Bulletin of the Korean Chemical Society

Korean Chemical Society (대한화학회)
권호 표지
DOI QR코드

DOI QR Code

Purification and Comparison of NADH-Cytochrome $b_5$ Reductase from Mitochondrial Outer Membrane of Bovine Heart and Turnip

  • Published : 1998.02.20
Download PDF
⟨ Previous Next ⟩

Abstract

The NADH-cytochrome b5 reductase (NCBR), a mitochondrial external electron carrier, was purified from bovine heart and turnip and their properties were examined. The mitochondrial outer membranes separated were subjected to NCBR isolation through DEAE-Cellulose ion exchange, DEAE-Sephadex gel chromatography, and hydroxyapatite adsorption chromatography. These processes yielded the purification folds of 88 and 42 and the recovery percentages of 0.2%, 5.67% for turnip and bovine heart, respectively. The molecular weight of the NCBR from the two sources was estimated to be 35,000 using SDS polyacrylamide gel electrophoresis. The Michaelis constant Km and maximum velocity Vmax were determined by measuring the NADH-ferricyanide redox system as well as the NADPH-ferricyanide redox system. The kinetics showed that both NCBRs had higher affinities for NADH than artificial electron-acceptor substrate ferricyanide. Although NADPH had a lower affinity for the enzymes than NADH, this study showed the 2'-phosphate dinucleotide could be used as a substrate.

Keywords

References

  1. FEBS Lett. v.61 Nicholls, D. G.
  2. Biochim. Biophys. Acta v.549 no.1 Nicholls, D. G.
  3. Physiol. Rev. v.64 Nicholls, D. G.;Locke, R. M.
  4. Membrane Bioenergetics Sli;acjev. V. P.
  5. J. Biol. Chem. v.190 Lehninger, A. F.
  6. Biochim. Biophys. Acta v.501 Mokhova, E. N.;Skulachev, V. P.;Zhigacheva, I. V.
  7. Biochim. Biophys. Acta v.806 Kirillova, G. P.;Ablyayeva, N. A.;Mokhova, E. N.
  8. Fed. Pro. v.22 Beyer, R. E.
  9. J. Biol. Chem. v.256 Bernardi, P.;Azzone, G. F.
  10. Eur. J. Biochem. v.132 Lederer, F.;Ghrir, R.;Guiard, B.;Cortial, S.;Ito, A.
  11. Biochem. Biophys. Res. Commun. v.96 Kuwahara, S.;Omura, T.
  12. J. Biol. Chem. v.259 Pompon, D.;Coon, M. J.
  13. Pharmacal. Ther. v.2 Oshino, N.
  14. J. Biol. Chem. v.221 Strittmatter, P.;Velick, S. F.
  15. J. Biol. Chem. v.228 Strittmatter, P.;Velick, S. F.
  16. Biochem. Biphys. Res. Commun. v.40 Takesue, S.;Omura, T.
  17. Arch. Biochem. Biophys. v.171 Day, D. A.;Wiskich, J. T.
  18. Mitochondria Ragan, C. I.;Wilson, M. T.;Darley-Usmar, V. M.;Lowe, P. N.
  19. Methods in Enzymology v.55 Comte, J.;Gautheron, D. C.
  20. The Tools of Biochemistry Cooper, T. G.
  21. Methods in Enzymology v.5 Levin, O.
  22. Korean Biochem. J. v.27 Lee, S. J.;Lee, J. Y.
  23. Methods in Enzymology v.52 Mihara, K.;Sato, R.
  24. Nature v.277 Laemmli, U. K.
  25. Gel Electrophoresis of Proteins Hames, B. D.
  26. Anal. Biochem. v.72 Bradford, M. M.
  27. Methods in Enzymology v.182 Stoscheck, C.
  28. J. Biol. Chem. v.243 Ito, A.;Sato, R.
  29. J. Biol. Chem. v.248 Spartz, L.;Strittmatter, P.
  30. Biochemistry (4th.) Stryer, L.