Purification and characterization of frog .alpha.-macroglobulin: receptor recognition of an amphibian glycoprotein

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Abstract
Frog .alpha.-macroglobulin [M] was purified to apparent homogeneity by Ni2+ chelate affinity chromatography. Frog .alpha.-M migrated as an .alpha.1-globulin in cellulose acetate electrophoresis. A MW of 730,000 was obtained by equilibrium sedimentation, and in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the protein migrated as a single band of MW .apprx. 360,000 before reduction and MW .apprx. 180,000 after reduction. Treatment with trypsin resulted in subunit cleavage to yield a fragment of MW .apprx. 90,000. After being heated, the protein fragmented, migrating in SDS-PAGE as 2 bands of MW .apprx. 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.-M. Typical slow to fast conformational change was not observed in native PAGE with this preparation. The protein incorporated .apprx. 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 similar changes, but of smaller magnitude. The inhibitor bound .apprx. 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 [mean survival time] > 2 h. After reaction with trypsin, the protein was removed in a 1st-order reaction with t1/2 < 3 min. The clearance was significantly inhibited only by coinjection of a combination of human .alpha.2M-methylamine, asialoorosomucoid, and lactoferrin; suggesting recognition by multiple receptor systems. Native frog .alpha.1M did not inhibit the binding of 0.2 nM 125I-labeled human .alpha.2M-methylamine to mouse peritoneal macrophages in vitro, but frog .alpha.1M-trypsin inhbited the binding by 50% at a concentration of 0.6 nM with a Ki of 0.4 nM.