Metabolism of Metolachlor by a Microsomal Fraction Isolated from Grain Sorghum ( Sorghum bicolor) Shoots

1990 ◽  
Vol 45 (5) ◽  
pp. 558-564 ◽  
Author(s):  
Donald E. Moreland ◽  
Frederick T. Corbin ◽  
William P. Novitzky ◽  
Carol E. Parker ◽  
Kenneth B. Tomer
Keyword(s):  
Sorghum Bicolor ◽  
Microsomal Fraction ◽  
Grain Sorghum ◽  
Side Chain ◽  
Type I ◽  
Difference Spectra ◽  
Content Type ◽  
Actinic Light ◽  
Tlc Plates ◽  
Cytochrome P 450

A microsomal fraction isolated from the shoots of 3- to 4-day-old, dark-grown, grain sorghum (Sorghum bicolor cv. Funk G 522 D R ) seedlings was characterized. The preparations had a cytochrome P-450 content that varied from approximately 90 to 150 pmol P-450/mg protein with cytochrome P-420 varying from 0 to 3% of the P-450 content. Type I difference spectra were formed with cinnamic acid and metolachlor, and a type II spectrum was formed with tetcyclacis. In short-term assays with [14C]metolachlor as substrate, the preparations produced a single time-dependent product that separated on silica gel TLC plates developed in benzene/acetone (2:1, v/v). RF values for metolachlor and the metabolite were approximately 0.70 and 0.48, respectively. The microsomal reaction required N A D P H and oxygen, and was inhibited by carbon monoxide, with the inhibition being partially reversed by actinic light. Compounds known to inhibit the activity of cytochrome P-450 monooxygenases (piperonyl butoxide, tetcyclacis, and tridiphane) also prevented formation of the metabolite. Identity of the metabolite was confirmed by TLC and positive ion thermospray LC/MS to be 2-chloro-N-(2-ethyl-6- methylphenyl)-N-(2-hydroxy-l-methylethyl)acetamide. Hence, the reaction catalyzed by the sorghum microsomes involved O-demethylation of the methoxypropyl side chain of metolachlor.

scholarly journals Mechanism of inhibition by carbonyl cyanide m-chlorophenylhydrazone and sodium deoxycholate of cytochrome P-450-catalysed hepatic microsomal drug metabolism

1976 ◽  
Vol 160 (1) ◽  
pp. 75-83 ◽  
Author(s):  
I B Tsyrlov ◽  
O A Gromova ◽  
V V Lyakhovich
Keyword(s):  
Sodium Deoxycholate ◽  
Protonated Form ◽  
Microsomal Membrane ◽  
Limiting Factor ◽  
Type I ◽  
Difference Spectra ◽  
Nadph Cytochrome ◽  
Carbonyl Cyanide ◽  
Rate Limiting ◽  
Cytochrome P 450

1. Treatment of liver microsomal fraction with 0.03-0.12% sodium deoxycholate and 0.005-0.06 mM carbonyl cyanide m-chlorophenylhydrazone decreases phospholipid-dependent hydrophobicity of the microsomal membrane, assayed by the kinetics of 8-anilinonaphthalene-1-sulphonate binding and ethyl isocyanide difference spectra. 2. Sodium deoxycholate at a concentration of 0.01% lacks its detergent properties, but competitively inhibits aminopyrine binding and activates the initial rate of NADPH-cytochrome P-450 reductase. In the presence of 0.03-0.09% sodium deoxycholate the rate-limiting factor in p-hydroxylation of aniline is the content of cytochrome P-450. and that for N-demethylation of aminopyrine is the activity of NADPH-cytochrome P-450 reductase. 3. Carbonyl cyanide m-chlorophenylhydrazone has no effect on the binding and metabolism of aniline; investigation of its inhibiting effect on aminopyrine N-demethylase established that the rate-limiting reaction is the dissociation of the enzyme-substrate complex in the microsomal preparations. 4. In the mechanism of action of carbonyl cyanide m-chlorophenylhydrazone the key step may be the electrostatic interaction of its protonated form and one of the forms of activated oxygen at the catalytic centre of cytochrome P-450. 5. at least two different phospholipid-dependent hydrophobic zones are assumed to exist in the microsomal membrane, both coupled with cytochrome P-450. One of them reveals selective sensitivity to the protonation action of carbonyl cyanide m-chlorophenylhydrazone and contains the ‘binding protein’ for type I substrates and NADPH-cytochrome P-450 reductase; the other contains the cytochrome P-450 haem group and binding sites for type II substrates.

scholarly journals The effect of ethanol on drug oxidations in vitro and the significance of ethanol–cytochrome P-450 interaction

1973 ◽  
Vol 134 (2) ◽  
pp. 367-375 ◽  
Author(s):  
Dominick L. Cinti ◽  
Robert Grundin ◽  
Sten Orrenius
Keyword(s):  
Microsomal Fraction ◽  
Ethanol Concentration ◽  
Inhibitory Effect ◽  
Type I ◽  
Spectral Change ◽  
High Concentration ◽  
Liver Slices ◽  
Liver Microsomal Fraction ◽  
Cytochrome P 450 ◽  
High Ethanol

The effect of ethanol on N-demethylation of aminopyrine in rat liver slices and in the microsomal fraction and on microsomal hydroxylation of pentobarbital and aniline was studied. With liver slices N-demethylation of aminopyrine was stimulated by 35–40% at low ethanol concentrations (2mm), whereas no stimulation occurred at high concentrations (100mm). With the liver microsomal fraction, an inhibitory effect was observed only at high ethanol concentrations (100mm). This was also observed with the other drugs studied. In agreement with these results, only at a high concentration did ethanol interfere with the binding of drug substrates to cytochrome P-450. Further, as previously reported, ethanol produced a reverse type I spectral change when added to the liver microsomal fraction. Evidence that this spectral change is due to removal of substrate, endogenously bound to cytochrome P-450, is reported. A dual effect of ethanol is assumed to explain the present findings; in liver slices, at a low ethanol concentration, the enhanced rate of drug oxidation is the result of an increased NADH concentration, whereas the inhibitory effect observed with the microsomal fraction at high ethanol concentration is due to the interference by ethanol with the binding of drug substrates to cytochrome P-450.

scholarly journals The metabolism in vitro and hepatic microsomal interactions of some enantiomeric drug substrates

1970 ◽  
Vol 117 (5) ◽  
pp. 833-841 ◽  
Author(s):  
David S. Hewick ◽  
James R. Fouts
Keyword(s):  
Type I ◽  
Type Ii ◽  
Difference Spectra ◽  
Microsomal Cytochrome ◽  
Hepatic Microsomes ◽  
Michaelis Constants ◽  
Nadph Oxidation ◽  
Identical Type ◽  
Cytochrome P 450

1. The metabolism in vitro and microsomal interactions of (+)-amphetamine, (−)-amphetamine, (+)-benzphetamine and (−)-benzphetamine were studied with hepatic microsomes from phenobarbitone-pretreated male rabbits. 2. (+)-Benzphetamine was N-demethylated 30–35% faster than (−)-benzphetamine, but the apparent Michaelis constants for the two enantiomers were similar. 3. (−)-Amphetamine was deaminated about 200% faster than (+)-amphetamine. 4. The benzphetamine enantiomers gave qualitatively and quantitatively identical type I microsomal difference spectra (peak, 390nm; trough, 425nm) indicating identical apparent binding affinities for microsomes and identical spectral changes at maxima (ΔEmax. values). 5. The amphetamine enantiomers gave qualitatively identical type II microsomal difference spectra (peak, 433nm; trough, 395nm). However, the type II spectral data indicated that (+)-amphetamine had a markedly higher apparent binding affinity than (−)-amphetamine for microsomes. The amphetamine enantiomers gave identical ΔEmax. values. 6. The benzphetamine enantiomers (0.5mm) enhanced the rate of microsomal cytochrome P-450 reduction by NADPH by 400–500%, (+)-benzphetamine enhancing the rate 20–25% more than (−)-benzphetamine. 7. The amphetamine enantiomers decreased the rate of microsomal cytochrome P-450 reduction by NADPH. At a concentration of 2mm, (+)-amphetamine decreased the rate more than (−)-amphetamine. 7. All four enantiomers enhanced microsomal NADPH oxidation.

scholarly journals Azole Affinity of Sterol 14α-Demethylase (CYP51) Enzymes from Candida albicans and Homo sapiens

2012 ◽  
Vol 57 (3) ◽  
pp. 1352-1360 ◽  
Author(s):  
Andrew G. Warrilow ◽  
Josie E. Parker ◽  
Diane E. Kelly ◽  
Steven L. Kelly
Keyword(s):  
Candida Albicans ◽  
Dissociation Constants ◽  
Homo Sapiens ◽  
Type I ◽  
Binding Affinities ◽  
Cytochrome P450 Reductase ◽  
Difference Spectra ◽  
Content Type ◽  
Redox Partners ◽  
Uv Visible

ABSTRACTCandida albicansCYP51 (CaCYP51) (Erg11), full-lengthHomo sapiensCYP51 (HsCYP51), and truncated Δ60HsCYP51 were expressed inEscherichia coliand purified to homogeneity. CaCYP51 and both HsCYP51 enzymes bound lanosterol (Ks, 14 to 18 μM) and catalyzed the 14α-demethylation of lanosterol usingHomo sapienscytochrome P450 reductase and NADPH as redox partners. Both HsCYP51 enzymes bound clotrimazole, itraconazole, and ketoconazole tightly (dissociation constants [Kds], 42 to 131 nM) but bound fluconazole (Kd, ∼30,500 nM) and voriconazole (Kd, ∼2,300 nM) weakly, whereas CaCYP51 bound all five medical azole drugs tightly (Kds, 10 to 56 nM). Selectivity for CaCYP51 over HsCYP51 ranged from 2-fold (clotrimazole) to 540-fold (fluconazole) among the medical azoles. In contrast, selectivity for CaCYP51 over Δ60HsCYP51 with agricultural azoles ranged from 3-fold (tebuconazole) to 9-fold (propiconazole). Prothioconazole bound extremely weakly to CaCYP51 and Δ60HsCYP51, producing atypical type I UV-visible difference spectra (Kds, 6,100 and 910 nM, respectively), indicating that binding was not accomplished through direct coordination with the heme ferric ion. Prothioconazole-desthio (the intracellular derivative of prothioconazole) bound tightly to both CaCYP51 and Δ60HsCYP51 (Kd, ∼40 nM). These differences in binding affinities were reflected in the observed 50% inhibitory concentration (IC50) values, which were 9- to 2,000-fold higher for Δ60HsCYP51 than for CaCYP51, with the exception of tebuconazole, which strongly inhibited both CYP51 enzymes. In contrast, prothioconazole weakly inhibited CaCYP51 (IC50, ∼150 μM) and did not significantly inhibit Δ60HsCYP51.

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