Biosynthesis of bikunin (urinary trypsin inhibitor) in rat hepatocytes.
One of the major sulfated proteins secreted by rat hepatocytes contains a low-sulfated chondroitin sulfate chain and its apparent molecular mass upon sodium dodecyl sulfate/polyacrylamide gel electrophoresis shifts from 40 to 28 kDa upon chondroitinase ABC treatment (E. M. Sjöberg and E. Fries, 1990, Biochem. J. 272, 113-118). These properties suggest that this protein is the rat homologue of the major trypsin inhibitor of human urine which was recently named bikunin. In serum, bikunin occurs mainly as a subunit of the pre-alpha-inhibitor and the inter-alpha-inhibitor; in these proteins it is covalently linked to the other polypeptides through its chondroitin sulfate chain. Bikunin has been shown to be synthesized by liver cells as a 42-kDa precursor, in which it is linked to alpha 1-microglobulin by two basic amino acids. We have isolated bikunin from rat urine and prepared antibodies against it. In rat hepatocytes pulse-labeled with [35S]methionine, these antibodies precipitated a labeled protein of 42 kDa. Upon chase, three different labeled proteins were recognized by the antibodies in the medium: one protein of 40 kDa (free bikunin), one of 125 kDa (presumably pre-alpha-inhibitor), and one greater than 240 kDa (possibly a protein related to the inter-alpha-inhibitor). Pulse-chase experiments with [35S]sulfate showed that these proteins occurred intracellularly as precursors containing alpha 1-microglobulin. These results demonstrate that the completion of the chondroitin sulfate chain and its coupling to other polypeptide chains occur before the cleavage of the alpha 1-microglobulin/bikunin precursor.
Sjöberg
E M
EM
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Fries
E
E
D000818:Animals; D002478:Cells, Cultured; D002808:Chondroitin Lyases / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000096:biosynthesis* / Q000378:metabolism; D008099:Liver / Q000166:cytology / Q000378:metabolism*; D008297:Male; D008562:Membrane Glycoproteins; D008715:Methionine / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013431:Sulfates / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014360:Trypsin Inhibitor, Kunitz Soybean; D014361:Trypsin Inhibitors / Q000096:biosynthesis* / Q000652:urine
D006023:Glycoproteins; D008562:Membrane Glycoproteins; C109148:SPINT2 protein, human; D013431:Sulfates; D013462:Sulfur Radioisotopes; D014361:Trypsin Inhibitors; D014360:Trypsin Inhibitor, Kunitz Soybean; D008715:Methionine; D002808:Chondroitin Lyases