Purification and characterization of a "half-molecule" alpha 2-macroglobulin from the southern grass frog: absence of binding to the mammalian alpha 2-macroglobulin receptor.
An alpha-macroglobulin (alpha 2M), which is a dimer consisting of two non-disulfide-bonded subunits, was identified and purified from frog plasma by Ni2+ chelate affinity chromatography. This frog "half-molecule" alpha-macroglobulin migrated as an alpha 2-globulin in cellulose-acetate electrophoresis rather than as the previously described frog alpha 1M, which exists as a tetramer formed by the noncovalent association of disulfide-bonded pairs. A molecular weight of approximately 380 000 was obtained by gel-filtration high-pressure liquid chromatography, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) the protein migrated as a single band of Mr approximately 180 000 before and after reduction. No evidence was obtained for association of this protein to a higher molecular weight species. After the preparation was heated, additional bands were obtained in SDS-PAGE with Mr approximately 60 000 and 12 000. The additional bands were not obtained after heating methylamine-treated preparations. The circular dichroic spectrum of frog alpha 2M exhibits negative ellipticity over the region 205-250 nm with a minimum at 216 nm. After reaction with proteinase, a decrease in the absolute mean residue rotation was obtained. Amino acid analysis demonstrated that frog alpha 2M and alpha 1M are similar in composition to avian and mammalian alpha-macroglobulins; however, there are sufficient differences in the composition of these two amphibian alpha-macroglobulins to support the conclusion that they are distinct proteins. Frog alpha 2M bound approximately 0.5 mol of trypsin/mol of inhibitor. This binding was abolished by pretreatment with methylamine.(ABSTRACT TRUNCATED AT 250 WORDS)
Feldman
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Pizzo
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D008958:Models, Molecular; D008970:Molecular Weight; D011487:Protein Conformation; D011894:Rana pipiens; D011971:Receptors, Immunologic / Q000378:metabolism*; D013045:Species Specificity; D000511:alpha-Macroglobulins / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins