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. 1981 May 1;89(2):246–255. doi: 10.1083/jcb.89.2.246

Orientation of glycoprotein galactosyltransferase and sialyltransferase enzymes in vesicles derived from rat liver Golgi apparatus

PMCID: PMC2111678  PMID: 6788776

Abstract

UDP-galactose: N-acetylglucosamine galactosyltransferase (GT) and CMP- sialic:desialylated transferrin sialyltransferse (ST) activities of rat liver Golgi apparatus are membrane-bound enzymes that can be released by treatment with Triton X-100. When protein substrates are used to assay these enzymes in freshly prepared Golgi vesicles, both activities are enhanced about eightfold by the addition of Triton X-100. When small molecular weight substrates are used, however, both activities are only enhanced about twofold by the addition of detergent. The enzymes remain inaccessible to large protein substrates even after freezing and storage of the Golgi preparation for 2 mo in liquid nitrogen. Accessibility to small molecular and weight substrates increases significantly after such storage. GT and ST activities in Golgi vesicles are not destroyed by treatment with trypsin, but are destroyed by this treatment if the vesicles are first disrupted with Triton X-100. Treatment of Golgi vesicles with low levels of filipin, a polyene antibiotic known to complex with cholesterol in biological membranes, also results in enhanced trypsin susceptibility of both glycosyltransferases. Maximum destruction of the glycosyltransferase activities by trypsin is obtained at filipin to total cholesterol weight ratios of approximately 1.6 or molar ratios of approximately 1. This level of filipin does not solubilize the enzymes but causes both puckering of Golgi membranes visible by electron microscopy and disruption of the Golgi vesicles as measured by release of serum albumin. When isolated Golgi apparatus is fixed with glutaraldehyde to maintain the three-dimensional orientation of cisternae and secretory vesicles, and then treated with filipin, cisternal membranes on both cis and trans faces of the apparatus as well as secretory granule membranes appear to be affected about equally. These results indicate that liver Golgi vesicles as isolated are largely oriented with GT and ST on the luminal side of the membranes, which corresponds to the cisternal compartment of the Golgi apparatus in the hepatocyte. Cholesterol is an integral part of the membrane of the Golgi apparatus and its distribution throughout the apparatus is similar to that of both transferases.

