 ns for the rotation of cytochrome P-450 in buffer solution is consistent with the fact that the purified free enzyme occurs as an oligomeric (6-8 monomers) aggregate. Further, the clusters rotate almost isotropically and therefore are in a first approximation spherically shaped. The effective correlation time of about 180 microseconds observed strong immobilization thus evidencing protein aggregation within the membrane. The anisotropic character of the spectra indicates a nonspherical shape and/or anisotropic rotational motion of the cluster. The results are compared with corresponding data from cytochrome P-450 in microsomal form.

Animals↗