BINDING OF TRANSFORMING GROWTH FACTOR-BETA-1 TO METHYLAMINE-MODIFIED ALPHA-2-MACROGLOBULIN AND TO BINARY AND TERNARY ALPHA-2-MACROGLOBULIN PROTEINASE COMPLEXES
The binding of I-125-labelled transforming growth factor-beta-1 (TGF-beta-1) to human alpha(2)-macroglobulin (alpha(2)M) was studied by native PAGE and autoradiography. TGF-beta-1 bound preferentially to alpha(3)M-methylaminc and minimally, if at all, to native alpha(2)M. Preparations of alpha(2)M protcinase complex were generated by incubating a standard concentration of alpha(g)M (0.4-mu-M) with different concentrations of trypsin, chymotrypsin or neutrophil clastase (0.04-2.0-mu-M). The I-125-TGF-beta-1-binding activity depended on the initial ratio of active proteinase to alpha(2)M, or r value, used to form the alpha(2)M-proteinase complex. With all three proteinases, r values of 2 or greater yielded preparations with unchanged or decreased TGF-beta-1-binding activity relative to native alpha(2)M. By contrast, r values near 1 yielded preparations with significantly increased TGF-beta-1-binding activity. The results of [H-3]thymidine-incorporation studies performed in mouse keratinocytes were consistent with the I-125-TGF-beta-binding experiments. Alpha(2)M trypsin and alpha(2)M-chymotrypsin prepared at an r value of 1.0 counteracted the activity of TGF-beta-1, whereas the equivalent complexes prepared at an r value of 3.0 had no effect. As determined by SDS/PAGE, I-125-TGF-beta-1 binding to alpha(2)m methylamine was at least 80% non-covalent. Reaction of alpha(2)M-methylamine with iodoacctamide or 5,5'-dithiobis-(2-nitrobenzoic acid) decreased the percentage of covalent binding but had no effect on total binding. Neuraminidase treatment had no effect on the binding of I-125-TGF-beta-1 to alpha(2)M-methylamine. Cleavage of the 'bait regions' in alpha(2)M-methylamine by prolonged treatment with trypsin also had no effect. These studies suggest that TGF-beta-1 binding to alpha(2)M is enhanced by conformational change in the proteinase inhibitor resulting from reaction with proteinase or aminc. If both proteinase-binding sites in a single alpha(2)M molecule are occupied, TGF-beta-1-binding activity is decreased or perhaps eliminated.