Mechanism to study 1:1 stoichiometry of NADPH and alkoxyphenoxazones metabolism spectrophotometrically in subcellular biological preparations.
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.
Das
Mukul
M
Food Toxicology Laboratory, Industrial Toxicology Research Centre, Mahatma Gandhi Marg, PO Box 80, Lucknow-226001, India. mditrc@rediffmail.com
Rastogi
Shipra
S
Khanna
Subhash K
SK
D000478:Alkylation; D000818:Animals; D065607:Cytochrome P-450 Enzyme Inhibitors; D003600:Cytosol / Q000378:metabolism; D001728:Dicumarol / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D010078:Oxazines / Q000378:metabolism*; D010084:Oxidation-Reduction; D051381:Rats; D013053:Spectrophotometry; D013347:Subcellular Fractions
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D010078:Oxazines; D009249:NADP; C007649:ethoxyresorufin; D001728:Dicumarol; C044943:pentoxyresorufin