CYP78A1 Preferentially Expressed in Developing Inflorescences of Zea mays Encoded a Cytochrome P450-Dependent Lauric Acid 12-Monooxygenase

Cytochrome P450 (P450 or CYP) monooxygenases play an important role in the oxidation of a number of lipophilic substrates including secondary metabolites in higher plants. Larkin reported that CYP78A1 was preferentially expressed in developing inflorescences of Zea mays (Larkin, Plant Mol. Biol. 25: 343-353, 1994). However, the enzymatic function of CYP78A1 hasn’t been clarified yet. To characterized the enzymatic activity of CYP78A1, in this study, CYP78A1 cDNA and tobacco or yeast NADPH-cytochrome P450 oxidoreductase (P450 reductase) was expressed in the yeast Saccharomyces cerevisiae AH22 cells under the control of alcohol dehydrogenase promoter I and terminator. The reduced CO-difference spectrum of a microsomal fraction prepared from the transformed yeast cells expressing CYP78A1 and yeast P450 reductase showed a peak at 449 nm. Based on the spectrum, the content of a P450 molecule was estimated to be 45 pmol P450 equivalent/mg of protein in the microsomal fraction. The recombinant yeast microsomes containing CYP78A1 and yeast P450 reductase were found to catalyze 12-monooxygenation of lauric acid. Based on these results, CYP78A1 preferentially expressed in developing inflorescences of Zea mays appeared to have participated in the monooxygenation of fatty acids.     

Cytochrome P-450 polypeptides in pulmonary microsomes from rats

Pulmonary microsomal polypeptides from different strains of rats were resolved using two-dimensional electrophoresis and were further characterized by in situ peptide mapping. Triton X-114 detergent separation was used to enrich cytochromes P-450 (P-450) and other integral membrane proteins from pulmonary microsomes, and these were directly compared with corresponding polypeptides from hepatic microsomes. The results demonstrated that P-450b and epoxide hydrolase were present in the lungs of male and female rats and that their expression in this tissue was independent of phenobarbital treatment. P-450e, which is co-induced with P-450b in the liver, was not detected in pulmonary microsomes under any condition. Four other pulmonary microsomal polypeptides were characterized and preliminary evidence suggested that they represent unique isozymic forms of P-450 with three of them being related to P-450b.     

Cytochrome P-450 mediated biotransformation of organic nitrates

The vascular biotransformation of organic nitrates appears to be a prerequisite for their action as vasodilators. In the current study, we assessed the involvement of cytochrome P-450 in the denitration of glyceryl trinitrate and the enantiomers of isoidide dinitrate. Denitration of organic nitrates by the microsomal fraction of rat liver was NADPH dependent and followed apparent first-order kinetics. Under aerobic conditions, the t1/2 of D-isoidide dinitrate was significantly shorter than that of L-isoidide dinitrate (11.9 vs. 14.1 min, p less than or equal to 0.05), which is consistent with the greater potency of the D-enantiomer for vasodilation. Under anaerobic conditions, the denitration of glyceryl trinitrate was very rapid (t1/2 approximately 30 s). Organic nitrate biotransformation was inhibited by carbon monoxide, SKF 525A, and dioxygen. This suggests that the biotransformation of organic nitrates can occur through the direct interaction with the heme moiety of cytochrome P-450. The biotransformation of glyceryl trinitrate was catalyzed preferentially by those isoenzymes induced by phenobarbital. The biotransformation of glyceryl trinitrate was regioselective for 1,3-glyceryl dinitrate formation except in phenobarbital-induced microsomes under aerobic conditions, in which preferential formation of 1,2-glyceryl dinitrate occurred. These data suggest that cytochrome P-450 is involved in the biotransformation of organic nitrates and raises the possibility that vascular cytochrome P-450 may play a role in the mechanism-based biotransformation of organic nitrates, the result of which is vascular smooth muscle relaxation.     

