Heart diaphorase

The low molecular weight form of mitochondrial NADH dehydrogenase was first isolated from pig heart muscle by Edelhoch et al. (60) and Mahler and his associates 57 in 1952. The mitochondrial origin of the enzyme was demonstrated by de Bernard 81. These and similar preparations reported subsequently by Mackler 63, Kumar et al. 63, and Pharo et al. 64 were isolated by extracting the source material (heart muscle or various submitochondrial preparations) with 9-11% ethanol at pH 4.8-5.S and 43°-45°, a procedure originally devised for isolation of the Straub diaphorase (lipoyl dehydrogenase) 85. Two other preparations of basically similar composition and catalytic proper-... 

A more precise method of calibration, especially for complex solutions, is described in reference 14. This method involves complete oxidation of a known amount of NADH (spectrophotometrically determined) by an oxygen generating enzyme system such as heart mitochondria electron transport particles or diaphorase. 

H. H., Murad, F., Fdrstermann, U., and Burnstock, G. (1992). Nitric oxide synthase immunoreactivity and NADPH-diaphorase activity in a subpopulation of intrinsic neurones of the guinea-pig heart. Neurosci. Lett. 143, 65-68. 

Searls and Sanadi (1959, 1960a) and Massey et al. (1960) observed an increase in absorbancy in the region between 500 and 600 mju (maximum at 530 m/i), concomitant with a decrease at 455 mju, on reduction of pig heart dihydrolipoic dehydrogenase with DPNH and with dihydrolipoic acid.A similar effect was noted previously by Savage (1957) on reduction of Straub s diaphorase with DPNH. These results recalled similar observations by Beinert (1957) with other flavoproteins which were attributed to the formation of a flavin semiquinone. Massey ei al. (1960) have made a detailed study of the stoichiometry of formation of the 530-mju band and the kinetics of its formation and disappearance under a variety of conditions, and attributed it to a flavin semiquinone which is an obligatory intermediate in the catalytic cycle of the enzyme. Addition of p-chloro-mercuriphenyl sulfonate to the partially reduced flavoprotein resulted in disappearance of the red color and further reduction of the flavin. This observation was interpreted as indicating that the flavin semiquinone is stabilized by interaction with a protein sulfhydryl group. 

At first, the enzymes involved were found to be flavoproteins, capable of transferring an electron from NADH or NADPH to an electron acceptor. NADH and NADPH cytochrome c reductase, as well as NADH diaphorase, were found to have transhydro-genase properties. The flavin nucleotide is not involved in the reaction, because it can be extracted from the protein without interfering with the transhydrogenation. Later, true transhydrogenases were found. A transhydrogenase has been purified from the ammonium sulfate fraction of rat liver mitochondria. Kaplan and his associates purified a transhydrogenase from heart mitochondria. 

In 1929, Straub prepared from the Keilin-Hartree heart muscle preparation an enzyme that contained flavin adenine dinucleotide in its prosthetic group, and that catalyzed the oxidation of NADH by dyes, not by ferrous cytochrome c. This enzyme was called dia-phorase. Besides the fact that diaphorase reduces only dyes, the enzyme differs from the NADH cytochrome c reductase in a variety of ways among them are its solubility and its iron content. 

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