Microtubules and the regulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase.
(33/898)
Cultured C-6 glial cells were utilized to evaluate the effect of antimicrotubular drugs on 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and cholesterol synthesis. Colchicine, Colcemid, and vinblastine (1.0 muM) caused a marked reduction in HMG-CoA reductase activity and, as a consequence, the rate of cholesterol synthesis in these cells. No effect was observed with lumicolchicine, a mixture of colchicine isomers with no effect on microtubules. The effect of colchicine was apparent within 1 h after addition to the culture medium, and, after 6 h, HMG-CoA reductase activity in treated cells was only approximately 15 to 30% of that in untreated cells. Reductase activity was very sensitive to the concentration of drug added, i.e. cells treated with just 0.1 muM colchicine for 6 h exhibited a 50% lower enzymatic activity than did untreated cells. The lack of a generalized, nonspecific toxic effect on the cells was indicated by the finding of no change in the activities of fatty acid synthetase and NADPH-cytochrome c reductase and the rate of total protein synthesis in cells treated with colchicine (1 muM) for 6 h. A close temporal and quantitative correlation was observed between the effects of colchicine on HMG-CoA reductase and on a parameter of microtubular function, i.e. maintenance of glial cell shape. The data suggest that microtubules are involved in the regulation of HMG-CoA reductase and cholesterol synthesis in C-6 glial cells. (+info)
Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase.
(34/898)
A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6. 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R. (+info)
Effects of the flavoprotein inhibitor, diphenyleneiodonium sulfate, on ex vivo organic nitrate tolerance in the rat.
(35/898)
The flavoprotein inhibitor, diphenyleneiodonium (DPI), inhibits the action of glyceryl trinitrate (GTN) and the D-enantiomer of isoidide dinitrate (IIDN), but not the L-enantiomer (L-IIDN), in isolated rat aorta via inhibition of the bioactivation of these prodrugs. Paradoxically, a vascular NAD(P)H oxidase, which also is inhibited by DPI, has been proposed to generate superoxide that quenches nitric oxide (NO) produced during GTN biotransformation, and increased oxidase levels are proposed to contribute to the phenomenon of organic nitrate tolerance. We examined the effect of DPI on isolated rat aorta using an in vivo model of organic nitrate tolerance. The EC(50) values for GTN-, D-IIDN-, and L-IIDN-induced relaxation of aorta from GTN-tolerant rats were increased 4.5- to 7.5-fold. Treatment of blood vessels with DPI (0.3 microM) increased the EC(50) values for GTN and D-IIDN by the same magnitude in control and tolerant aortae, a result that would not be predicted if DPI and GTN tolerance affected common targets. The expression of NADPH-cytochrome P450 reductase (CPR) during in vivo tolerance was assessed by NADPH-dependent cytochrome c reductase activity of aortic microsomes, immunoblotting, and Northern analysis. By all three determinants, CPR expression was unchanged in aorta from GTN-tolerant rats. Superoxide dismutase-inhibitable NADPH-dependent cytochrome c reductase activity (a measure of superoxide generation) of tolerant rat aortic microsomes was not different from that of controls. Superoxide dismutase-inhibitable NADH-dependent cytochrome c reductase activity was detected only in microsomes from tolerant animals. DPI caused a modest increase in the sensitivity for relaxation by the NO donor DEA NONOate to an equal extent in tolerant and nontolerant tissues, whereas the superoxide scavenger, 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), had no effect on the sensitivity for relaxation by GTN. These results would not be expected if tolerance-induced increases in superoxide were a causative factor for the reduced relaxation response in tolerance. We conclude that neither reduced flavoprotein-dependent metabolic activation of organic nitrates, such as that mediated by CPR, nor increased superoxide due to increased NAD(P)H oxidase activity can account for the development of in vivo tolerance to GTN. (+info)
Flexibility and stability of the structure of cytochromes P450 3A4 and BM-3.