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Selected References

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  1. Bergeron J. J., Borts D., Cruz J. Passage of serum-destined proteins through the Golgi apparatus of rat liver. An examination of heavy and light Golgi fractions. J Cell Biol. 1978 Jan;76(1):87–97. doi: 10.1083/jcb.76.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blau L., Bittman R. Cholesterol distribution between the two halves of the lipid bilayer of human erythrocyte ghost membranes. J Biol Chem. 1978 Dec 10;253(23):8366–8368. [PubMed] [Google Scholar]
  3. Blobel G., Walter P., Chang C. N., Goldman B. M., Erickson A. H., Lingappa V. R. Translocation of proteins across membranes: the signal hypothesis and beyond. Symp Soc Exp Biol. 1979;33:9–36. [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. Brew K., Vanaman T. C., Hill R. L. The role of alpha-lactalbumin and the A protein in lactose synthetase: a unique mechanism for the control of a biological reaction. Proc Natl Acad Sci U S A. 1968 Feb;59(2):491–497. doi: 10.1073/pnas.59.2.491. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bélanger L., Fleischer B., Fleischer S., Guillouzo A., Lemonnier M., Chiu J. F. Subcellular distribution and molecular heterogeneity of alpha 1-fetoprotein in newborn rat liver. Biochemistry. 1979 May 15;18(10):1962–1968. doi: 10.1021/bi00577a018. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Edwards K., Fleischer B., Dryburgh H., Fleischer S., Schreiber G. The distribution of albumin precursor protein and albumin in liver. Biochem Biophys Res Commun. 1976 Sep 7;72(1):310–318. doi: 10.1016/0006-291x(76)90995-5. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. 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]
  12. 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]
  13. Fleischer B. Isolation and characterization of Golgi apparatus and membranes from rat liver. Methods Enzymol. 1974;31:180–191. doi: 10.1016/0076-6879(74)31020-8. [DOI] [PubMed] [Google Scholar]
  14. Fleischer B., Smigel M. Solubilization and properties of galactosyltransferase and sulfotransferase activities of Golgi membranes in Triton X-100. J Biol Chem. 1978 Mar 10;253(5):1632–1638. [PubMed] [Google Scholar]
  15. GOLDFISCHER S., ESSNER E., NOVIKOFF A. B. THE LOCALIZATION OF PHOSPHATASE ACTIVITIES AT THE LEVEL OF ULTRASTRUCTURE. J Histochem Cytochem. 1964 Feb;12:72–95. doi: 10.1177/12.2.72. [DOI] [PubMed] [Google Scholar]
  16. Helenius A., Simons K. The binding of detergents to lipophilic and hydrophilic proteins. J Biol Chem. 1972 Jun 10;247(11):3656–3661. [PubMed] [Google Scholar]
  17. Hino Y., Asano A., Sato R., Shimizu S. Biochemical studies of rat liver Golgi apparatus. I. Isolation and preliminary characterization. J Biochem. 1978 Apr;83(4):909–923. doi: 10.1093/oxfordjournals.jbchem.a132018. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Keenan T. W., Morré D. J. Phospholipid class and fatty acid composition of golgi apparatus isolated from rat liver and comparison with other cell fractions. Biochemistry. 1970 Jan 6;9(1):19–25. doi: 10.1021/bi00803a003. [DOI] [PubMed] [Google Scholar]
  21. Kinsky S. C., Haxby J., Kinsky C. B., Demel R. A., van Deenen L. L. Effect of cholesterol incorporation on the sensitivity liposomes to the polyene antibiotic, filipin. Biochim Biophys Acta. 1968 Jan 10;152(1):174–185. doi: 10.1016/0005-2760(68)90019-2. [DOI] [PubMed] [Google Scholar]
  22. Kuhn N. J., White A. The role of nucleoside diphosphatase in a uridine nucleotide cycle associated with lactose synthesis in rat mammary-gland Golgi apparatus. Biochem J. 1977 Dec 15;168(3):423–433. doi: 10.1042/bj1680423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kuhn N. J., White A. The topography of lactose synthesis. Biochem J. 1975 Apr;148(1):77–84. doi: 10.1042/bj1480077. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. Merritt W. D., Morré D. J. A glycosyl transferase of high specific activity in secretory vesicles from isolated Golgi apparatus of rat liver. Biochim Biophys Acta. 1973 Apr 28;304(2):397–407. doi: 10.1016/0304-4165(73)90259-6. [DOI] [PubMed] [Google Scholar]
