Tyrosine O-sulfation of the fibrinogen gamma B chain in primary cultures of rat hepatocytes.
Sulfation of fibrinogen was studied in a primary culture of rat hepatocytes. After cells were incubated with [35S]sulfate, 35S-labeled fibrinogen was obtained from the medium by immunoprecipitation and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis/fluorography. It was demonstrated that [35S]sulfate is exclusively incorporated into the gamma B chain, which is a minor variant form found in rat fibrinogen, in addition to a major gamma A chain. When the purified 35S-gamma B chain was digested with carboxypeptidase Y, the radioactivity was almost completely released from the protein, and the labeled product released was identified as tyrosine O-sulfate. Based on the available primary structure of the gamma B chain, the results suggest that sulfation occurs on the tyrosine residue at the second position from its COOH terminus. Pulse-chase experiments using both [3H]leucine and [35S]sulfate showed that 35S-labeled fibrinogen is secreted into the medium much faster than the 3H-labeled molecule. Incubation of cells with monensin, an inhibitor of Golgi function, strongly inhibited the sulfation of fibrinogen. In addition, in vitro sulfation experiments demonstrated that sulfotransferase activity is localized in the Golgi fraction. These results indicate that the sulfation of fibrinogen takes place in the Golgi complex, especially in the trans Golgi region, just before its secretion.
Hirose
S
S
Department of Biochemistry, Fukuoka University School of Medicine, Japan.
Oda
K
K
Ikehara
Y
Y
D000818:Animals; D002478:Cells, Cultured; D005340:Fibrinogen / Q000096:biosynthesis / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D046911:Macromolecular Substances; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013431:Sulfates / Q000378:metabolism*; D013462:Sulfur Radioisotopes; D014443:Tyrosine
D046911:Macromolecular Substances; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014443:Tyrosine; D005340:Fibrinogen