Abstract
Cytochemical methods involving metal chelation of the formazan of an N-thiazol-2-yl tetrazolium salt are described for the localization of diphosphopyridine nucleotide diaphorase (DPND) and triphosphopyridine nucleotide diaphorase (TPND) in mitochondria. These methods utilize the reduced coenzymes DPNH or TPNH as substrate. The reaction involves a direct transfer of electrons from reduced coenzyme to the respective diaphorase which in turn transfers the electrons to tetrazolium salt, reducing it to the insoluble formazan. Competition for electrons by preferential acceptors in the respiratory chain was prevented by various inhibitors. In the presence of respiratory inhibitors the rate of tetrazolium reduction was markedly increased. The greatest reduction was observed when amytal was used. Sites of diaphorase activity appeared as deposits of blue-black metal formazan chelate measuring 0.2 to 0.3 µ in diameter. Small mitochondria contained 2 deposits, while larger ones contained up to 6. Considerable differences were observed in the rate of tetrazolium reduction and cellular localization of diaphorase activity when DPNH was used as substrate as compared to TPNH. In each instance DPNH was oxidized more rapidly by tissues than TPNH. These findings support the concept that the oxidation of coenzymes I and II is mediated through separate diaphorases.
Full Text
The Full Text of this article is available as a PDF (725.6 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- BALTSCHEFFSKY H. DPNH-oxidase activity in fragments of rat liver mitochondria. Exp Cell Res. 1957 Dec;13(3):630–632. doi: 10.1016/0014-4827(57)90101-5. [DOI] [PubMed] [Google Scholar]
- BRODIE A. F., GOTS J. S. The reduction of tetrazolium salts by an isolated bacterial flavoproteins. Science. 1952 Nov 28;116(3022):588–589. doi: 10.1126/science.116.3022.588. [DOI] [PubMed] [Google Scholar]
- BRODY T. M., BAIN J. A. Effect of barbiturates on oxidative phosphorylation. Proc Soc Exp Biol Med. 1951 May;77(1):50–53. doi: 10.3181/00379727-77-18674. [DOI] [PubMed] [Google Scholar]
- ESTABROOK R. W. Kinetic properties of a reduced diphosphopyridine nucleotide cytochrome c reductase from heart muscle. J Biol Chem. 1957 Aug;227(2):1093–1108. [PubMed] [Google Scholar]
- FARBER E., STERNBERG W. H., DUNLAP C. E. Histochemical localization of specific oxidative enzymes. I. Tetrazolium stains for diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase. J Histochem Cytochem. 1956 May;4(3):254–265. doi: 10.1177/4.3.254. [DOI] [PubMed] [Google Scholar]
- Green D. E., Dewan J. G., Leloir L. F. The beta-hydroxybutyric dehydrogenase of animal tissues. Biochem J. 1937 Jun;31(6):934–949. doi: 10.1042/bj0310934. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HESS R., SCARPELLI D. G., PEARSE A. G. Cytochemical localization of pyridine nucleotide-linked dehydrogenases. Nature. 1958 May 31;181(4622):1531–1532. doi: 10.1038/1811531a0. [DOI] [PubMed] [Google Scholar]
- LEHNINGER A. L. Phosphorylation coupled to oxidation of dihydrodiphosphopyridine nucleotide. J Biol Chem. 1951 May;190(1):345–359. [PubMed] [Google Scholar]
- NACHLAS M. M., WALKER D. G., SELIGMAN A. M. A histochemical method for the demonstration of diphosphopyridine nucleotide diaphorase. J Biophys Biochem Cytol. 1958 Jan 25;4(1):29–38. doi: 10.1083/jcb.4.1.29. [DOI] [PMC free article] [PubMed] [Google Scholar]
- NASON A., DONALDSON K. O., LEHMAN I. R. The role of vitamin E at the enzymatic level. Trans N Y Acad Sci. 1957 Nov;20(1):27–50. doi: 10.1111/j.2164-0947.1957.tb00571.x. [DOI] [PubMed] [Google Scholar]
- PEARSE A. G. Intracellular localisation of dehydrogenase systems using monotetrazolium salts and metal chelation of their formazans. J Histochem Cytochem. 1957 Sep;5(5):515–527. doi: 10.1177/5.5.515. [DOI] [PubMed] [Google Scholar]
- Straub F. B. Isolation and properties of a flavoprotein from heart muscle tissue. Biochem J. 1939 May;33(5):787–792. doi: 10.1042/bj0330787. [DOI] [PMC free article] [PubMed] [Google Scholar]