Demonstration and Biochemical Characterisation of Rat Brain NADPH-Dependent Diaphorase
- 1 April 1988
- journal article
- research article
- Published by Wiley in Journal of Neurochemistry
- Vol. 50 (4) , 1017-1025
- https://doi.org/10.1111/j.1471-4159.1988.tb10567.x
Abstract
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 utilized. 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 .mu.M; Km NADPH = 1.9 .mu.M, Vmax = 30.8 .mu.mol 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.This publication has 20 references indexed in Scilit:
- Selective Sparing of a Class of Striatal Neurons in Huntington's DiseaseScience, 1985
- NADPH-diaphorase: A selective histochemical marker for the cholinergic neurons of the pontine reticular formationNeuroscience Letters, 1983
- NADPH‐diaphorase: A selective histochemical marker for striatal neurons containing both somatostatin‐ and avian pancreatic polypeptide (APP)‐like immunoreactivitiesJournal of Comparative Neurology, 1983
- Histochemical demonstration of separate populations of somatostatin and cholinergic neurons in the rat striatumNeuroscience Letters, 1983
- A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye BindingAnalytical Biochemistry, 1976
- Terminal electron-transport mechanisms in the tubercle bacillus. A reduced diphosphopyridine nucleotide-nitro-blue tetrazolium diaphoraseBiochimica et Biophysica Acta, 1962
- Oxidation of reduced pyridine nucleotides in Pasteurella tularensisBiochimica et Biophysica Acta, 1961
- The relation of diaphorase and cytochrome reductaseBiochimica et Biophysica Acta, 1959
- Syntheses of Some p-Nitrophenyl Substituted Tetrazolium Salts as Electron Acceptors for the Demonstration of Dehydrogenases1Journal of the American Chemical Society, 1956
- STUDIES IN HISTOCHEMISTRY: XLII FURTHER STUDIES ON THE DETERMINATION OF SUCCINIC DEHYDROGENASE IN MICROGRAM AMOUNTS OF TISSUE AND DISTRIBUTION OF THE ACTIVITY IN THE BOVINE ADRENALJournal of Histochemistry & Cytochemistry, 1956