Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1980 Jun 1;85(3):516–526. doi: 10.1083/jcb.85.3.516

Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. II. Evidence that a single enzyme accounts for the activity in its various subcellular locations

PMCID: PMC2111440  PMID: 7391132

Abstract

NADH-cytochrome b5 reductases of rat liver microsomes, mitochondria, and heavy and light Golgi fractions (GF3 and GF 1+2) were compared by antibody inhibition and competition experiments, by peptide mapping, and by CNBr fragment analysis. The water-soluble portion of the microsomal enzyme, released by lysosomal digestion and purified by a published procedure, was used to raise antibodies in rabbits. Contaminant antimicrosome antibodies were removed from immune sera by immunoadsorption onto the purified antigen, and the F(ab')2 fragments of the pure antireductase antibody thus obtained were found to inhibit the NADH-cytochrome c reductase activity equally well in the four membrane fractions investigated, with similar dose-response relationships. Moreover, the purified water-soluble fragment of microsomal reductase, which by itself is very inefficient in reducing cytochrome c, competed for antibody binding with the membrane-bound enzymes, and therefore prevented the inhibition of their activity not only in microsomes but also in the other fractions. The reductases isolated from detergent-solubilized microsomes, mitochondria, GF3, and GF1+2 by immunoadsorption had identical mobilities in SDS polyacrylamide gels. The corresponding bands were eluted from gels, fragmented with pepsin or CNBr treatment, and the two families of peptides thus obtained were analyzed by two-dimensional mapping and SDS polyacrylamide gel electrophoresis, respectively. Both analyses failed to reveal differences among reductases of the four fractions. These findings support the hypothesis that NADH-cytochrome b5 reductase in its various subcellular locations is molecularly identical.

Full Text

The Full Text of this article is available as a PDF (922.8 KB).

