Abstract
The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.3:1 (thrombin:alpha 2M). Measurements of the ability of alpha 2M to bind trypsin after prior reaction with thrombin indicated that thrombin binds rapidly at one site on alpha 2M, but occupies the second site with some difficulty. Intrinsic fluorescence studies of trypsin binding to alpha 2M at pH 5.0, 6.5, and 8.0 not only revealed striking differences in trypsin's behavior over this pH range, but also some similarities between the behavior of thrombin and trypsin not heretofore recognized. Structural studies, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to measure alpha 2M-125I-thrombin covalent complex formation, indicated that covalency reached a maximum at a mole ratio of approximately 1.5:1. At this ratio, only 1 mol of thrombin is bound covalently per mol of alpha 2M. These gel studies and those of proteolytic digests of denatured alpha 2M-125I-trypsin and alpha 2M-125I-thrombin complexes suggest that proteinases form covalent bonds with uncleaved alpha 2M subunits. The sum of our results is consistent with a mechanism of proteinase binding to alpha 2M in which the affinity of the proteinase for alpha 2M during an initial reversible interaction determines its binding ratio to the inhibitor.
Highlights
From the Howard Hughes Medical Institute Laboratories, the Departmentof Medicine and the Departmentof Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
The interaction between azM and thrombin waalso assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digestsof denatured ~y~M-’~~I-throm and azM-’261-trypsincomplexes
The posure caused by methylaminwe,e found that thrombin precise structural aspectsof the proteinasebinding sites have produced its maximum effectsa t a mole ratio of -1.3:l not been defined, it is generally accepted that azM has two
Summary
Perature was controlled at 25 "C using a Lauda K-2/RD circulating water bath. Reaction of azM with Methylaminaefter Treatment with Thrombin ThiolEsterExposurewhena2M Is Reacted withDifferent or Trypsin-apM (0.89 p M ) was incubated with thrombin (0-2.3 p M ) MoleRatiosofThrombin-The reaction of thrombin with overnight a t 4 "C in 0.05 M Tris, 1%PEG at pH 8.0 and with a2M and the subsequentcleavage of thiol ester bonds in azM the thiol reagent 2,2'-dipyridyl disulfide (0.07 mM) for 1 h at room temperature. The Effect of ThrombinReaction with aaM on Subsequent Binding of Trypsin-Increasing concentrations of thrombin (0-1.3 p ~ we) re reacted overnight with azM (0.43 p ~ a t) 4 'C in 0.05 M Tris, 0.1 M tometer and reacted with methylamine (0.1 M, 25 "C) until a maximum fluorescence change was reached. Importantly,ATIII(unlike soybean trypsininhibitor) does bin:azM complexes [14].We attempted to study this not bind to the a,M-proteinase complex [27, 28] and, under in more detail to determine the extent wtohich such covalent our conditions, the ATIII-trypscinomplexes do notdissociate bond formation is involved in the mechanism of thrombin's during the assay [29].Fig. 2 shows results of an experi- reaction with aaM.In accord with earlier findings, the a2M-.
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