Characterisation of neuronal nadph-diaphorase activity

Kemp, Martyn Charles (1991) Characterisation of neuronal nadph-diaphorase activity. University of Southampton, Doctoral Thesis.

Abstract

The object of this work is to identify the enzyme responsible for the staining of the sparsely distributed medium aspiny NADPH-diaphorase positive neurons in rat brain. In human brain these diaphorase positive neurons have been shown to be selectively spared in Huntington's disease. This is a fully penetrative, progressive, hereditary neurodegenerative disease which usually first manifests itself in the fourth or fifth decade of life and causes increasingly severe disturbance of the control of voluntary muscles, cognisance and eventually death. This NADPH dependent diaphorase enzyme has oxido-reductive properties and other characteristics akin to the xenobiotic metabolising enzyme cytochrome P₄₅₀ reductase. If cytochrome P₄₅₀ reductase is the enzyme responsible for the diaphorase staining, it could, due to it's known xenobiotic metabolising action, impart some protection to its host cell against the as yet unknown causative agent of Huntington's disease. This work details the experiments performed to isolate, purify and characterise an enzyme from rat brain and liver using electrophoretic, ion-exchange and affinity column techniques amongst others. This enzyme is demonstrated to have very similar physical and biochemical characteristics as those published for the enzyme cytochrome P₄₅₀ reductase (EC 1.6.4.2). The important corresponding features being the molecular mass (146kD, with two subunits of 73kD), flavin content (equimolar FAD and FMN with the subunit), kinetics (Km= 1.7 μm ctochrome c, 1.15μm NADPH), activity towards substrates, inhibitors and inducers. The effects of inhibitors and inducers being demonstrated both in vitro and histochemically. Antibodies have been raised in rabbits against the purified enzyme and characterised to demonstrate their specificity to the antigen alone using electroblotting techniques. These antibodies have been subsequently used to demonstrate, immunocytochemically, that the NADPH-diaphorase positive neurons may contain amounts of cytochrome P₄₅₀ reductase which could be responsible for the diaphorase staining. These findings and their bearing on the possible reason why the NADPH-diaphorase neurons are selectively spared in Huntington's disease is fully discussed as is the possible aetiology of Huntington's disease, potential neurotoxic causative agents and alternative possibilities for the identity of the enzyme responsible for the NADPH-diaphorase staining in neurons.

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