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Identification of "embryonin" as bovine alpha 2-macroglobulin
Abstract:
Pedersen fetuin contains a contaminant, previously named "embryonin" that exhibits immuno-cross reactivity with human alpha2-macroglobulin (alpha2Mh). In the present study, it is demonstrated that this protein coelutes with alpha2Mh in gel filtration chromatography and can be purified to homogeneity by Zn2+ chelate chromatography. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this contaminant exhibited similar subunit size, protease-induced cleavage fragments, and heat fragmentation as alpha2Mh. 125I-trypsin and 125I-chymotrypsin each bound at a ratio of 0.9 mol/mol to this fetuin-derived native alpha2M (alpha2Mf) and at a ratio of less than 0.2 mol/mol to methylamine-treated alpha2Mf. As determined by SDS-PAGE, 1:1 molar ratio of protease to alpha2Mf cleaved each alpha2Mf subunit to fragments of Mr approximately 72,000. At a 0.2:1 molar ratio of trypsin to alpha2Mf-methylamine, every alpha2Mf-methylamine subunit was cleaved to polypeptide chains of Mr approximately 72,000 and 110,000. In native PAGE, alpha2Mf and alpha2Mf-methylamine migrated with the same mobility; after reaction with trypsin their mobilities increased similarly. 125I-alpha2Mf cleared from the circulation of mice with a t1/2 of 30 min. The trypsin or methylamine derivative of 125I-alpha2Mf cleared with t1/2 of less than 5 min and clearance was competable when the ligand was co-injected with a large molar excess of unlabeled alpha2Mh-methylamine. alpha2Mf, 0.3 nM, treated with trypsin or methylamine, inhibited 50% of the binding of 0.1 nM 125I-alpha2Mh-methylamine to specific receptors on mouse peritoneal macrophages in vitro. Native alpha2Mf did not inhibit significantly the binding of the ligand at this concentration. Bovine alpha2M (alpha2Mb) was purified from plasma by Ni2+ chelate chromatography. By SDS-PAGE, amino acid analysis, and cyanogen bromide peptide mapping, it was indistinguishable from the alpha2M purified from fetuin. It is concluded that embryonin is bovine alpha2M.