In view of the extensive interest in microsomal NADH and NADPH oxidases -for example, in the relationship of NADPH oxidase to drug-metabolizing enzymes-optimal conditions for the assay of these enzymes were established. Optimal pH was found to depend upon the buffer employed. Variation of microsomal concentration did not give strictly linear rates; high concentration of microsomes caused a decrease in observed reaction rate. In high concentration, both substrates were inhibitory. The reported ADP-stimulated oxidation of NADPH by rat liver microsomes could be confirmed only when particular reaction conditions were employed. The buffer used is crucial, and manometric and spectrophotometric assays may yield divergent results. Poor correlation was found between the extent of metabolism of drugs by NADPH-linked microsomal enzymes and the ability of the drugs to block the stimulation by ADP of oxygen uptake by microsomes in the presence of NADPH. This uptake has previously been suggested to involve the peroxidation of microsomal lipid.
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