Structural characterization of human alpha2-macroglobulin subunits.

Abstract

Human crz-macroglobulin (azM) (>98% a single component) was isolated in gram quantities using polyethylene glycol, ammonium sulfate, and ion exchange fractionation of outdated human plasma.To determine the number and similarity of subunits in the native protein, quantitative repetitive Edman degradation was performed.The single, unique sequence was found to be: NHz-Ser-Val-Ser-Gly-Lys-Pro-Ghr-Tyr-Met-Val-Leu-Val-Pro-.The sequence was quantitated to 3.2 mol of NH&erminal residues/M, = 725,000.Carboxypeptidase Y digestion of lvzM released seven different amino acids in a time-dependent pattern and in quantities approaching 4 mol of amino acid/M= = 725,000.The following COOH-terminal sequence was assigned from the results, -Be-Phe-(Ala,Tyr)-(Glu,Val)-Leu-COOH.Radioautography of tryptic peptide maps of [2-14C]carboxymethylated aaM revealed 20 to 23 radiodense spots, which is consistent with a single subunit ofM, = 185,000 containing 28 cysteinyl residues (one-fourth of the native molecular weight).These data, for the first time, provide chemical evidence that azM is composed of four identical, M, = 185,000 peptide chains.Using our highly purified arzM and carefully quantitated trypsin, LY~M was found to inhibit 1.7 to 2.1 mol of trypsin/Cchain native aaM.Concomitant with trypsin binding, all four peptide chains were hydrolyzed to two M, = 85,000 fragments.This indicates that two peptide chains are cleaved per molecule of trypsin bound.