Purification and characterization of frog alpha-macroglobulin: receptor recognition of an amphibian glycoprotein.
Frog alpha-macroglobulin was purified to apparent homogeneity by Ni2+ chelate affinity chromatography. Frog alpha-macroglobulin migrated as an alpha 1-globulin in cellulose acetate electrophoresis. A molecular weight of 730 000 was obtained by equilibrium sedimentation, and in sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE), the protein migrated as a single band of Mr approximately 360 000 before reduction and Mr approximately 180 000 after reduction. Treatment with trypsin resulted in subunit cleavage to yield a fragment of Mr approximately 90 000. After being heated, the protein fragmented, migrating in SDS-PAGE as two bands of Mr approximately 120 000 and 60 000. This fragmentation was inhibited by prior reaction of the protein with methylamine. In native pore-limit electrophoresis the protein exhibited the characteristic "slow" to "fast" conformational change of protease-treated alpha-macroglobulins. In contrast, typical "slow" to "fast" conformational change was not observed in native PAGE with this preparation. Moreover, the protein incorporated approximately 2 mol of [14C]methylamine/mol of inhibitor without demonstrating a change in mobility in native PAGE. In circular dichroism studies, the protein exhibited a spectrum similar to that of human alpha 2M. Reaction with trypsin resulted in a broadening and decrease in the magnitude of the spectrum. Reaction with methylamine resulted in similar changes, but of smaller magnitude. The inhibitor bound approximately 0.7 mol of trypsin in both radiolabeled protease binding and amidolytic titration studies. 125I-Labeled native frog alpha 1M was removed slowly from the circulation of mice with a t1/2 greater than 2h.(ABSTRACT TRUNCATED AT 250 WORDS)
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Feldman
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Pizzo
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001667:Binding, Competitive; D002241:Carbohydrates / Q000032:analysis; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D006358:Hot Temperature; D006801:Humans; D007700:Kinetics; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D008744:Methylamines / Q000494:pharmacology; D008970:Molecular Weight; D011487:Protein Conformation; D011894:Rana pipiens; D011971:Receptors, Immunologic / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D002241:Carbohydrates; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D046911:Macromolecular Substances; D008744:Methylamines; D011971:Receptors, Immunologic; D000511:alpha-Macroglobulins; C027451:methylamine