Etude du complexe plasmine-α2-macroglobuline de porc

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)

Section: Discussionsupporting

confidence: 92%

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

Steiner¹,

Bhattacharya²,

Strickland³

1985

“…Studies examining the ability of trypsin to bind to the complex confirm that this is indeed the case and that the a2M-plasmin complex is capable of binding 0.4 mol of trypsin. These studies are in agreement with those of Jacquot-Armand and Guinand (1976) but appear to differ from those of Gonias and Pizzo (1983b), who examined the ability of the a2M-plasmin complex to bind trypsin and essentially found no binding. In this case, the initial a2M-plasmin complex was prepared by incubating a 4.4-fold molar excess of plasmin with the inhibitor for an hour.…”

Section: Discussionsupporting

confidence: 84%

Characterization of the reaction of plasmin with .alpha.2-macroglobulin: effect of antifibrinolytic agents

Steiner¹,

Migliorini²,

Strickland³

1987

The reaction of several plasmin derivatives with alpha 2-macroglobulin (alpha 2M) has been investigated. Titration experiments measuring conformational changes in alpha 2M, changes in the number of sulfhydryl groups available for titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and changes in the ability of alpha 2M to protect bound plasmin from inhibition by soybean trypsin inhibitor all suggested that between 1.3 and 1.5 mol of plasmin was bound per mole of inhibitor. Under experimental conditions where [plasmin] greater than [alpha 2M], the conformational change occurring in the inhibitor and thiol group appearance displayed biphasic kinetics. Examination of the extent of subunit cleavage by plasmin revealed that the rapid phase was associated with cleavage of approximately two to three of the four alpha 2M subunits, while cleavage of the remaining subunits occurred during the slow phase of the reaction. Binary (1:1) alpha 2M-plasmin complexes were prepared by reacting a large excess of alpha 2M with plasmin and purifying the resultant complex by immunoaffinity chromatography using a monoclonal antibody specific for a neoantigen on alpha 2M that is generated when the inhibitor reacts with proteases or with methylamine. Characterization of the purified complex revealed that two of the four subunits were cleaved, and the conformational change, measured by alterations in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS), was approximately 50% of that measured for a 2:1 complex. Thus it appears that proteolysis and conformational alterations associated with the binding of 1 mol of plasmin to alpha 2M are limited to one of two functional units in the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

“…The results outlined above, supporting the model of Pochon et al (1981), are also consistent with this second model. A number of laboratories have reported, however, that 1:1 complexes of a2M with plasmin or a-chymotrypsin dimer retain the capacity to bind small proteases such as trypsin or a-chymotrypsin monomer (Ganrot, 1967a;Pochon et al, 1981;Jacquat-Armand & Guinand, 1976). These results are difficult to reconcile with a model of a2M-protease binding stoichiometry based on reaction rate alone.…”

Section: Discussionmentioning

confidence: 96%

Reaction of human .alpha.2-macroglobulin half-molecules with plasmin as a probe of protease binding site structure

Gonias¹,

Pizzo²

1983

Human alpha 2-macroglobulin (alpha 2M) half-molecules were prepared by limited reduction and alkylation of the native protein. Reaction with plasmin resulted in nearly quantitative cleavage of the half-molecule Mr approximately 180000 subunits into Mr approximately 90000 fragments. Subunit cleavage was significantly less complete when plasmin was reacted with alpha 2M whole molecules. The plasmin and trypsin binding capacities of the two forms of alpha 2M were compared by using radioiodinated proteases. alpha 2M half-molecules bound an equivalent number of moles of plasmin or trypsin. Native unreduced alpha 2M bound only half as much plasmin as trypsin. These data are consistent with the hypothesis that the two protease binding sites are adjacent in native alpha 2M. alpha 2M half-molecule-plasmin complexes reassociated less readily than half-molecule-trypsin complexes, supporting this interpretation. The frequency of covalent bond formation between plasmin and alpha 2M was considerably higher than that previously observed with other proteases. Approximately 80-90% of the plasmin that reacted with alpha 2M whole molecules or half-molecules became covalently bound. The reactivities of purified alpha 2M-plasmin complexes were compared with small and large substrates. Equivalent kcat/Km values were determined at 22 degrees C for the hydrolysis of H-D-Val-Leu-Lys-p-nitroanilide dihydrochloride by whole molecule-plasmin complex and half-molecule-plasmin complex (40 mM-1 s-1 and 39 mM-1 s-1, respectively, compared with 66 mM-1 s-1 determined for free plasmin).(ABSTRACT TRUNCATED AT 250 WORDS)