Temperature and pH dependence of the association rate constant of elastase with alpha 2-macroglobulin.

Bieth, J G; Meyer, Jean-François

doi:10.1016/s0021-9258(17)47240-1

Cited by 18 publications

(1 citation statement)

References 25 publications

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“…While the interactions of several different enzymes with this inhibitor have been characterized (Gonias et al, 1982;Bjork & Fish, 1982;Barrett et al, 1979;Harpel, 1973;Straight & McKee, 1982; Bieth & Meyer, 1984; Virca & Travis, 1984), several questions remain regarding the reaction mechanism, including the detailed sequence of events that occurs upon proteolysis of a2M which leads to inhibition of the protease. In the present study, the reaction of thrombin with a2M has been characterized by monitoring conformational changes and by quantitating the appearance of free thiol groups in a2M.…”

mentioning

confidence: 99%

Thrombin-induced conformational changes of human .alpha.2-macroglobulin: evidence for two functional domains

Steiner¹,

Bhattacharya²,

Strickland³

1985

Biochemistry

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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)

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