Cytochrome P-450 catalyzed redox cycling of orthophenylphenol

o-Phenylphenol was converted to 2,5-dihydroxy biphenyl (phenylhydroquinone) by microsomal P-450. Depending on the cofactor used, microsomal enzymes catalyzed oxidation and/or reduction of phenylhydroquinone. Phenylhydroquinone was oxidized to phenyl 2,5'-p-quinone by cumene hydroperoxide-supported microsomal P-450. Phenyl 2,5'-p-quinone was reduced to phenylhydroquinone by cytochrome P-450 reductase. This study provides direct evidence of cytochrome P-450 catalyzed redox cycling of o-phenylphenol. It is postulated that redox cycling of o-phenylphenol may play a role in o-phenylphenol-caused bladder cancer.     

Cytochrome P-450 and transformation from 18-hydroxycorticosterone to aldosterone]

The authors have studied the in vitro conversion of 18 hydroxycorticostérone to aldosterone (18 oxidation) by duck adrenal subcellular fractions. Considering the new hypothesis about the mechanism of this step (hydroxylation mechanism) the authors have investigated a possible relationship between this reaction and cytochrome P450. With experimental conditions described, data show that metyrapone, a cytochrome P450 competitive inhibitor does not inhibit 18 oxidation. In contrast, 18 oxidation is inhibited by spirolactones (spironolactones, canrenone, potassium canrenoate). These compounds act at the cytochrome P450 level but have also an uncoupling effect which has been recently discovered. The effects of metyrapone and spirolactones on 18 oxidation as well as the different behaviour between biologicaly and organically synthetised 18 hydroxycorticosterone allow us to propose hypotheses for the mechanism of this step.     

Cytochrome P-450-catalyzed dehydrogenation of 1,4-dihydropyridines

A variety of different 4-substituted 1,4-dihydropyridine Hantzsch esters are substrates for ring dehydrogenation by a cytochrome P-450 (P-450) enzyme (P-450 UT-A); the substitutent could be varied from a hydrogen to a naphthalenyl, but a pyrenyl derivative was not dehydrogenated. When a 4-alkyl group is present, both the P-450 which oxidizes the substrate and other P-450s can be inactivated (by putative alkyl radicals). P-450s did not discriminate with regard to removal of the 4-H atoms from an enantiomeric pair of dihydropyridines. Losses of the 4-proton and N-methyl from a N-methyl-1,4-dihydropyridine occur at similar rates. The calculated intrinsic kinetic hydrogen isotope effect (Dk) for dehydrogenation of 1,4-dihydro-2,6-dimethyl-4-phenyl-3,5-pyridinedicarboxylic acid dimethyl ester was 2.9 in a reconstituted P-450 UT-A enzyme system. No significant kinetic hydrogen isotope effect was observed in microsomal incubations for the dehydrogenation of this compound or 1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylic acid diethyl ester in a variety of competitive and noncompetitive experiments. In light of previous studies on the magnitude of kinetic hydrogen isotope effects in P-450 systems (e.g. Miwa et al., 1983 (Miwa, G. T., Walsh, J. S., Kedderis, G. L., and Hollenberg, P. F. (1983) J. Biol. Chem. 258, 14445-14449], the mechanistic proposals of Augusto et al., 1982 (Augusto, O., Beilan, H. S., and Ortiz de Montellano, P. R. (1982) J. Biol. Chem. 257, 11288-11295)) for enzyme inactivation by 4-alkyl-substituted Hantzsch pyridine esters, and other precedents for sequential electron transfer in amine oxidation by P-450s, we interpret these results as being consistent with P-450-mediated 1-electron oxidation of dihydropyridines followed by the facile loss of the 4-proton, with subsequent electron transfer to complete the reaction.     

Cytochrome P-450-mediated redox cycling of estrogens

The cytochrome P-450-mediated reactions of the synthetic stilbene estrogen (E)-diethylstilbestrol (DES) and of 2-hydroxyestradiol have been investigated in vitro. Depending on the cofactor used, microsomal enzymes catalyzed reductions and/or oxidations of the estrogens: Phenobarbital-induced rat liver microsomes catalyzed the oxidation of DES to 4',4"-diethylstilbestrol quinone (DES quinone) with cumene hydroperoxide as cofactor. The quinone was unstable and spontaneously rearranged to (Z,Z)-dienestrol. DES quinone was reduced to a mixture of E- and Z-isomers of DES by NADPH catalyzed by purified cytochrome P-450 reductase. After rearrangement of the quinone to (Z,Z)-dienestrol, reduction reactions did not proceed. Rat liver microsomes and NADPH catalyzed the conversion of DES to (Z,Z)-dienestrol and (Z)-DES, but DES quinone could not be detected. The reactions described provide direct evidence for microsome-mediated redox cycling of estrogens. Although DES quinone could not be detected in the incubation of DES, microsomes, and NADPH as cofactor, the intermediacy of the quinone is demonstrated by the formation of (Z,Z)-dienestrol, the marker product for oxidation. The quinone could not be detected because it was rapidly reduced to DES and its Z-isomer. Microsome-mediated redox cycling between 2-hydroxyestradiol and the corresponding quinone was also demonstrated. Using cumene hydroperoxide as cofactor, the oxidation to the quinone was favored, while with NADPH as cofactor the reduction to 2-hydroxyestradiol was preferred. It is postulated that microsome-mediated redox cycling of estrogens plays a role in hormonal carcinogenesis.     

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)

The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450.     

Lauric Acid Alleviates Neuroinflammatory Responses by Activated Microglia: Involvement of the GPR40-Dependent Pathway

In several neurodegenerative diseases such as Alzheimer’s disease (AD), microglia are hyperactivated and release nitric oxide (NO) and proinflammatory cytokines, resulting its neuropathology. Mounting evidence indicates that dietary supplementation with coconut oil (CNO) reduces the cognitive deficits associated with AD; however, the precise mechanism(s) underlying the beneficial effect of CNO are unknown. In the present study, we examined the effects of lauric acid (LA), a major constituent of CNO, on microglia activated experimentally by lipopolysaccharide (LPS), using primary cultured rat microglia and the mouse microglial cell line, BV-2. LA attenuated LPS-stimulated NO production and the expression of inducible NO synthase protein without affecting cell viability. In addition, LA suppressed LPS-induced reactive oxygen species and proinflammatory cytokine production, as well as phosphorylation of p38-mitogen activated protein kinase and c-Jun N-terminal kinase. LA-induced suppression of NO production was partially but significantly reversed in the presence of GW1100, an antagonist of G protein-coupled receptor (GPR) 40, which is an LA receptor on the plasma membrane. LA also decreased LPS-induced phagocytosis, which was completely reversed by co-treatment with GW1100. Moreover, LA alleviated amyloid-β-induced enhancement of phagocytosis. These results suggest that attenuation of microglial activation by LA may occur via the GPR40-dependent pathway. Such effects of LA may reduce glial activation and the subsequent neuronal damage in AD patients who consume CNO.     

Cytochrome P-450 and hydroxylating activity of microsomal preparations from whole houseflies

1. A new microsomal preparation, obtained from whole houseflies is described in terms of its cytochrome P-450 content and its hydroxylating activity. 2. Microsomes prepared from whole-fly brei, obtained with the aid of a mortar (a procedure that avoids the destruction of sarcosomes), contain 0.265nmol of cytochrome P-450 and hydroxylate naphthalene at a rate of 28.5nmol/mg of microsomal protein in 30min at 30 degrees C. This corresponds to 104nmol of naphthalene hydroxylated/nmol of cytochrome P-450. This is the highest rate ever reported for housefly and rat liver microsomal preparations. 3. Microsomal fractions prepared by procedures that do not retain the integrity of sarcosomes show the presence in the CO-difference spectrum of a 428nm peak. This cytochrome is associated with sarcosomal microsomes and it may be involved in the inhibition of insect microsomal mixed-function oxidases, although other factors cannot be discarded at present. 4. The inability to show cytochrome P-450 in microsomal fractions isolated from whole houseflies by other procedures may be at least partially due to a masking effect brought about by contamination with the sarcosomal cytochrome.