(36/898)
The flexibility of the structure and compressibility of the respective active site of cytochromes P450 3A4 (CYP3A4) and BM-3 (CYP102) were studied using absorption spectroscopy in the ultraviolet and visual regions. Conformational changes in the overall protein structures of both CYP3A4 and CYP102 due to the effects of temperature and pressure are reversible. However, the enzymes differ in the properties of their active sites. The CYP3A4 enzyme denatures to the inactive P420 form relatively easy, at 3000 bar over half is converted to P420. The compressibility of its active site is lower than that of CYP102 and is greater with the substrate bound, which is in line with the observed lack of a stabilizing effect of the substrate on its conformation under pressure. In contrast, CYP102, although having the most compressible active site among the P450s, possesses a structure that does not denature easily to the inactive (P420) form under pressure. In this respect, it resembles the P450 isolated from acidothermophilic archaebacteria [McLean, M.A., Maves, S.A., Weiss, K.E., Krepich, S. & Sligar, S.G. (1998) Biochem. Biophys. Res. Commun. 252, 166-172]. (+info)
Conversion of pregnenolone to DHEA by human 17alpha-hydroxylase/17, 20-lyase (P450c17). Evidence that DHEA is produced from the released intermediate, 17alpha-hydroxypregnenolone.
(37/898)
Most previous studies using reconstituted systems and fast kinetics suggest that the conversion of pregnenolone to dehydroepiandrosterone (DHEA; the precursor of androgen and estrogen biosynthesis) by P450c17 does not require the release of the intermediate 17alpha-OHPreg (a precursor of cortisol biosynthesis). With such a mechanism, it is difficult to conceive how high amounts of DHEA may be produced in some cells or tissues, such as the testis and cells from the adrenal reticularis, while in other tissues such as the fasciculata zone, high levels of 17alpha-OHPreg are synthesized. In this report, we address this matter using intact transfected cells, which better reflect the actual cellular conditions. Furthermore, by using transfected cells, we can conveniently analyze human enzymes, as we are not restricted by the availability of human tissues as in the case of methods using purified or partially purified enzymes. Using intact HEK-293 cells transfected with human P450c17 in culture, we showed, in a time course study of the transformation of pregnenolone, that there is an accumulation of 17alpha-OHPreg, and that, subsequently, the accumulated 17alpha-OHPreg decreases with a concomitant increase in DHEA production. The DHEA/17alpha-OHPreg ratio changes from 0.1 :1 after 1 h incubation to 50 : 1 after 20 h. This result strongly suggests that the transformation of Preg to DHEA proceeds through two steps in which DHEA is produced from the released intermediate 17alpha-OHPreg. We also show that high levels of substrate vs. enzyme concentration will lead to high hydroxylase activity whereas the reverse will increase the lyase activity. The result is in good agreement with recent observations suggesting that surrounding enzymes and steroids could modulate the lyase activity. Cotransfection of vectors expressing cytochrome b5 and NADPH cytochrome P450 reductase indicates that both are required for an optimum production of DHEA. (+info)
Inhibitory effects of 1,4-naphthoquinone derivatives on rat cytochrome P4501A1-dependent monooxygenase activity in recombinant yeast microsomes.
