Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1981 Jun 1;89(3):456–474. doi: 10.1083/jcb.89.3.456

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

PMCID: PMC2111792  PMID: 7251662

Abstract

Preparations enriched with plasmalemmal, outer mitochondrial, or Golgi complex membranes from rat liver were subfractionated by isopycnic centrifugation, without or after treatment with digitonin, to establish the subcellular distribution of a variety of enzymes. The typical plasmalemmal enzymes 5'-nucleotidase, alkaline phosphodiesterase I, and alkaline phosphatase were markedly shifted by digitonin toward higher densities in all three preparations. Three glycosyltransferases, highly purified in the Golgi fraction, were moderately shifted by digitonin in both this Golgi complex preparation and the microsomal fraction. The outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the outer mitochondrial membrane marker, monoamine oxidase, was not affected by digitonin in the out mitochondrial membrane preparation, in agreement wit its behavior in microsomes. With the exception of NADH cytochrome c reductase (which was concentrated in the outer mitochondrial membrane preparation), typical microsomal enzymes (glucose-6-phosphatase, esterase, and NADPH cytochrome c reductase) displayed low specific activities in the three preparations; except for part of the glucose-6-phosphatase activity in the plasma membrane preparation, their density distributions were insensitive to digitonin, as they were in microsomes. The influence of digitonin on equilibrium densities was correlated with its morphological effects. Digitonin induced pseudofenestrations in plasma membranes. In Golgi and outer mitochondrial membrane preparations, a few similarly altered membranes were detected in subfractions enriched with 5'-nucleotidase and alkaline phosphodiesterase I. The alterations of Golgi membranes were less obvious and seemingly restricted to some elements in the Golgi preparation. No morphological modification was detected in digitonin-treated outer mitochondrial membranes. These results indicate that each enzyme is associated with the same membrane entity in all membrane preparations and support the view that there is little overlap in the enzymatic equipment of the various types of cytomembranes.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

