Subcellular localization of the sulphation reaction of heparan sulphate synthesis and transport of the proteoglycan to the cell surface in rat liver.
We report on the incorporation of radiolabelled sulphate into proteoglycan in the 'in situ'-perfused rat liver. After 5 min virtually all of the [35S]sulphate was incorporated into heparan sulphate; no partially sulphated precursors were detected. Pulse-chase experiments, followed by centrifugation in gradients of sucrose and metrizamide, showed that, at 5 min, the heparan sulphate was associated predominantly with the Golgi membranes. Over the next 20 min, intact proteoglycan appeared at the plasma membrane. At intermediate times the heparan sulphate was detected simultaneously in two distinct populations of membrane vesicles. Whether the heparan sulphate in these two populations has two different destinies (e.g. plasma membrane or secretion) is not yet clear. Subfractionation of the Golgi membranes showed that the N-sulphotransferase co-purified with the heparan [35S]sulphate and was separable from the galactosyltransferase of glycoprotein synthesis, confirming that the Golgi membrane system is functionally segregated. Subfractionation also permitted an almost 100-fold purification of the N-sulphotransferase over the homogenate: this will provide an excellent starting material for isolation and further characterization of the enzyme.
Graham
J M
JM
Department of Biochemistry, St. George's Hospital Medical School, London, U.K.
Winterbourne
D J
DJ
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D006025:Glycosaminoglycans / Q000096:biosynthesis*; D006056:Golgi Apparatus / Q000201:enzymology / Q000378:metabolism; D006497:Heparan Sulfate / Q000096:biosynthesis*; D007425:Intracellular Membranes / Q000378:metabolism; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism; D046911:Macromolecular Substances; D008297:Male; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*; D013466:Sulfurtransferases / Q000378:metabolism
D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D046911:Macromolecular Substances; D011509:Proteoglycans; D013431:Sulfates; D013466:Sulfurtransferases