Open AccessIntracellular modifications of rat α 1 inhibitor 3
Author(s) -
SJÖBERG Mathilda,
ESNARD Frédéric,
FRIES Erik
Publication year - 1991
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1991.tb15882.x
Subject(s) - endoplasmic reticulum , intracellular , chemistry , cleavage (geology) , monomer , sulfation , immunoprecipitation , cell fractionation , methionine , biochemistry , stereochemistry , alpha (finance) , gel electrophoresis , biophysics , enzyme , biology , amino acid , paleontology , organic chemistry , fracture (geology) , gene , polymer , medicine , construct validity , nursing , patient satisfaction
α 1 Inhibitor 3 (α 1 I 3 ) 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 S]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 α 1 I 3 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 α 1 I 3 migrated with a mobility lower than that of the reduced protein, half‐maximal conversion occurring after about 10 min. This finding suggests that α 1 I 3 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 α 1 I 3 is sulphated, presumably at Tyr618.