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. 1976 Nov 1;71(2):535–550. doi: 10.1083/jcb.71.2.535

Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions

PMCID: PMC2109757  PMID: 791954

Abstract

The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.

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Selected References

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  1. Allmann D. W., Bachmann E., Orme-Johnson N., Tan W. C., Green D. E. Membrane systems of mitochondria. VI. Membranes of liver mitochondria. Arch Biochem Biophys. 1968 Jun;125(3):981–1012. doi: 10.1016/0003-9861(68)90537-7. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Amar-Costesec A., Wibo M., Thinès-Sempoux D., Beaufay H., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. IV. Biochemical, physical, and morphological modifications of microsomal components induced by digitonin, EDTA, and pyrophosphate. J Cell Biol. 1974 Sep;62(3):717–745. doi: 10.1083/jcb.62.3.717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Axén R., Porath J., Ernback S. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides. Nature. 1967 Jun 24;214(5095):1302–1304. doi: 10.1038/2141302a0. [DOI] [PubMed] [Google Scholar]
  5. Baudhuin P., Evrard P., Berthet J. Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles. J Cell Biol. 1967 Jan;32(1):181–191. doi: 10.1083/jcb.32.1.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bergeron J. J., Ehrenreich J. H., Siekevitz P., Palade G. E. Golgi fractions prepared from rat liver homogenates. II. Biochemical characterization. J Cell Biol. 1973 Oct;59(1):73–88. doi: 10.1083/jcb.59.1.73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bieglmayer C., Graf J., Ruis H. Membranes of glyoxysomes from castor-bean endosperm. Enzymes bound to purified-membrane preparations. Eur J Biochem. 1973 Sep 3;37(3):553–562. doi: 10.1111/j.1432-1033.1973.tb03018.x. [DOI] [PubMed] [Google Scholar]
  8. CHANCE B., WILLIAMS G. R. Kinetics of cytochrome b5 in rat liver microsomes. J Biol Chem. 1954 Aug;209(2):945–951. [PubMed] [Google Scholar]
  9. De Duve C. Tissue fractionation. Past and present. J Cell Biol. 1971 Jul;50(1):20d–55d. [PubMed] [Google Scholar]
  10. Donaldson R. P., Tolbert N. E., Schnarrenberger C. A comparison of microbody membranes with microsomes and mitochondria from plant and animal tissue. Arch Biochem Biophys. 1972 Sep;152(1):199–215. doi: 10.1016/0003-9861(72)90208-1. [DOI] [PubMed] [Google Scholar]
  11. FARR R. S. A quantitative immunochemical measure of the primary interaction between I BSA and antibody. J Infect Dis. 1958 Nov-Dec;103(3):239–262. doi: 10.1093/infdis/103.3.239. [DOI] [PubMed] [Google Scholar]
  12. Fleischer S., Fleischer B., Azzi A., Chance B. Cytochrome b5 and P-450 in liver cell fractions. Biochim Biophys Acta. 1971 Feb 2;225(2):194–200. doi: 10.1016/0005-2736(71)90212-4. [DOI] [PubMed] [Google Scholar]
  13. Fukushima K., Ito A., Omura T., Sato R. Occurrence of different types of cytochrome b 5 -like hemoprotein in liver mitochondria and their intramitochondrial localization. J Biochem. 1972 Mar;71(3):447–461. [PubMed] [Google Scholar]
  14. Green D. E., Allmann D. W., Harris R. A., Tan W. C. Enzyme localization in the inner and outer mitochondrial membranes. Biochem Biophys Res Commun. 1968 May 10;31(3):368–378. doi: 10.1016/0006-291x(68)90485-3. [DOI] [PubMed] [Google Scholar]
  15. Hong R., Nisonoff A. Relative labilities of the two types of interchain disulfide bond of rabbit gamma G-immunoglobulin. J Biol Chem. 1965 Oct;240(10):3883–3891. [PubMed] [Google Scholar]
