Rat brain NADPH-dependent diaphorase. A possible relationship to cytochrome P450 reductase.

An enzyme (NADPH-dependent diaphorase) present in rat brain microsomes has been solubilised and shown to utilise both nitrobluetetrazolium and cytochrome c as electron acceptors, when reduced by NADPH. The kinetics of the enzyme have been determined using cytochrome c (Km = 1.3 microM), NADPH (Km = 1.4 microM) and the Vmax (4.7 nmol/min/mg solubilised microsome protein). The subunit Mr is approximately 73,000 D and that of the native enzyme is 170,000-180,000 D, indicating that the enzyme is probably a dimer. Evidence is also provided to show that the enzyme is a flavoprotein, and that it has equimolar amounts of FAD and FMN with respect to the subunit concentration. It seems a possibility that the rat brain diaphorase enzyme may be cytochrome P450 reductase, EC 1.6.2.4.

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.

Solubilisation of a glutamate binding protein from rat brain.

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.

Purification and analysis of mitochondrial membrane proteins on nondenaturing gradient polyacrylamide gels.

A nondenaturing gradient polyacrylamide gel electrophoresis method is described for the resolution of membrane proteins. Bovine heart inner mitochondrial membranes were solubilized in Triton X-100 and individual complexes were identified by staining for activity and protein. Succinate dehydrogenase was isolated by band excision and shown by electrophoresis under denaturing conditions to be highly purified. In addition, the electrophoretic transfer of NADH dehydrogenase to nitrocellulose was demonstrated. The enzyme was identified on the resulting blot by activity staining and the binding of monospecific antibodies.