ENZYMES CATALYZING SEQUENTIAL REACTIONS IN MOUSE BRAIN AND LIVER SUPERNATANT FRACTIONS: I. DIFFERENTIAL USE OF JANUS GREEN B AND PHENAZINE METHOSULFATE

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Abstract
(1) Janus green B and phenazine methosulfate were used to determine the presence of single enzymes, or enzyme systems, which catalyze sequential reactions in brain and liver supernatants. (2) Spectrophotometric studies on the reduction of Janus green B by supernatants demonstrated the absence of functional flavoprotein in brain supernatants and their presence in liver supernatants, corroborating results obtained by manometry. (3) Brain supernatants contained at least 0.5 microgram of total flavin per ml equivalent; liver supernatant contained about 7 micrograms per ml equivalent. Therefore, the inability of brain supernatant to react with Janus green B was due not to the absence of riboflavin or flavin-adenine dinucleotide, but to the absence of a functional flavoprotein. (4) The absence of a functional flavoprotein did not eliminate the activity of the glycolytic enzyme system in brain supernatants as measured by substrate-dependent lactate accumulation. (5) A flavin inhibitor, acriflavin, did not significantly block glycolysis in brain or liver supernatants, either in the presence or absence of Janus green B. (6) There was no evidence for the presence of a soluble flavin inhibitor in brain supernatants. (7) Reduced diphosphopyridine nucleotide, used as substrate, was oxidized endogenously at a slow rate by both supernatants. This rate was increased by Janus green B in liver but not in brain supernatants. (8) In the presence of pyruvate and Janus green B, both supernatants rapidly oxidized reduced diphosphopyridine nucleotide, but the dye remained oxidized. This indicates that the flavindye-oxygen electron transport chain was inoperative in the presence of the lactic dehydrogenase-pyruvate system of both supernatants. (9) Where the absence of functional flavoprotein prevented the use of Janus green B for the demonstration of enzyme systems, phenazine methosulfate was used. Oxygen uptake in the presence of this dye was a measure of reduced diphosphopyridine nucleotide formed. Thus, it was a measure of the activity of enzymes or enzyme systems, of which diphosphopyridine nucleotide-requiring dehydrogenases are components. (10) By supplying phenazine methosulfate as terminal electron transport agent, both supernatants were shown to contain enzyme systems which metabolized fructose diphosphate, lactate, and malate. (11) The results are discussed with reference to the thesis that the enzyme systems occurring in supernatants represent fractions of those retained by the corresponding mitochondria.

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