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Mouse liver microsomal hexose-6-phosphate dehydrogenase. NADPH generation and utilization in monooxygenation
Abstract:
Hexose-6-phosphate dehydrogenase (H6PD) activity in washed hepatic microsomes from male ICR mice, when assayed with NADP+ and deoxyglucose-6-phosphate, was partially latent. Brief sonication or detergents activated H6PD causing an approximately 4- and 8.5-fold increase in NADPH generation respectively. The sonicated microsomes exhibited H6PD-linked N-demethylase activity toward aminopyrine. This activity was best sustained in the presence of deoxyglucose-6-phosphate, while galactose-6-phosphate, glucose-6-phosphate, and glucose were less effective. Reaction media containing sonicated microsomes, NADP+ and deoxyglucose-6-phosphate also catalyzed N-demethylation of p-chloro-N-methylaniline, N,N-dimethylaniline and nicotine, O-demethylation of p-nitroanisole, p-hydroxylation of aniline, ring hydroxylation of biphenyl at the 2- and 4-positions, dearylation of parathion, and the N-oxidation of N,N-dimethylaniline. In general, the hexose-6-phosphate dehydrogenase-linked monooxygenation rates were 60% or more of those observed in the presence of exogenous NADPH.
Insights
Hexose-6-phosphate dehydrogenase (H6PD) activity in mouse liver microsomes is latent but can be activated. Activated H6PD supports various metabolic reactions, including N-demethylation and monooxygenation.
Area of Science:
- Biochemistry
- Enzymology
- Drug Metabolism
Background:
- Hepatic microsomes contain enzymes crucial for drug metabolism.
- Hexose-6-phosphate dehydrogenase (H6PD) is an enzyme found in liver microsomes.
- The activity of H6PD can be influenced by assay conditions.
Purpose of the Study:
- To investigate the latent activity of Hexose-6-phosphate dehydrogenase (H6PD) in mouse hepatic microsomes.
- To determine the effect of activation on H6PD activity and its role in metabolic reactions.
- To explore the substrate specificity of H6PD-linked enzymatic activities.
Main Methods:
- Washed hepatic microsomes from male ICR mice were used.
- H6PD activity was assayed using NADP+ and deoxyglucose-6-phosphate.
- Microsomes were activated by sonication or detergents.
- Various N-demethylation, O-demethylation, hydroxylation, dearylation, and N-oxidation reactions were monitored.
Main Results:
- H6PD activity in microsomes was partially latent, with activation yielding 4- to 8.5-fold increases in NADPH generation.
- Activated H6PD supported N-demethylation of aminopyrine, with deoxyglucose-6-phosphate being the most effective cofactor.
- H6PD-linked activities included N-demethylation, O-demethylation, hydroxylation, dearylation, and N-oxidation, with rates comparable to exogenous NADPH.
Conclusions:
- H6PD is a latent enzyme in hepatic microsomes that can be activated to support NADPH-dependent metabolic reactions.
- Deoxyglucose-6-phosphate is a preferred substrate for H6PD-linked N-demethylation.
- Activated H6PD contributes significantly to hepatic monooxygenase activities.