Structure of alpha 2-macroglobulin-protease complexes. Methylamine competition shows that proteases bridge two disulfide-bonded half-molecules.
alpha 2-Macroglobulin (alpha 2M) forms several different covalent complexes with proteases. These include unusual forms in which more than one of the four identical subunits of alpha 2M are cross-linked by amide bonds to more than one lysyl amino group of the bound protease. The structure of these complexes and the question of how the identical subunits are arranged to form two protease binding sites are matters of current controversy. The 185-kDa subunits are arranged into two disulfide-bonded half-molecules which are, in turn, noncovalently associated. We have provided evidence that, in the major multivalent cross-linked form, proteases can span the two half-molecules, forming a covalently bonded tetramer [Wang, D., Yuan, A. I., & Feinman, R. D. (1984) Biochemistry 23, 2807-2811]. An alternative theory has recently been proposed in which the major high molecular weight form has two bonds to protease that are within half-molecules--a multivalent cross-linked dimer [Sottrup-Jensen, L., Hansen, H. F., Pedersen, H. S., & Kristensen, L. (1990) J. Biol. Chem. 265, 17727-17737]. To resolve this conflict, experiments were carried out to determine the structure of one of the high molecular weight bands (band 3) seen on SDS-PAGE. Band 3 has anomalous migration, corresponding to markers of apparent molecular mass of 550 kDa (between the tetramer and dimer). In the experiments described here, reactions of thrombin with alpha 2M were run in the presence of methylamine, which competes for one of the two thrombin-alpha 2M covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)
Chen
B J
BJ
Department of Biochemistry, State University of New York Health Science Center, Brooklyn 11203.
Wang
D
D
Yuan
A I
AI
Feinman
R D
RD
D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000648:ultrastructure*; D006801:Humans; D046911:Macromolecular Substances; D008744:Methylamines / Q000737:chemistry*; D008970:Molecular Weight; D011485:Protein Binding; D013917:Thrombin / Q000737:chemistry; D000511:alpha-Macroglobulins / Q000648:ultrastructure*
D004220:Disulfides; D046911:Macromolecular Substances; D008744:Methylamines; D000511:alpha-Macroglobulins; C027451:methylamine; D010450:Endopeptidases; D013917:Thrombin