title: Nonproteolytic incorporation of protein ligands into human alpha 2-macroglobulin: implications for the binding mechanism of alpha 2-macroglobulin.
abstract: alpha 2-Macroglobulin (alpha 2M) is a complex tetrameric protein of 718 kDa. In native alpha 2M, each of the four subunits contains a thiol ester between the side chains of Cys949 and Gln952. Cleavage of the thiol ester with small nucleophiles destabilizes the native conformation and causes a major conformational change in alpha 2M, which leads to exposure of receptor binding sites and a change in electrophoretic mobility. Recently it has been shown that nucleophilic cleavage of the four thiol esters in alpha 2M is a reversible process with energy requirements dependent on the nucleophile [Grøn, H., Thøgersen, I. B., Enghild, J. J., and Pizzo, S. V. (1996) Biochem. J. 318, 539-545]. The present study is a further investigation of the properties of alpha 2M with cleaved thiol esters and the potential for incorporation of protein ligands at the site of the thiol ester. The thiol ester in alpha 2M was cleaved by NH3. After removal of excess NH3, the alpha 2M derivative was incubated with excess protein ligand (hen egg lysozyme or bovine insulin) at 23, 37, or 50 degreesC, leading to covalent incorporation of the ligands in alpha 2M as analyzed by SDS-PAGE, gel filtration, and centrifugal microfiltration. Receptor binding studies and native pore-limit PAGE confirmed that the alpha 2M derivatives with ligand incorporated remained in the receptor-recognized, "fast" migrating conformation. This is the first demonstration of nonproteolytic, covalent incorporation of protein ligands into receptor-recognized alpha 2M.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, North Carolina 27710, USA.
authors.forename: H
authors.initials: H
authors.lastname: Grøn
authors.forename: S V
authors.initials: SV
authors.lastname: Pizzo
mesh_terms: D000818:Animals; D001665:Binding Sites; D002645:Chickens; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D008024:Ligands; D046911:Macromolecular Substances; D017737:Macrophages, Peritoneal / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009113:Muramidase / Q000378:metabolism; D011973:Receptors, LDL / Q000378:metabolism; D013696:Temperature; D000511:alpha-Macroglobulins / Q000378:metabolism*
chemical_list: D007328:Insulin; D008024:Ligands; D046911:Macromolecular Substances; D011973:Receptors, LDL; D000511:alpha-Macroglobulins; D009113:Muramidase; D010450:Endopeptidases