Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1984 Dec 1;224(2):525–534. doi: 10.1042/bj2240525

Photoaffinity labelling of mitochondrial NADH dehydrogenase with arylazidoamorphigenin, an analogue of rotenone.

F G Earley, C I Ragan
PMCID: PMC1144461  PMID: 6517863

Abstract

A photoaffinity-labelling analogue of the respiratory inhibitor rotenone was synthesized from the naturally occurring rotenoid amorphigenin. The analogue inhibits NADH-ubiquinone oxidoreductase activity at concentrations comparable with those of rotenone. Photolysis of the radiolabelled analogue bound to isolated NADH-ubiquinone oxidoreductase resulted in preferential incorporation of radioactivity into a polypeptide of Mr 33 000, particularly at low concentrations of the inhibitor. Preparations of the enzyme differ in a parallel fashion in the content of this polypeptide, the degree of photolabelling by the analogue and their sensitivity to rotenone, providing further evidence that the 33 000-Mr protein forms part of the rotenone-binding site.

Full text

PDF
525

Images in this article

531Fig. 6.
on p.531
531Fig. 7.
on p.531

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bayley H., Knowles J. R. Photogenerated reagents for membranes: selective labeling of intrinsic membrane proteins in the human erythrocyte membrane. Biochemistry. 1980 Aug 19;19(17):3883–3892. doi: 10.1021/bi00558a001. [DOI] [PubMed] [Google Scholar]
  2. Beinert H., Albracht S. P. New insights, ideas and unanswered questions concerning iron-sulfur clusters in mitochondria. Biochim Biophys Acta. 1982 Dec 31;683(3-4):245–277. doi: 10.1016/0304-4173(82)90003-9. [DOI] [PubMed] [Google Scholar]
  3. De Vries S., Albracht S. P., Berden J. A., Slater E. C. The pathway of electrons through OH2:cytochrome c oxidoreductase studied by pre-steady -state kinetics. Biochim Biophys Acta. 1982 Jul 22;681(1):41–53. doi: 10.1016/0005-2728(82)90276-6. [DOI] [PubMed] [Google Scholar]
  4. Earley F. G., Ragan C. I. Identification of the subunits of bovine heart mitochondrial NADH dehydrogenase that are exposed to the phospholipid bilayer by photo-labelling with 5-iodonaphth-1-yl azide. Biochem J. 1980 Nov 1;191(2):429–436. doi: 10.1042/bj1910429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fleet G. W., Knowles J. R., Porter R. R. The antibody binding site. Labelling of a specific antibody against the photo-precursor of an aryl nitrene. Biochem J. 1972 Jul;128(3):499–508. doi: 10.1042/bj1280499. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Galante Y. M., Hatefi Y. Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase. Arch Biochem Biophys. 1979 Feb;192(2):559–568. doi: 10.1016/0003-9861(79)90126-7. [DOI] [PubMed] [Google Scholar]
  7. Guillory R. J., Jeng S. J. Arylazido nucleotide analogs in a photoaffinity approach to receptor site labeling. Methods Enzymol. 1977;46:259–288. doi: 10.1016/s0076-6879(77)46029-4. [DOI] [PubMed] [Google Scholar]
  8. Gutman M., Coles C. J., Singer T. P., Casida J. E. On the functional organization of the respiratory chain at the dehydrogenase-coenzyme Q junction. Biochemistry. 1971 May 25;10(11):2036–2043. doi: 10.1021/bi00787a011. [DOI] [PubMed] [Google Scholar]
  9. Gutman M., Singer T. P., Casida J. E. Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase. XVII. Reaction sites of piericidin A and rotenone. J Biol Chem. 1970 Apr 25;245(8):1992–1997. [PubMed] [Google Scholar]