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

  1. Amar-Costesec A. Analytical study of microsomes and isolated subcellular membranes from rat liver. VII. Distribution of protein-bound sialic acid. J Cell Biol. 1981 Apr;89(1):62–69. doi: 10.1083/jcb.89.1.62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amar-Costesec A., Beaufay H. A structural basis of enzymic heterogeneity within liver endoplasmic reticulum. J Theor Biol. 1981 Mar 21;89(2):217–230. doi: 10.1016/0022-5193(81)90309-x. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. 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]
  5. Avruch J., Wallach D. F. Preparation and properties of plasma membrane and endoplasmic reticulum fragments from isolated rat fat cells. Biochim Biophys Acta. 1971 Apr 13;233(2):334–347. doi: 10.1016/0005-2736(71)90331-2. [DOI] [PubMed] [Google Scholar]
  6. BEAUFAY H., HERS H. G., BERTHET J., DE DUVE C. Le sustème hexose-phosphatasique. V. Influence de divers agents sur l'activité et la stabilité de la glucose-6-phosphatase. Bull Soc Chim Biol (Paris) 1954;36(11-12):1539–1550. [PubMed] [Google Scholar]
  7. 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]
  8. Beaufay H., Amar-Costesec A., Thinès-Sempoux D., Wibo M., Robbi M., Berthet J. Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients. J Cell Biol. 1974 Apr;61(1):213–231. doi: 10.1083/jcb.61.1.213. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Beaufay H., Jacques P., Baudhuin P., Sellinger O. Z., Berthet J., De Duve C. Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients. Biochem J. 1964 Jul;92(1):184–205. doi: 10.1042/bj0920184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Bergeron J. J. Golgi fractions from livers of control and ethanol-intoxicated rats. Enzymic and morphologic properties following rapid isolation. Biochim Biophys Acta. 1979 Aug 23;555(3):493–503. doi: 10.1016/0005-2736(79)90402-4. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Bretz R., Bretz H., Palade G. E. Distribution of terminal glycosyltransferases in hepatic Golgi fractions. J Cell Biol. 1980 Jan;84(1):87–101. doi: 10.1083/jcb.84.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cassidy J. T., Jourdian G. W., Roseman S. The sialic acids. VI. Purification and properties of sialidase from Clostridium perfringens. J Biol Chem. 1965 Sep;240(9):3501–3506. [PubMed] [Google Scholar]
  15. Cheng H., Farquhar M. G. Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. II. Cytochemical localization within Golgi and ER membranes. J Cell Biol. 1976 Sep;70(3):671–684. doi: 10.1083/jcb.70.3.671. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Claude A. Growth and differentiation of cytoplasmic membranes in the course of lipoprotein granule synthesis in the hepatic cell. I. Elaboration of elements of the Golgi complex. J Cell Biol. 1970 Dec;47(3):745–766. doi: 10.1083/jcb.47.3.745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Colbeau A., Nachbaur J., Vignais P. M. Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim Biophys Acta. 1971 Dec 3;249(2):462–492. doi: 10.1016/0005-2736(71)90123-4. [DOI] [PubMed] [Google Scholar]
  18. DE DUVE C., PRESSMAN B. C., GIANETTO R., WATTIAUX R., APPELMANS F. Tissue fractionation studies. 6. Intracellular distribution patterns of enzymes in rat-liver tissue. Biochem J. 1955 Aug;60(4):604–617. doi: 10.1042/bj0600604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. DePierre J. W., Karnovsky M. L. Plasma membranes of mammalian cells: a review of methods for their characterization and isolation. J Cell Biol. 1973 Feb;56(2):275–303. doi: 10.1083/jcb.56.2.275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. 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]
  21. Elias P. M., Goerke J., Friend D. S., Brown B. E. Freeze-fracture identification of sterol-digitonin complexes in cell and liposome membranes. J Cell Biol. 1978 Aug;78(2):577–596. doi: 10.1083/jcb.78.2.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Evans W. H. Fractionation of liver plasma membranes prepared by zonal centrifugation. Biochem J. 1970 Mar;116(5):833–842. doi: 10.1042/bj1160833. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Farquhar M. G., Bergeron J. J., Palade G. E. Cytochemistry of Golgi fractions prepared from rat liver. J Cell Biol. 1974 Jan;60(1):8–25. doi: 10.1083/jcb.60.1.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Fleischer B., Fleischer S., Ozawa H. Isolation and characterization of Golgi membranes from bovine liver. J Cell Biol. 1969 Oct;43(1):59–79. doi: 10.1083/jcb.43.1.59. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Fowler S., Remacle J., Trouet A., Beaufay H., Berthet J., Wibo M., Hauser P. 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. J Cell Biol. 1976 Nov;71(2):535–550. doi: 10.1083/jcb.71.2.535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. GOLDBARG J. A., RUTENBURG A. M. The colorimetric determination of leucine aminopeptidase in urine and serum of normal subjects and patients with cancer and other diseases. Cancer. 1958 Mar-Apr;11(2):283–291. doi: 10.1002/1097-0142(195803/04)11:2<283::aid-cncr2820110209>3.0.co;2-8. [DOI] [PubMed] [Google Scholar]
  28. Hino Y., Asano A., Sato R. Biochemical studies on rat liver Golgi apparatus. II. Further characterization of isolated Golgi fraction. J Biochem. 1978 Apr;83(4):925–934. doi: 10.1093/oxfordjournals.jbchem.a132019. [DOI] [PubMed] [Google Scholar]
  29. Hino Y., Asano A., Sato R. Biochemical studies on rat liver Golgi apparatus. III. Subfractionation of fragmented Golgi apparatus by counter-current distribution. J Biochem. 1978 Apr;83(4):935–942. doi: 10.1093/oxfordjournals.jbchem.a132020. [DOI] [PubMed] [Google Scholar]
  30. House P. D., Poulis P., Weidemann M. J. Isolation of a plasma-membrane subfraction from rat liver containing an insulin-sensitive cyclic-AMP phosphodiesterase. Eur J Biochem. 1972 Jan 21;24(3):429–437. doi: 10.1111/j.1432-1033.1972.tb19703.x. [DOI] [PubMed] [Google Scholar]
  31. Howell K. E., Ito A., Palade G. E. Endoplasmic reticulum marker enzymes in Golgi fractions--what does this mean? J Cell Biol. 1978 Nov;79(2 Pt 1):581–589. doi: 10.1083/jcb.79.2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Huang C. M., Goldenberg H., Frantz C., Morré D. J., Keenan T. W., Crane F. L. Comparison of NADH-linked cytochrome C reductases of endoplasmic reticulum, golgi apparatus and plasma membrane. Int J Biochem. 1979;10(8):723–731. doi: 10.1016/0020-711x(79)90218-0. [DOI] [PubMed] [Google Scholar]
  33. Ito A. Cytochrome b5-like hemoprotein of outer mitochondrial membrane; OM cytochrome b. I. Purification of OM cytochrome b from rat liver mitochondria and comparison of its molecular properties with those of cytochrome b5. J Biochem. 1980 Jan;87(1):63–71. doi: 10.1093/oxfordjournals.jbchem.a132753. [DOI] [PubMed] [Google Scholar]
  34. Ito A., Palade G. E. Presence of NADPH-cytochrome P-450 reductase in rat liver Golgi membranes. Evidence obtained by immunoadsorption method. J Cell Biol. 1978 Nov;79(2 Pt 1):590–597. doi: 10.1083/jcb.79.2.590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Little J. S., Widnell C. C. Evidence for the translocation of 5'-nucleotidase across hepatic membranes in vivo. Proc Natl Acad Sci U S A. 1975 Oct;72(10):4013–4017. doi: 10.1073/pnas.72.10.4013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Losa G. A., Weibel E. R., Bolender R. P. Integrated stereological and biochemical studies on hepatocytic membranes. III. Relative surface of endoplasmic reticulum membranes in microsomal fractions estimated on freeze-fracture preparations. J Cell Biol. 1978 Aug;78(2):289–308. doi: 10.1083/jcb.78.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsuura S., Tashiro Y. Immunoelectron-microscopic studies of endoplasmic reticulum-Golgi relationships in the intracellular transport process of lipoprotein particles in rat hepatocytes. J Cell Sci. 1979 Oct;39:273–290. doi: 10.1242/jcs.39.1.273. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Morre J., Merlin L. M., Keenan T. W. Localization of glycosyl transferase activities in a Golgi apparatus-rich fraction isolated from rat liver. Biochem Biophys Res Commun. 1969 Nov 20;37(5):813–819. doi: 10.1016/0006-291x(69)90964-4. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Morré D. J., Kartenbeck J., Franke W. W. Membrane flow and intercoversions among endomembranes. Biochim Biophys Acta. 1979 Apr 23;559(1):71–52. doi: 10.1016/0304-4157(79)90008-x. [DOI] [PubMed] [Google Scholar]
  43. NEVILLE D. M., Jr The isolation of a cell membrane fraction from rat liver. J Biophys Biochem Cytol. 1960 Oct;8:413–422. doi: 10.1083/jcb.8.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Novikoff P. M., Yam A. Sites of lipoprotein particles in normal rat hepatocytes. J Cell Biol. 1978 Jan;76(1):1–11. doi: 10.1083/jcb.76.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Palade G. Intracellular aspects of the process of protein synthesis. Science. 1975 Aug 1;189(4200):347–358. doi: 10.1126/science.1096303. [DOI] [PubMed] [Google Scholar]
  46. 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]
  47. 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]
  48. Schachter H., Jabbal I., Hudgin R. L., Pinteric L., McGuire E. J., Roseman S. Intracellular localization of liver sugar nucleotide glycoprotein glycosyltransferases in a Golgi-rich fraction. J Biol Chem. 1970 Mar 10;245(5):1090–1100. [PubMed] [Google Scholar]
  49. Schilling E. E., Goldenberg H., Morré D. J., Crane F. L. Distribution of insulin receptors among mouse liver endomembranes. Biochim Biophys Acta. 1979 Aug 23;555(3):504–511. doi: 10.1016/0005-2736(79)90403-6. [DOI] [PubMed] [Google Scholar]
  50. Schnaitman C., Erwin V. G., Greenawalt J. W. The submitochondrial localization of monoamine oxidase. An enzymatic marker for the outer membrane of rat liver mitochondria. J Cell Biol. 1967 Mar;32(3):719–735. doi: 10.1083/jcb.32.3.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. 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]
  53. Tartakoff A. M. The Golgi complex: crossroads for vesicular traffic. Int Rev Exp Pathol. 1980;22:227–251. [PubMed] [Google Scholar]
  54. Thines-Sempoux D., Amar-Costesec A., Beaufay H., Berthet J. The association of cholesterol, 5'-nucleotidase, and alkaline phosphodiesterase I with a distinct group of microsomal particles. J Cell Biol. 1969 Oct;43(1):189–192. doi: 10.1083/jcb.43.1.189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Toda G., Oka H., Oda T., Ikeda Y. Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface. Biochim Biophys Acta. 1975 Nov 17;413(1):52–64. doi: 10.1016/0005-2736(75)90058-9. [DOI] [PubMed] [Google Scholar]
  56. WEIMER H. E., MEHL J. W., WINZLER R. J. Studies on the mucoproteins of human plasma. V. Isolation and characterization of a homogeneous mucoprotein. J Biol Chem. 1950 Aug;185(2):561–568. [PubMed] [Google Scholar]
  57. 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]
  58. Wisher M. H., Evans W. H. Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte. Biochem J. 1975 Feb;146(2):375–388. doi: 10.1042/bj1460375. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES