title: alpha-Macroglobulin domain structure studied by specific limited proteolysis.
abstract: Limited proteolysis was used to probe the domain structures of rat alpha 1-inhibitor 3, human pregnancy zone protein, and rat alpha 1-macroglobulin representing monomeric, dimeric, and tetrameric members, respectively, of the alpha-macroglobulin family. Specific limited digestion with trypsin, chymotrypsin, elastase, subtilisin, or Staphylococcus aureus V8 proteinase produced well-defined fragments as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragments were electroblotted to polyvinylidene difluoride membranes and subjected to NH2-terminal sequence analysis to locate the cleavage sites by comparison with the known primary structures of these proteins. Based on these results and the sizes of the generated fragments the 180-kDa alpha-macroglobulin subunit is proposed to contain seven relatively large domains resistant to proteolytic digestion, constituted by approximate residues 1-200 (I), 290-400 (II), 415-660 (III), 710-860 (IVa), 920-1160 (IVb), 1203-1305 (Va), and 1314-1451 (Vb) (human alpha 2-macroglobulin numbering). The overall domain organization is similar to that recently proposed by D. S. Rubenstein, J. J. Enghild, and S. V. Pizzo (1991, J. Biol. Chem. 266, 11252-11261) from studies of rat alpha 1-inhibitor 3, but the present results suggest that the large domains IV and V proposed by these authors are each composed of two domains. The present study emphasizes that domain Vb contains the determinants necessary for receptor recognition.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Denmark.
authors.forename: N K
authors.initials: NK
authors.lastname: Thomsen
authors.forename: L
authors.initials: L
authors.lastname: Sottrup-Jensen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004220:Disulfides / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011257:Pregnancy Proteins / Q000737:chemistry* / Q000302:isolation & purification; D051381:Rats; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000302:isolation & purification
chemical_list: D004220:Disulfides; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011257:Pregnancy Proteins; D000511:alpha-Macroglobulins; D010450:Endopeptidases