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. 1986 Oct 15;239(2):393–403. doi: 10.1042/bj2390393

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

N Borgese, G Pietrini
PMCID: PMC1147293  PMID: 3814080

Abstract

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.

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Selected References

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

  1. Amar-Costesec A., Beaufay H., Wibo M., Thinès-Sempoux D., Feytmans E., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. II. Preparation and composition of the microsomal fraction. J Cell Biol. 1974 Apr;61(1):201–212. doi: 10.1083/jcb.61.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Beaufay H., Amar-Costesec A., Feytmans E., Thinès-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. I. Biochemical methods. J Cell Biol. 1974 Apr;61(1):188–200. doi: 10.1083/jcb.61.1.188. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bensadoun A., Weinstein D. Assay of proteins in the presence of interfering materials. Anal Biochem. 1976 Jan;70(1):241–250. doi: 10.1016/s0003-2697(76)80064-4. [DOI] [PubMed] [Google Scholar]
  4. Borgese N., Gaetani S. In vitro synthesis and post-translational insertion into microsomes of the integral membrane protein, NADH-cytochrome b5 oxidoreductase. EMBO J. 1983;2(8):1263–1269. doi: 10.1002/j.1460-2075.1983.tb01579.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Borgese N., Gaetani S. Site of synthesis of rat liver NADH--cytochrome b5 reductase, an integral membrane protein. FEBS Lett. 1980 Apr 7;112(2):216–220. doi: 10.1016/0014-5793(80)80183-9. [DOI] [PubMed] [Google Scholar]
  6. Borgese N., Macconi D., Parola L., Pietrini G. Rat erythrocyte NADH-cytochrome b5 reductase. Quantitation and comparison between the membrane-bound and soluble forms using an antibody against the rat liver enzyme. J Biol Chem. 1982 Nov 25;257(22):13854–13861. [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Ehrenreich J. H., Bergeron J. J., Siekevitz P., Palade G. E. Golgi fractions prepared from rat liver homogenates. I. Isolation procedure and morphological characterization. J Cell Biol. 1973 Oct;59(1):45–72. doi: 10.1083/jcb.59.1.45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  11. Fujiki Y., Hubbard A. L., Fowler S., Lazarow P. B. Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum. J Cell Biol. 1982 Apr;93(1):97–102. doi: 10.1083/jcb.93.1.97. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. GARFINKEL D. Studies on pig liver microsomes. I. Enzymic and pigment composition of different microsomal fractions. Arch Biochem Biophys. 1958 Oct;77(2):493–509. doi: 10.1016/0003-9861(58)90095-x. [DOI] [PubMed] [Google Scholar]
  13. Howell K. E., Palade G. E. Hepatic Golgi fractions resolved into membrane and content subfractions. J Cell Biol. 1982 Mar;92(3):822–832. doi: 10.1083/jcb.92.3.822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hubbard A. L., Wall D. A., Ma A. Isolation of rat hepatocyte plasma membranes. I. Presence of the three major domains. J Cell Biol. 1983 Jan;96(1):217–229. doi: 10.1083/jcb.96.1.217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ito A., Hayashi S., Yoshida T. Participation of a cytochrome b5-like hemoprotein of outer mitochondrial membrane (OM cytochrome b) in NADH-semidehydroascorbic acid reductase activity of rat liver. Biochem Biophys Res Commun. 1981 Jul 30;101(2):591–598. doi: 10.1016/0006-291x(81)91300-0. [DOI] [PubMed] [Google Scholar]
  16. Jarasch E. D., Kartenbeck J., Bruder G., Fink A., Morré D. J., Franke W. W. B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes. J Cell Biol. 1979 Jan;80(1):37–52. doi: 10.1083/jcb.80.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kay D. G., Khan M. N., Posner B. I., Bergeron J. J. In vivo uptake of insulin into hepatic Golgi fractions: application of the diaminobenzidine-shift protocol. Biochem Biophys Res Commun. 1984 Sep 28;123(3):1144–1148. doi: 10.1016/s0006-291x(84)80252-1. [DOI] [PubMed] [Google Scholar]
  18. Kim Y. S., Perdomo J., Nordberg J. Glycoprortein biosynthesis in small intestinal mucosa. I. A study of glycosyltransferases in microsomal subfractions. J Biol Chem. 1971 Sep 10;246(17):5466–5476. [PubMed] [Google Scholar]
  19. 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]
  20. 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]
  21. Lodish H. F., Braell W. A., Schwartz A. L., Strous G. J., Zilberstein A. Synthesis and assembly of membrane and organelle proteins. Int Rev Cytol Suppl. 1981;12:247–307. doi: 10.1016/b978-0-12-364373-5.50016-0. [DOI] [PubMed] [Google Scholar]
  22. Lévy M., Toury R., André J. Essai de séparation des deux membranes mitochondriales. C R Acad Sci Hebd Seances Acad Sci D. 1966 Apr 4;262(14):1593–1596. [PubMed] [Google Scholar]