  16. Hämmerling U., Aoki T., de Harven E., Boyse E. A., Old L. J. Use of hybrid antibody with anti-gamma-G and anti-ferritin specificities in locating cell surface antigens by electron microscopy. J Exp Med. 1968 Dec 1;128(6):1461–1473. doi: 10.1084/jem.128.6.1461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ichikawa Y., Yamano T. Cytochrome b5 and CO-binding cytochromes in the Golgi membranes of mammalian livers. Biochem Biophys Res Commun. 1970 Jul 27;40(2):297–305. doi: 10.1016/0006-291x(70)91009-0. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Leighton F., Poole B., Beaufay H., Baudhuin P., Coffey J. W., Fowler S., De Duve C. The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions. J Cell Biol. 1968 May;37(2):482–513. doi: 10.1083/jcb.37.2.482. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Margoliash E., Nisonoff A., Reichlin M. Immunological activity of cytochrome c. I. Precipitating antibodies to monomeric vertebrate cytochromes c. J Biol Chem. 1970 Mar 10;245(5):931–939. [PubMed] [Google Scholar]
  21. Morré D. J., Hamilton R. L., Mollenhauer H. H., Mahley R. W., Cunningham W. P., Cheetham R. D., Lequire V. S. Isolation of a Golgi apparatus-rich fraction from rat liver. I. Method and morphology. J Cell Biol. 1970 Mar;44(3):484–491. doi: 10.1083/jcb.44.3.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. NISONOFF A., DIXON D. J. EVIDENCE FOR LINKAGE OF UNIVALENT FRAGMENTS OR HALF-MOLECULES OF RABBIT GAMMA-GLOBULIN BY THE SAME DISULFIDE BOND. Biochemistry. 1964 Sep;3:1338–1342. doi: 10.1021/bi00897a025. [DOI] [PubMed] [Google Scholar]
  23. NISONOFF A., PALMER J. L. HYBRIDIZATION OF HALF MOLECULES OF RABBIT GAMMA GLOBULIN. Science. 1964 Jan 24;143(3604):376–379. doi: 10.1126/science.143.3604.376. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. REYNOLDS E. S. The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J Cell Biol. 1963 Apr;17:208–212. doi: 10.1083/jcb.17.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Remacle J., Fowler S., Beaufay H., Amarcostesec A., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. VI. Electron microscope examination of microsomes for cytochrome b5 by means of a ferritin-labeled antibody. J Cell Biol. 1976 Nov;71(2):551–564. doi: 10.1083/jcb.71.2.551. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Remacle J., Fowler S., Trouet A. Preparation at pureification d'anticorps hybrides anti-ferritine, anti-cytochrome b 5. Arch Int Physiol Biochim. 1973 Feb;81(1):197–199. [PubMed] [Google Scholar]
  29. Rosenstein R. W., Nisonoff A., Uhr J. W. Significance of bivalence of antibody in viral neutralization. J Exp Med. 1971 Dec 1;134(6):1431–1441. doi: 10.1084/jem.134.6.1431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. SINGER S. J. Preparation of an electron-dense antibody conjugate. Nature. 1959 May 30;183(4674):1523–1524. doi: 10.1038/1831523a0. [DOI] [PubMed] [Google Scholar]
  31. Sargent J. R., Vadlamudi B. P. Characterization and biosynthesis of cytochrome b(5) in rat liver microsomes. Biochem J. 1968 May;107(6):839–849. doi: 10.1042/bj1070839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Song C. S., Rubin W., Rifkind A. B., Kappas A. Plasma membranes of the rat liver. Isolation and enzymatic characterization of a fraction rich in bile canaliculi. J Cell Biol. 1969 Apr;41(1):124–132. doi: 10.1083/jcb.41.1.124. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Spatz L., Strittmatter P. A form of cytochrome b5 that contains an additional hydrophobic sequence of 40 amino acid residues. Proc Natl Acad Sci U S A. 1971 May;68(5):1042–1046. doi: 10.1073/pnas.68.5.1042. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Strittmatter C. F., Ball E. G. A Hemochromogen Component of Liver Microsomes. Proc Natl Acad Sci U S A. 1952 Jan;38(1):19–25. doi: 10.1073/pnas.38.1.19. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wibo M., Amar-Costesec A., Berthet J., Beaufay H. Electron microscope examination of subcellular fractions. 3. Quantitative analysis of the microsomal fraction isolated from rat liver. J Cell Biol. 1971 Oct;51(1):52–71. doi: 10.1083/jcb.51.1.52. [DOI] [PMC free article] [PubMed] [Google Scholar]

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