  10. Gutman M., Singer T. P. Spectrophotometric observations on the oxidation-reduction cycle of the respiratory chain-linked reduced nicotinamide-adenine dinucleotide dehydrogenase. Biochemistry. 1970 Nov 24;9(24):4750–4758. doi: 10.1021/bi00826a019. [DOI] [PubMed] [Google Scholar]
  11. HATEFI Y., HAAVIK A. G., GRIFFITHS D. E. Studies on the electron transfer system. XL. Preparation and properties of mitochondrial DPNH-coenzyme Q reductase. J Biol Chem. 1962 May;237:1676–1680. [PubMed] [Google Scholar]
  12. Hatefi Y., Stempel K. E. Isolation and enzymatic properties of the mitochondrial reduced diphosphopyridine nucleotide dehydrogenase. J Biol Chem. 1969 May 10;244(9):2350–2357. [PubMed] [Google Scholar]
  13. Heron C., Smith S., Ragan C. I. An analysis of the polypeptide composition of bovine heart mitochondrial NADH-ubiquinone oxidoreductase by two-dimensional polyacrylamide-gel electrophoresis. Biochem J. 1979 Aug 1;181(2):435–443. doi: 10.1042/bj1810435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Horgan D. J., Singer T. P., Casida J. E. Studies on the respiratory chain-linked reduced nicotinamide adenine dinucleotide dehydrogenase. 13. Binding sites of rotenone, piericidin A, and amytal in the respiratory chain. J Biol Chem. 1968 Feb 25;243(4):834–843. [PubMed] [Google Scholar]
  15. LINDAHL P. E., OBERG K. E. The effect of rotenone on respiration and its point of attack. Exp Cell Res. 1961 Mar;23:228–237. doi: 10.1016/0014-4827(61)90033-7. [DOI] [PubMed] [Google Scholar]
  16. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  17. Ragan C. I., Bloxham D. P. Specific labelling of a constituent polypeptide of bovine heart mitochondrial reduced nicotinamide-adenine dinucleotide-ubiquinone reductase by the inhibitor diphenyleneiodonium. Biochem J. 1977 Jun 1;163(3):605–615. doi: 10.1042/bj1630605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ragan C. I., Galante Y. M., Hatefi Y., Ohnishi T. Resolution of mitochondrial NADH dehydrogenase and isolation of two iron-sulfur proteins. Biochemistry. 1982 Feb 2;21(3):590–594. doi: 10.1021/bi00532a027. [DOI] [PubMed] [Google Scholar]
  19. Ragan C. I., Galante Y. M., Hatefi Y. Purification of three iron-sulfur proteins from the iron-protein fragment of mitochondrial NADH-ubiquinone oxidoreductase. Biochemistry. 1982 May 11;21(10):2518–2524. doi: 10.1021/bi00539a035. [DOI] [PubMed] [Google Scholar]
  20. Ragan C. I., Heron C. The interaction between mitochondrial NADH-ubiquinone oxidoreductase and ubiquinol-cytochrome c oxidoreductase. Evidence for stoicheiometric association. Biochem J. 1978 Sep 15;174(3):783–790. doi: 10.1042/bj1740783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ragan C. I. The structure and subunit composition of the particulate NADH-ubiquinone reductase of bovine heart mitochondria. Biochem J. 1976 Feb 15;154(2):295–305. doi: 10.1042/bj1540295. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Singer T. P., Gutman M. The DPNH dehydrogenase of the mitochondrial respiratory chain. Adv Enzymol Relat Areas Mol Biol. 1971;34:79–153. doi: 10.1002/9780470122792.ch3. [DOI] [PubMed] [Google Scholar]
  23. Smith S., Ragan C. I. The organization of NADH dehydrogenase polypeptides in the inner mitochondrial membrane. Biochem J. 1980 Feb 1;185(2):315–326. doi: 10.1042/bj1850315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suzuki H., King T. E. Evidence of an ubisemiquinone radical(s) from the NADH-ubiquinone reductase of the mitochondrial respiratory chain. J Biol Chem. 1983 Jan 10;258(1):352–358. [PubMed] [Google Scholar]
  25. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES