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. 1973 Oct 1;59(1):45–72. doi: 10.1083/jcb.59.1.45

GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES

I. Isolation Procedure and Morphological Characterization

J H Ehrenreich 1, J J M Bergeron 1, P Siekevitz 1, G E Palade 1
PMCID: PMC2110914  PMID: 4356571

Abstract

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes ∼90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at ∼13 x 106 g·min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation.

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Selected References

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  1. Beams H. W., Kessel R. G. The Golgi apparatus: structure and function. Int Rev Cytol. 1968;23:209–276. doi: 10.1016/s0074-7696(08)60273-9. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Bruni C., Porter K. R. The Fine Structure of the Parenchymal Cell of the Normal Rat Liver: I. General Observations. Am J Pathol. 1965 May;46(5):691–755. [PMC free article] [PubMed] [Google Scholar]
  4. CARO L. G., PALADE G. E. PROTEIN SYNTHESIS, STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL. AN AUTORADIOGRAPHIC STUDY. J Cell Biol. 1964 Mar;20:473–495. doi: 10.1083/jcb.20.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Castle J. D., Jamieson J. D., Palade G. E. Radioautographic analysis of the secretory process in the parotid acinar cell of the rabbit. J Cell Biol. 1972 May;53(2):290–311. doi: 10.1083/jcb.53.2.290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chapman M. J., Mills G. L., Taylaur C. E. Lipoprotein particles from the Golgi apparatus of guinea-pig liver. Biochem J. 1972 Jul;128(4):779–787. doi: 10.1042/bj1280779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Droller M. J., Roth T. F. An electron microscope study of yolk formation during oogenesis in Lebistes reticulatus guppyi. J Cell Biol. 1966 Feb;28(2):209–232. doi: 10.1083/jcb.28.2.209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Fleischer B., Fleischer S. Preparation and characterization of golgi membranes from rat liver. Biochim Biophys Acta. 1970 Dec 1;219(2):301–319. doi: 10.1016/0005-2736(70)90209-9. [DOI] [PubMed] [Google Scholar]
  11. Friend D. S., Brassil G. E. Osmium staining of endoplasmic reticulum and mitochondria in the rat adrenal cortex. J Cell Biol. 1970 Aug;46(2):252–266. doi: 10.1083/jcb.46.2.252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friend D. S., Farquhar M. G. Functions of coated vesicles during protein absorption in the rat vas deferens. J Cell Biol. 1967 Nov;35(2):357–376. doi: 10.1083/jcb.35.2.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Friend D. S. The fine structure of Brunner's glands in the mouse. J Cell Biol. 1965 Jun;25(3):563–576. doi: 10.1083/jcb.25.3.563. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Grove S. N., Bracker C. E., Morré D. J. Cytomembrane differentiation in the endoplasmic reticulum-Golgi apparatus-vesicle complex. Science. 1968 Jul 12;161(3837):171–173. doi: 10.1126/science.161.3837.171. [DOI] [PubMed] [Google Scholar]
  15. Haddad A., Smith M. D., Herscovics A., Nadler N. J., Leblond C. P. Radioautographic study of in vivo and in vitro incorporation of fucose-3H into thyroglobulin by rat thyroid follicular cells. J Cell Biol. 1971 Jun;49(3):856–877. doi: 10.1083/jcb.49.3.856. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hamilton R. L., Regen D. M., Gray M. E., LeQuire V. S. Lipid transport in liver. I. Electron microscopic identification of very low density lipoproteins in perfused rat liver. Lab Invest. 1967 Feb;16(2):305–319. [PubMed] [Google Scholar]
  17. Hicks R. M. The function of the golgi complex in transitional epithelium. Synthesis of the thick cell membrane. J Cell Biol. 1966 Sep;30(3):623–643. doi: 10.1083/jcb.30.3.623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Holtzman E., Novikoff A. B., Villaverde H. Lysosomes and GERL in normal and chromatolytic neurons of the rat ganglion nodosum. J Cell Biol. 1967 May;33(2):419–435. doi: 10.1083/jcb.33.2.419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jamieson J. D., Palade G. E. Intracellular transport of secretory proteins in the pancreatic exocrine cell. II. Transport to condensing vacuoles and zymogen granules. J Cell Biol. 1967 Aug;34(2):597–615. doi: 10.1083/jcb.34.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jones A. L., Ruderman N. B., Herrera M. G. Electron microscopic and biochemical study of lipoprotein synthesis in the isolated perfused rat liver. J Lipid Res. 1967 Sep;8(5):429–446. [PubMed] [Google Scholar]
