Demonstration and Biochemical Characterisation of Rat Brain NADPH-Dependent Diaphorase

An enzyme responsible for the NADPH-dependent reduction of nitroblue tetrazolium HCl (NBT) has been isolated from rat brain. Although other tetrazolium salts could be utilised, NBT was the preferred substrate, and the enzyme had an absolute requirement for NADPH. An in vitro assay was developed and used to determine the kinetic constants: Km NBT = 17.3 microM; Km NADPH = 1.9 microM, Vmax = 30.8 mumol product produced/min/mg protein. Substrate inhibition by NADPH was observed in some instances. Brain subcellular fractionation indicated highest enzyme activities in the microsomal fraction. Activity was present in all brain regions and in a variety of peripheral tissues. Relative molecular mass determinations of the native enzyme yielded an Mr = 170-180,000. It seems likely that the enzyme activity described in this study relates directly to the histochemical demonstration of brain NADPH-diaphorase-positive neurons. As yet, the natural substrate for the enzyme is unknown. However, the isolation and purification of NADPH-dependent diaphorase may be anticipated to assist in the elucidation of its function in the brain, and in the special characteristics of those neurons that contain the enzyme in abundance.