Functional modifications of alpha 2-macroglobulin by primary amines. I. Characterization of alpha 2 M after derivatization by methylamine and by factor XIII.

Human at-macroglobulin (azM) was isolated after derivatization with monodansylcadaverine by endogenous fibrin stabilizing factor activity in clottingplasma.Spectrophotometric analysis indicated two y-glutamyl residues/molecule in az-macroglobulin dansylcadaverine (azM.DC).This derivative was compared to azM isolated from human plasma with respect to trypsin binding and methylamine inactivation and was further characterized in the following systems: rate electrophoresis in 5% polyacrylamide gels under native conditions, isoelectric focusing, and recognition by the fibroblast azM-receptor.Trypsin treatment and inactivation with methylamine of a2M and a2M.DC resulted in a higher electrophoretic mobility in polyacrylamide gels, confirming literature data.In addition, isoelectric focusing indicated this to be accompanied by an increase in PI and a dramatic increase in the complexity of the pattern.The focusing patterns were similar for both azM and azM= DC before and after trypsin or methylamine treatment.Incorporation of ["C]methylamine in azM was studied as a function of time.Under the conditions specified, the overall time course was biphasic.Incorporation of methylamine and the ensuing conformational change of azM could be clearly separated in time by analytical examination of labeled slow and fast forms of azM during the course of the reaction.Complete inactivation of azM with methylamine resulted in modified azM effectively recognized by the fibroblast receptor.Kinetic analysis of endocytosis indicated this form to behave exactly as azM-protease complexes.These results indicate that the receptor recognizes the typical conformational state of atM, excluding primary changes resulting from proteolytic attack to be involved in receptor binding.Although the analytical systems were not able to detect differences in aZM* DC when compared to plasma azM, azM*DC was unable to inhibit receptor-mediated endocytosis of '261-labeled a2M.trypsin complexes by fibroblasts in culture.This indicates that a2M-DC, after trypsin or methylamine treatment, is not effectively recognized by the cellular receptor.We have previously demonstrated that normal human skin fibroblasts in culture are capable of internalizing large