  23. Meldolesi J., Corte G., Pietrini G., Borgese N. 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. J Cell Biol. 1980 Jun;85(3):516–526. doi: 10.1083/jcb.85.3.516. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mihara K., Blobel G., Sato R. In vitro synthesis and integration into mitochondria of porin, a major protein of the outer mitochondrial membrane of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7102–7106. doi: 10.1073/pnas.79.23.7102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Munro H. N., Fleck A. Recent developments in the measurement of nucleic acids in biological materials. A supplementary review. Analyst. 1966 Feb;91(79):78–88. doi: 10.1039/an9669100078. [DOI] [PubMed] [Google Scholar]
  26. Negishi M., Kreibich G. Coordinated polypeptide synthesis and insertion of protoheme in cytochrome P-450 during development of endoplasmic reticulum membranes. J Biol Chem. 1978 Jul 10;253(13):4791–4797. [PubMed] [Google Scholar]
  27. Okada Y., Frey A. B., Guenthner T. M., Oesch F., Sabatini D. D., Kreibich G. Studies on the biosynthesis of microsomal membrane proteins. Site of synthesis and mode of insertion of cytochrome b5, cytochrome b5 reductase, cytochrome P-450 reductase and epoxide hydrolase. Eur J Biochem. 1982 Feb;122(2):393–402. doi: 10.1111/j.1432-1033.1982.tb05894.x. [DOI] [PubMed] [Google Scholar]
  28. Omura T., Siekevitz P., Palade G. E. Turnover of constituents of the endoplasmic reticulum membranes of rat hepatocytes. J Biol Chem. 1967 May 25;242(10):2389–2396. [PubMed] [Google Scholar]
  29. Parsons D. F., Williams G. R., Chance B. Characteristics of isolated and purified preparations of the outer and inner membranes of mitochondria. Ann N Y Acad Sci. 1966 Jul 14;137(2):643–666. doi: 10.1111/j.1749-6632.1966.tb50188.x. [DOI] [PubMed] [Google Scholar]
  30. ROY A. B. The sulphatase of ox liver. 7. The intracellular distribution of sulphatases A and B. Biochem J. 1960 Nov;77:380–386. doi: 10.1042/bj0770380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rachubinski R. A., Verma D. P., Bergeron J. J. Synthesis of rat liver microsomal cytochrome b5 by free ribosomes. J Cell Biol. 1980 Mar;84(3):705–716. doi: 10.1083/jcb.84.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rouser G., Fkeischer S., Yamamoto A. Two dimensional then layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots. Lipids. 1970 May;5(5):494–496. doi: 10.1007/BF02531316. [DOI] [PubMed] [Google Scholar]
  33. Sabatini D. D., Kreibich G., Morimoto T., Adesnik M. Mechanisms for the incorporation of proteins in membranes and organelles. J Cell Biol. 1982 Jan;92(1):1–22. doi: 10.1083/jcb.92.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Shawver L. K., Seidel S. L., Krieter P. A., Shires T. K. An enzyme-linked immunoadsorbent assay for measuring cytochrome b5 and NADPH-cytochrome P-450 reductase in rat liver microsomal fractions. Evidence for functionally inactive protein. Biochem J. 1984 Feb 1;217(3):623–632. doi: 10.1042/bj2170623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shore G. C., Tata J. R. Two fractions of rough endoplasmic reticulum from rat liver. I. Recovery of rapidly sedimenting endoplasmic reticulum in association with mitochondria. J Cell Biol. 1977 Mar;72(3):714–725. doi: 10.1083/jcb.72.3.714. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Sottocasa G. L., Kuylenstierna B., Ernster L., Bergstrand A. An electron-transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol. 1967 Feb;32(2):415–438. doi: 10.1083/jcb.32.2.415. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Strittmatter P., Rogers M. J., Spatz L. The binding of cytochrome b 5 to liver microsomes. J Biol Chem. 1972 Nov 25;247(22):7188–7194. [PubMed] [Google Scholar]
  38. 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]
  39. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. WURTMAN R. J., AXELROD J. A SENSITIVE AND SPECIFIC ASSAY FOR THE ESTIMATION OF MONOAMINE OXIDASE. Biochem Pharmacol. 1963 Dec;12:1439–1441. doi: 10.1016/0006-2952(63)90215-6. [DOI] [PubMed] [Google Scholar]
  41. Wattiaux R., Wattiaux-De Coninck S., Ronveaux-dupal M. F., Dubois F. Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient. J Cell Biol. 1978 Aug;78(2):349–368. doi: 10.1083/jcb.78.2.349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wibo M., Thinès-Sempoux D., Amar-Costesec A., Beaufay H., Godelaine D. Analytical study of microsomes and isolated subcellular membranes from rat liver VIII. Subfractionation of preparations enriched with plasma membranes, outer mitochondrial membranes, or Golgi complex membranes. J Cell Biol. 1981 Jun;89(3):456–474. doi: 10.1083/jcb.89.3.456. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wickner W. T., Lodish H. F. Multiple mechanisms of protein insertion into and across membranes. Science. 1985 Oct 25;230(4724):400–407. doi: 10.1126/science.4048938. [DOI] [PubMed] [Google Scholar]

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