(38/898)
We reported previously that various naphthoquinone derivatives inhibited cytochrome P450-dependent monooxygenase of liver and placenta microsomes [Muto, N. et al. (1987) Biochem. Biophys. Res. Commun. 146, 487-494]. To understand the complex inhibitory behaviors that were observed, it is desirable to study the relationship between structure and inhibitory activity of naphthoquinones in a simplified system containing a single P450 species. In the present study, the inhibitory effects of six derivatives of 1,4-naphthoquinone (hereafter referred to as NQ) on rat cytochrome P4501A1-dependent 7-ethoxycoumarin O-deethylation were examined using yeast microsomes containing overexpressed rat P4501A1. Of these, 2-methyl-5-hydroxy-NQ, 2-methyl-NQ, 2-hydroxy-NQ, and NQ showed competitive inhibition, whereas 5,8-dihydroxy-NQ and 5-hydroxy-NQ showed noncompetitive inhibition. Judging from the inhibitor constant (K(i)), the binding affinity of the four competitive inhibitors for the substrate-binding pocket of P4501A1 is in the order: 2-CH(3)-5-OH-NQ > 2-CH(3)-NQ > NQ >> 2-OH-NQ. On binding with P4501A1, 2-CH(3)-5-OH-NQ, 2-CH(3)-NQ, and NQ induced distinct Type II, Type I, and reverse Type I spectra, respectively. These results indicate that methyl and hydroxyl groups introduced into NQ have unique effects on their binding mode and binding affinity. (+info)
Characterization of human liver leukotriene B(4) omega-hydroxylase P450 (CYP4F2).
(39/898)
We previously reported the cloning of a human liver leukotriene B(4) (LTB(4)) omega-hydroxylase P450 designated CYP 4F2 [Kikuta et al. (1994) FEBS Lett. 348, 70-74]. However, the properties of CYP 4F2 remain poorly defined. The preparation solubilized using n-octyl-beta-D-glucopyranoside from microsomes of CYP 4F2-expressing yeast cells catalyzes v- hydroxylation of LTB(4), 6-trans-LTB(4), lipoxin A(4), 8-hydroxyeicosatetraenoate, 12-hydroxyeicosatetraenoate, and 12-hydroxystearate in the presence of rabbit liver NADPH-P450 reductase. In addition, the enzyme shows ethoxycoumarin O-deethylase and p-nitroanisole O-demethylase activities. The enzyme was purified to apparent electrophoretic homogeneity from yeast cells by sequential chromatography of solubilized microsomes through amino-n-hexyl-Sepharose 4B, DEAE-HPLC, and hydroxylapatite HPLC columns. The final preparation showed a specific content of 11.1 nmol of P450/mg of protein, with an apparent molecular mass of 56.3 kDa. CYP 4F2 was distinguished from the closely homologous CYP 4F3 (human neutrophil LTB(4) omega-hydroxylase) by its much higher K(m) for LTB(4), inability to omega-hydroxylate lipoxin B(4), and extreme instability. (+info)
Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase.
(40/898)
A Salmonella typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 together with human NADPH-cytochrome P450 reductase (OR) was established. The mutagen-activating capacity of human CYP2E1 for N-alkylnitrosamines was compared with that of CYP2A6 using the YG7108 2E1/OR and the YG7108 2A6/OR strains of SALMONELLA: Salmonella YG7108 2A6/OR is a derivative of YG7108 co-expressing CYP2A6 together with OR. Eight N-alkylnitrosamines, including N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA), N-nitrosomethylphenylamine (NMPhA), N-nitrosopyrrolidine (NPYR), N-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were examined. CYP2E1 expressed in the YG7108 2E1/OR cells showed mutagen-activating capacity, as indicated by induced revertants/min/pmol cytochrome P450, for NDMA, NDEA, NDPA, NDBA, NPYR and NNK, but not NMPhA and NNN. CYP2A6 activated NDMA, NDEA, NDPA, NDBA, NMPhA, NPYR, NNN and NNK. The ratio of the mutagen-activating capacity seen with CYP2A6 to that seen with CYP2E1 was calculated for each N-alkylnitrosamine. In the case of NDMA, NPYR and NDEA, the ratio was under 1.0, while the ratio was over 1.0 with NDPA, NDBA, NNK, NMPhA and NNN. We conclude that human CYP2E1 is mainly responsible for the metabolic activation of N-nitrosamines with a relatively short alkyl chain(s), whereas CYP2A6 was predominantly responsible for the metabolic activation of N-alkylnitrosamines possessing a relatively bulky alkyl chain(s). (+info)