  27. Nelson B. D., Mendel-Hartvig I. Immunological studies on beef-heart ubiquinol--cytochrome c reductase (complex III) Eur J Biochem. 1977 Oct 17;80(1):267–274. doi: 10.1111/j.1432-1033.1977.tb11879.x. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Norman A. W., Demel R. A., de Kruyff B., van Deenen L. L. Studies on the biological properties of polyene antibiotics. Evidence for the direct interaction of filipin with cholesterol. J Biol Chem. 1972 Mar 25;247(6):1918–1929. [PubMed] [Google Scholar]
  30. Paulson J. C., Rearick J. I., Hill R. L. Enzymatic properties of beta-D-galactoside alpha2 leads to 6 sialytransferase from bovine colostrum. J Biol Chem. 1977 Apr 10;252(7):2363–2371. [PubMed] [Google Scholar]
  31. Peters T., Jr, Fleischer B., Fleischer S. The biosynthesis of rat serum albumin. IV. Apparent passage of albumin through the Golgi apparatus during secretion. J Biol Chem. 1971 Jan 10;246(1):240–244. [PubMed] [Google Scholar]
  32. Robinson J. M., Karnovsky M. J. Evaluation of the polyene antibiotic filipin as a cytochemical probe for membrane cholesterol. J Histochem Cytochem. 1980 Feb;28(2):161–168. doi: 10.1177/28.2.6766487. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Schachter H. The subcellular sites of glycosylation. Biochem Soc Symp. 1974;(40):57–71. [PubMed] [Google Scholar]
  35. Schanbacher F. L., Ebner K. E. Galactosyltransferase acceptor specificity of the lactose synthetase A protein. J Biol Chem. 1970 Oct 10;245(19):5057–5061. [PubMed] [Google Scholar]
  36. Schroeder F., Holland J. F., Bieber L. L. Fluorometric investigations of the interaction of polyene antibiotics with sterols. Biochemistry. 1972 Aug 1;11(16):3105–3111. doi: 10.1021/bi00766a026. [DOI] [PubMed] [Google Scholar]
  37. Tanford C., Nozaki Y., Reynolds J. A., Makino S. Molecular characterization of proteins in detergent solutions. Biochemistry. 1974 May 21;13(11):2369–2376. doi: 10.1021/bi00708a021. [DOI] [PubMed] [Google Scholar]
  38. Waechter C. J., Lennarz W. J. The role of polyprenol-linked sugars in glycoprotein synthesis. Annu Rev Biochem. 1976;45:95–112. doi: 10.1146/annurev.bi.45.070176.000523. [DOI] [PubMed] [Google Scholar]
  39. Weeke B. Rocket immunoelectrophoresis. Scand J Immunol Suppl. 1973;1:37–46. doi: 10.1111/j.1365-3083.1973.tb03777.x. [DOI] [PubMed] [Google Scholar]
  40. Widnell C. C. Cytochemical localization of 5'-nucleotidase in subcellular fractions isolated from rat liver. I. The origin of 5'-nucleotidase activity in microsomes. J Cell Biol. 1972 Mar;52(3):542–558. doi: 10.1083/jcb.52.3.542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yamazaki M., Hayaishi O. Allosteric properties of nucleoside diphosphatase and its identity with thiamine pyrophosphatase. J Biol Chem. 1968 Jun 10;243(11):2934–2942. [PubMed] [Google Scholar]
  42. Zambrano F., Fleischer S., Fleischer B. Lipid composition of the Golgi apparatus of rat kidney and liver in comparison with other subcellular organelles. Biochim Biophys Acta. 1975 Mar 24;380(3):357–369. doi: 10.1016/0005-2760(75)90104-6. [DOI] [PubMed] [Google Scholar]
  43. de Kruijff B., Demel R. A. Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. 3. Molecular structure of the polyene antibiotic-cholesterol complexes. Biochim Biophys Acta. 1974 Feb 26;339(1):57–70. doi: 10.1016/0005-2736(74)90332-0. [DOI] [PubMed] [Google Scholar]
  44. de Kruijff B., Gerritsen W. J., Oerlemans A., Demel R. A., van Deenen L. L. Polyene antibiotic-sterol interactions in membranes of Acholeplasma laidlawii cells and lecithin liposomes. I. Specificity of the membrane permeability changes induced by the polyene antibiotics. Biochim Biophys Acta. 1974 Feb 26;339(1):30–43. doi: 10.1016/0005-2736(74)90330-7. [DOI] [PubMed] [Google Scholar]
  45. de Kruijff B., Gerritsen W. J., Oerlemans A., van Dijck P. W., Demel R. A., van Deenen L. L. Polyene antibiotic-sterol interactions in membranes of Acholesplasma laidlawii cells and lecithin liposomes. II. Temperature dependence of the polyene antibiotic-sterol complex formation. Biochim Biophys Acta. 1974 Feb 26;339(1):44–56. doi: 10.1016/0005-2736(74)90331-9. [DOI] [PubMed] [Google Scholar]

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