Selected References

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

  1. Bjerrum O. J. Immunochemical investigation of membrane proteins. A methodological survey with emphasis placed on immunoprecipitation in gels. Biochim Biophys Acta. 1977 Aug 9;472(2):135–195. doi: 10.1016/0304-4157(77)90016-8. [DOI] [PubMed] [Google Scholar]
  2. Borgese N., Meldolesi J. Immunological similarity of the NADH-cytochrome c electron transport system in microsomes, Golgi complex and mitochondrial outer membrane of rat liver cells. FEBS Lett. 1976 Apr 1;63(2):231–234. doi: 10.1016/0014-5793(76)80101-9. [DOI] [PubMed] [Google Scholar]
  3. Borgese N., Meldolesi J. Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. I. Distribution of the enzyme activity in microsomes, mitochondria, and golgi complex. J Cell Biol. 1980 Jun;85(3):501–515. doi: 10.1083/jcb.85.3.501. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Corte G., Tonda P., Cosulich E., Milstein C. P., Bargellesi A., Ferrarini M. Characterization of IgD. I. Isolation of two molecular forms from human serum. Scand J Immunol. 1979;9(2):141–149. doi: 10.1111/j.1365-3083.1979.tb02716.x. [DOI] [PubMed] [Google Scholar]
  5. Depierre J. W., Dallner G. Structural aspects of the membrane of the endoplasmic reticulum. Biochim Biophys Acta. 1975 Dec 29;415(4):411–472. doi: 10.1016/0304-4157(75)90006-4. [DOI] [PubMed] [Google Scholar]
  6. Fukushima K., Sato R. Purification and characterization of cytochrome b5-like hemoprotein associated with outer mitochondrial membrane of rat liver. J Biochem. 1973 Jul;74(1):161–173. doi: 10.1093/oxfordjournals.jbchem.a130219. [DOI] [PubMed] [Google Scholar]
  7. GREENWOOD F. C., HUNTER W. M., GLOVER J. S. THE PREPARATION OF I-131-LABELLED HUMAN GROWTH HORMONE OF HIGH SPECIFIC RADIOACTIVITY. Biochem J. 1963 Oct;89:114–123. doi: 10.1042/bj0890114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kraehenbuhl J. P., Racine L., Galardy R. E. Localization of secretory IgA, secretory component, and alpha chain in the mammary gland of lactating rabbits by immunoelectron microscopy. Ann N Y Acad Sci. 1975 Jun 30;254:190–202. doi: 10.1111/j.1749-6632.1975.tb29169.x. [DOI] [PubMed] [Google Scholar]
  9. Kuwahara S., Okada Y., Omura T. Evidence for molecular identity of microsomal and mitochondrial NADH-cytochrome b5 reductases of rat liver. J Biochem. 1978 Apr;83(4):1049–1059. doi: 10.1093/oxfordjournals.jbchem.a131993. [DOI] [PubMed] [Google Scholar]
  10. Lai C. Y. Studies on the structure of rabbit muscle aldolase. I. Cleavage with cyanogen bromide: an approach to the determination of the total primary structure. Arch Biochem Biophys. 1968 Oct;128(1):201–211. [PubMed] [Google Scholar]
  11. Mihara K., Sato R. Partial purification of NADH-cytochrome b 5 reductase from rabbit liver microsomes with detergents and its properties. J Biochem. 1972 Apr;71(4):725–735. [PubMed] [Google Scholar]
  12. Mihara K., Sato R., Sakakibara R., Wada H. Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase: location of the hydrophobic, membrane-binding region at the carboxyl-terminal end and the masked amino terminus. Biochemistry. 1978 Jul 11;17(14):2839–2834. doi: 10.1021/bi00607a020. [DOI] [PubMed] [Google Scholar]
  13. Mole L. R. A genetic marker in the variable region of rabbit immunoglobulin heavy chain. Biochem J. 1975 Nov;151(2):351–359. doi: 10.1042/bj1510351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Opheim D. J., Touster O. Lysosomal alpha-D-mannosidase of rat liver. Purification and comparison with the golgi and cytosolic alpha-D-mannosidases. J Biol Chem. 1978 Feb 25;253(4):1017–1023. [PubMed] [Google Scholar]
  15. Raftell M., Perlmann P. Antigen development in submicrosomal fractions of rat liver. Exp Cell Res. 1969 Sep;57(1):119–128. doi: 10.1016/0014-4827(69)90375-9. [DOI] [PubMed] [Google Scholar]
  16. Spatz L., Strittmatter P. A form of reduced nicotinamide adenine dinucleotide-cytochrome b 5 reductase containing both the catalytic site and an additional hydrophobic membrane-binding segment. J Biol Chem. 1973 Feb 10;248(3):793–799. [PubMed] [Google Scholar]
  17. Takesue S., Omura T. Immunological similarity between NADH-cytochrome c reductases of mitochondrial outer membrane and microsomes. Biochem Biophys Res Commun. 1970 Jul 27;40(2):396–401. doi: 10.1016/0006-291x(70)91022-3. [DOI] [PubMed] [Google Scholar]
  18. Takesue S., Omura T. Purification and properties of NADH-cytochrome b5 reductase solubilized by lysosomes from rat liver microsomes. J Biochem. 1970 Feb;67(2):267–276. doi: 10.1093/oxfordjournals.jbchem.a129250. [DOI] [PubMed] [Google Scholar]
  19. Takesue S., Omura T. Solubilization of NADH-cytochrome b5 reductase from liver microsomes by lysosomal digestion. J Biochem. 1970 Feb;67(2):259–266. doi: 10.1093/oxfordjournals.jbchem.a129249. [DOI] [PubMed] [Google Scholar]
  20. Tulsiani D. R., Six H., Touster O. Rat liver microsomal and lysosomal beta-glucuronidases differ in both carbohydrate and amino acid compositions. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3080–3084. doi: 10.1073/pnas.75.7.3080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Uemura T., Chiesara E. NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane. Eur J Biochem. 1976 Jul 1;66(2):293–307. doi: 10.1111/j.1432-1033.1976.tb10519.x. [DOI] [PubMed] [Google Scholar]
  22. Vannier C., Louvard D., Maroux S., Desnuelle P. Structural and topological homology between porcine intestinal and renal brush border aminopeptidase. Biochim Biophys Acta. 1976 Nov 11;455(1):185–199. doi: 10.1016/0005-2736(76)90163-2. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES