Mechanism to study 1:1 stoichiometry of NADPH and alkoxyphenoxazones metabolism spectrophotometrically in subcellular biological preparations

被引：11

作者：

Das, M ^{[1
]}

Rastogi, S ^{[1
]}

Khanna, SK ^{[1
]}

机构：

[1] Ind Toxicol Res Ctr, Food Toxicol Lab, Lucknow 226001, Uttar Pradesh, India

来源：

BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS | 2004年 / 1675卷 / 1-3期

关键词：

alkoxyphenoxazone; cytochrome P-450; ethoxyresorufin-O-dealkylase; pentoxyresorufin-O-dealkylase; stoichiometry;

D O I：

10.1016/j.bbagen.2004.07.007

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

Our prior studies have shown that pentoxyresorufin-O-dealkylation (PROD) can be measured spectrophotometrically with simultaneous monitoring of stoichiometry of NADPH/substrate and NADP/product as 10:1:10:1 [Rastogi et al. FEBS Letters 512 (2002) 121-124]. In the present investigation, mechanism of action of other enzymes in modulating the stoichiometry of alkoxyphenoxazones metabolism to 1:1 for electron donor/substrate and oxidized electron donor/product in the same incubation mixture was studied. The spectrophotometric analysis reveals 10:1 ratio between NADPH and pentoxyresorufin (PRF)-ethoxyresorufin (ERF) in microsomal system. The high ratio of electron donor to substrate is due to the presence of the other forms of P-450, which may participate in endogenous metabolism of compounds, thereby reducing the ratio to 4:1 and 7:1 for NADPH/PRF-ERF. Incubation of dicumarol in the microsomal PROD or ethoxyresorufin-O-dealkylase (EROD) assay led to significant decrease in the consumption of NADPH with a ratio of 4:1 and 7:1 for NADPH/PRF-ERF which is due to inhibition of NADPH cytochrome c (P-450) reductase. In post mitochondrial fraction (S-9), the ratio of 11:1 and 15:1 is seen for NADPH/PRF-ERF. The addition of dicumarol in S-9 fraction showed enhanced rate of alkoxyphenoxazone utilization, suggesting the possibility of reduced resorufin product as a feedback inhibitor. Equating the ratio of NADPH/substrate(s) derived after endogenous utilization of NADPH with the ratio after accounting for NADPH consumption following dicumarol addition in either S-9 or microsomal fraction, a 1:1 mol of NADPH/substrate(s) and oxidized electron donor/product is obtained. The results further suggest that cytosolic fraction may interfere in monitoring the formation of resorufin during dealkylation of alkoxyphenoxazones making dicumarol a mandatory cofactor. (C) 2004 Elsevier B.V. All rights reserved.

引用

页码：1 / 11

页数：11

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