Thrombin-induced conformational changes of human alpha 2-macroglobulin: evidence for two functional domains.
The interaction of thrombin with alpha 2-macroglobulin (alpha 2M) was characterized by monitoring conformational changes and measuring the increase of free sulfhydryl groups during the reaction. Under experimental conditions where [thrombin] greater than [alpha 2M], the conformational change, measured by increases in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate, and thiol group appearance displayed biphasic kinetics. The initial rapid phase results in the formation of a stable complex, the appearance of two sulfhydryl groups, the cleavage of approximately half of the Mr 180 000 subunits, and a conformational change that is not as extensive as that which occurs with trypsin. The slower phase is associated with the appearance of two additional sulfhydryl groups, increased cleavage of the Mr 180 000 subunit, and additional conformational changes. The available evidence suggests that the slow phase results from hydrolysis of the Mr 180 000 subunit(s) due to proteolysis of the alpha 2M-thrombin complex by free thrombin. Experiments with 125I-thrombin document the binding of 1 mol of thrombin/mol of alpha 2M that is not dissociated upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the complex. At higher ratios of thrombin to alpha 2M, a second mole of thrombin will reversibly associate with the 1:1 alpha 2M-thrombin complex. Under conditions where [thrombin] less than [alpha 2M], biphasic kinetics were not observed, and the conformational change, sulfhydryl appearance, and hydrolysis of the Mr 180 000 subunit were found to follow second-order kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)
J P
JP
Steiner
P
P
Bhattacharya
D K
DK
Strickland
D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005456:Fluorescent Dyes; D006801:Humans; D007700:Kinetics; D009282:Naphthalenesulfonates; D011485:Protein Binding; D011487:Protein Conformation; D013917:Thrombin / Q000502:physiology*; D000511:alpha-Macroglobulins / Q000378:metabolism*
D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D000511:alpha-Macroglobulins; C009497:2-(4-toluidino)-6-naphthalenesulfonic acid; D004228:Dithionitrobenzoic Acid; D013917:Thrombin