Covalent thrombin-alpha 2-macroglobulin complexes. Evidence for bivalent cross-linking of inhibitor chains by a single enzyme molecule.
Complexes formed between thrombin and alpha 2-macroglobulin (alpha 2M) were studied by polyacrylamide gel electrophoresis. The results provide evidence for the existence of a recently proposed novel enzyme-inhibitor species in which a single thrombin molecule forms two or more covalent bonds to two or more different alpha 2M chains. At least one of several slowly migrating bands (greater than 375K on nonreduced gels) that have previously been observed in the literature but not well characterized can be assigned to the new species. The involvement of the lysyl amino groups of thrombin is shown by the observation that methylation of these groups reduces the higher molecular weight bands. In addition, increasing the thrombin:alpha 2M ratio causes a relative decrease in the higher molecular weight species, suggesting that these complexes arise by intramolecular reactions that are susceptible to competition by solution thrombin. The data provide support for our previous proposal [Wang, D., Yuan, A., & Feinman, R.D. (1983) Ann. N.Y. Acad. Sci. 421, 90-97] that the 260K band seen in reduced gels is composed of two proteolyzed inhibitor subunits linked to one thrombin molecule. This intersubunit link maintains the integrity of the alpha 2M in sodium dodecyl sulfate, accounting for the high molecular weight bands under nonreducing conditions. Comparison with a synthetically cross-linked alpha 2M molecule allows a tentative but not unambiguous assignment of one of the bands to this novel structure.
D003432:Cross-Linking Reagents; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D011485:Protein Binding; D011725:Pyridines / Q000494:pharmacology; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013917:Thrombin / Q000378:metabolism*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D004220:Disulfides; D046911:Macromolecular Substances; D011725:Pyridines; D013439:Sulfhydryl Reagents; D000511:alpha-Macroglobulins; C007542:4,4'-dipyridyl disulfide; D013917:Thrombin