Cytochemical localization of certain oxidative enzymes.

Employing the technique of cell fractionation, Schneider and Hogeboom (’56) described the localization of certain oxidative enzymes. They reported that cytochrome oxidase and succinic dehydrogenase were found only in the mitochondrial fraction while isocitric dehydrogenase was present in the supernatant and DPN and TPN cytochrome c reductases were observed to be in both fractions. More recently Thorne (’60) has reported a mitochondrial and a supernatant malic dehydrogenase. In contrast to these reports on the localization of certain oxidative enzymes is the intracellular distribution as demonstrated by histochemical procedures. Scarpelli, Hess and Pearse (’58), Hess, Scarpelli and Pearse (’58), and Pearse (’60) reported that all pyridine nucleotide-linked dehydrogenases and DPN and TPN diaphorases were confined to the mitochondria. Nachlas, Walker and Seligman (’58) described the intramitochondrial localization of DPN diaphorase and extramitochondrial localization of TPN diaphorase. Becker (’61) observed the mitochondrial localization of TPN diaphorase and intraand extramitochondrial distribution of DPN diaphorase. The obvious discrepancy between the evidence gained by the biochemical and histochemical methods might appear unresolvable except that the reliability of certain of the techniques employed in the previous histochemical studies are subject to question. The current methods for the histochemical demonstration of oxidative enzyme activity depend upon the incubation of cells or tissues in a buffered medium containing the substrate, required cofactors, and a tetrazolium salt. In the presence of the enzyme the substrate is oxidized and the electrriis made available by the oxidation are transferred (see below) to the tetrazolium salt reducing it to an insoluble formazan and thus indicating the site of enzyme activity. Of prime importance in these methods are the cofactors, the electron transferring agents and the tetrazolium salt. Several tetrazolium compounds have been utilized for the demonstration of oxidative enzymes. Farber, Sternberg and Dunlap ( ’56) tested the available tetrazolium salts and reported that the best histological definition was obtained with neotetrazolium and blue tetrazolium. These compounds have subsequently been replaced by more sensitive salts. Nachlas et al. (’57) introduced the dye, Nitro-BT, ( 2,2’-di-p-nitropheny1-5,5’-diphenyl-3,3’( 3, 3’dimethoxy 4,4’biphenylene) ditetrazolium chloride) while Pearse (’57) advocated the use of MTT (3,5-diphenyl-2-(4, 5’-dimethylthiazol-2-yl) tetrazolium bromide) chelated with cobaltous ions. Novikoff, Shin and Drucker (’61) have reported that, of the two compounds, Nitro-BT is superior for critical studies of mitochondrial morphology and oxidative enzyme activity since the deposition of MTT-Co++ is determined by physicochemical factors other than enzyme localization. As demonstrated by Brodie and Gots (’51 ) the enzymatic reduction of the tetrazolium salt is dependent upon the presence of a flavoprotein, a diaphorase. In actuality with the methods employed, it is the diaphorase which is being localized and not the dehydrogenase (Farber, Sternberg and Dunlap, ’56; Nachlas, Walker and Seligman, ’58). Complications do not arise as long as the dehydrogenase and