  21. KARNOVSKY M. J. Simple methods for "staining with lead" at high pH in electron microscopy. J Biophys Biochem Cytol. 1961 Dec;11:729–732. doi: 10.1083/jcb.11.3.729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kessel R. G. An electron microscope study of differentiation and growth in oocytes of Ophioderma panamensis. J Ultrastruct Res. 1968 Jan;22(1):63–89. doi: 10.1016/s0022-5320(68)90050-6. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Mahley R. W., Bersot T. P., LeQuire V. S., Levy R. I., Windmueller H. G., Brown W. V. Identity of very low density lipoprotein apoproteins of plasma and liver Golgi apparatus. Science. 1970 Apr 17;168(3929):380–382. doi: 10.1126/science.168.3929.380. [DOI] [PubMed] [Google Scholar]
  25. Mahley R. W., Hamilton R. L., Lequire V. S. Characterization of lipoprotein particles isolated from the Golgi apparatus of rat liver. J Lipid Res. 1969 Jul;10(4):433–439. [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. NOVIKOFF A. B., GOLDFISCHER S. Nucleosidediphosphatase activity in the Golgi apparatus and its usefulness for cytological studies. Proc Natl Acad Sci U S A. 1961 Jun 15;47:802–810. doi: 10.1073/pnas.47.6.802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Neutra M., Leblond C. P. Radioautographic comparison of the uptake of galactose-H and glucose-H3 in the golgi region of various cells secreting glycoproteins or mucopolysaccharides. J Cell Biol. 1966 Jul;30(1):137–150. doi: 10.1083/jcb.30.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Novikoff A. B., Albala A., Biempica L. Ultrastructural and cytochemical observations on B-16 and Harding-Passey mouse melanomas. The origin of premelanosomes and compound melanosomes. J Histochem Cytochem. 1968 May;16(5):299–319. doi: 10.1177/16.5.299. [DOI] [PubMed] [Google Scholar]
  31. ROTH T. F., PORTER K. R. YOLK PROTEIN UPTAKE IN THE OOCYTE OF THE MOSQUITO AEDES AEGYPTI. L. J Cell Biol. 1964 Feb;20:313–332. doi: 10.1083/jcb.20.2.313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. SCHNEIDER W. C., KUFF E. L. On the isolation and some biochemical properties of the Golgi substance. Am J Anat. 1954 Mar;94(2):209–224. doi: 10.1002/aja.1000940203. [DOI] [PubMed] [Google Scholar]
  33. SIEKEVITZ P. Protoplasm: endoplasmic reticulum and microsomes and their properties. Annu Rev Physiol. 1963;25:15–40. doi: 10.1146/annurev.ph.25.030163.000311. [DOI] [PubMed] [Google Scholar]
  34. STEIN Y., STEIN O. Metabolic activity of rat epididymal fat pad labeled selectively by an in vivo incubation technique. Biochim Biophys Acta. 1961 Dec 23;54:555–571. doi: 10.1016/0006-3002(61)90096-8. [DOI] [PubMed] [Google Scholar]
  35. Stein O., Stein Y. Lipid synthesis, intracellular transport, storage, and secretion. I. Electron microscopic radioautographic study of liver after injection of tritiated palmitate or glycerol in fasted and ethanol-treated rats. J Cell Biol. 1967 May;33(2):319–339. doi: 10.1083/jcb.33.2.319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. VENABLE J. H., COGGESHALL R. A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol. 1965 May;25:407–408. doi: 10.1083/jcb.25.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. WATSON M. L. Staining of tissue sections for electron microscopy with heavy metals. J Biophys Biochem Cytol. 1958 Jul 25;4(4):475–478. doi: 10.1083/jcb.4.4.475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. WHALEY W. G., MOLLENHAUER H. H. The Golgi apparatus and cell plate formation--a postulate. J Cell Biol. 1963 Apr;17:216–221. doi: 10.1083/jcb.17.1.216. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Whur P., Herscovics A., Leblond C. P. Radioautographic visualization of the incorporation of galactose-3H and mannose-3H by rat thyroids in vitro in relation to the stages of thyroglobulin synthesis. J Cell Biol. 1969 Nov;43(2):289–311. doi: 10.1083/jcb.43.2.289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. YAMAMOTO T. On the thickness of the unit membrane. J Cell Biol. 1963 May;17:413–421. doi: 10.1083/jcb.17.2.413. [DOI] [PMC free article] [PubMed] [Google Scholar]

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