Intracellular modifications of rat alpha 1 inhibitor3. Formation of disulphides, internal thiolester and sulphation.
alpha 1 Inhibitor3 (alpha 1I3) is a monomeric protease inhibitor of about 190 kDa which is secreted by rodent hepatocytes. We have studied intracellular modifications of this protein in [35]methionine-labelled rat hepatocytes by pulse/chase experiments followed by immunoprecipitation and gel electrophoresis under reducing and nonreducing conditions. Directly after the pulse, most of the unreduced alpha 1I3 migrated faster than the reduced form, indicating that disulphide bridges are formed during or shortly after synthesis yielding a compact structure. With increasing chase time however, an increasing portion of the unreduced alpha 1I3 migrated with a mobility lower than that of the reduced protein, half-maximal conversion occurring after about 10 min. This finding suggests that alpha 1I3 undergoes a conformational change in the endoplasmic reticulum, possibly becoming more elongated. During 10-30 min of chase, the protein acquired the capacity to undergo autolytic cleavage upon heating, a property due to the existence of an internal thiolester bond [Howard, J. B., Vermeulen, M. & Swenson, R. P. (1980) J. Biol. Chem. 255, 3820-3823; Esnard, F., Gutman, N., El Moujahed, A. & Gauthier, F. (1985) FEBS Lett. 182, 125-129]. Analysis by subcellular fractionation indicated that this bond is formed in the endoplasmic reticulum. Finally, we show that secreted alpha 1I3 is sulphated, presumably at Tyr618.
Sjöberg
M
M
Department of Medical and Physiological Chemistry, University of Uppsala, Sweden.
Esnard
F
F
Fries
E
E
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography / Q000379:methods; D002478:Cells, Cultured; D004220:Disulfides / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008715:Methionine / Q000378:metabolism; D008744:Methylamines / Q000494:pharmacology; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000378:metabolism*; D051381:Rats; D013440:Sulfides / Q000378:metabolism; D013462:Sulfur Radioisotopes; D014316:Tritium
D000209:Acute-Phase Proteins; D004220:Disulfides; D008744:Methylamines; D011480:Protease Inhibitors; D013440:Sulfides; D013462:Sulfur Radioisotopes; C017894:alpha 1-inhibitor 3; D014316:Tritium; D008715:Methionine; C027451:methylamine