ENZYMES CATALYZING SEQUENTIAL REACTIONS IN MOUSE BRAIN AND LIVER SUPERNATANT FRACTIONS: II. THE USE OF PHENAZINE METHOSULFATE

JANE SHOWACRE ¹ and HERMAN G. DU BUY ¹

¹ National Institutes of Health, Bethesda 14, Maryland.

1 . Spectrophotometric and manometric studies of pertinent non-enzymic solutions showed that reduced diphosphopyridine nucleotide is rapidly oxidized by phenazine methosulfate. The reduced phenazine methosulfate (PMS) is gradually oxidized by molecular oxygen. H₂O₂ is formed. A portion of this H₂O₂ is spontaneously split into H₂O and O₂

2. In mouse brain and liver supernatants, with PMS as dye and reduced diphosphopyridine nucleotide (DPNH) as substrate, the same reactions occur. In addition, the formed H₂O₂ is split by catalase demonstrated to be present in the supernatants used. Th oxygen taken up was a direct measure of the DPNH oxidized.

3. Pyruvate was the main competing electron acceptor studied. When it was added to, or formed by the supernatants, the oxygen uptake during DPNH-oxidation through PMS was reduced, depending on the relative concentration of the electron transferring systems present. In that case the amount of oxygen consumed, when added to the amount of lactate accumulated, was a direct measure of the DPNH oxidized.

4. When oxidized diphosphopyridine nucleotide (DPN) and an appropriate substrate were added to supernatants, the reoxidation by PMS of the DPNH formed by the DPN-requiring dehydrogenase components of a given enzyme group was measured. The oxygen uptake was a measure of the amount of DPNH formed, and thus a measure of the enzymic breakdown of the substrate by the enzyme system under study.

5. When the metabolism of a substrate by the enzyme system under study included the formation of pyruvate, the PMS-dependent oxygen uptake, added to the lactate accumulated, was a measure of the activity of the enzyme system.

6. Homogenized brain tissue yielded a supernatant with fragmented enzyme groups lacking functional flavoprotein. By contrast, as shown previously, this tissue yielded a mitochondrial fraction which retained all of the enzynes necessary for an integrated glucose-metabolizing enzyme system.

7. Homogenized liver tissue yielded a supernatant with flavoprotein linked enzyme systems less fragmented than those found in brain supernatants. At the same time it yields a mitochondrial fraction with an incomplete glycolytic enzyme system.

8. The data support the thesis proposed previously, that the enzymes found in the supernatant fraction of brain and liver cells represent mainly enzymes washed from the mitochondria, and therefore complementary to those retained by these organelles.

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