摘要：

The reaction between bovine antithrombin III (AT) and bovine trypsin was'studied and compared to the inactivation of thrombin by the inhibitor in order to elucidate general aspects of the mechanism of AT action. AT and trypsin formed inactive 1:1 complexes, as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS/ PAGE) and trypsin activity measurements. In the absence of heparin, the reaction was about 100-fold faster than the AT-thrombin reaction. The reaction rate increased when AT was preincubated with heparin before trypsin was added. Purified AT-trypsin complex dissociated at pH 10 into free enzyme and two proteolytically modified forms of AT, while no intact AT appeared. In SDS/PAGE, the two modified inhibitors gave bands corresponding to apparent molecular weights of 52000 and 48000 under reducing conditions, while both forms co-migrated with intact AT (mol wt 56000) under non-reducing conditions. This indicates that each of the two modified forms of AT had been cleaved into two or more chains held together by disulfide bonds. Under reducing conditions, the larger of the modified AT chains co-migrated grated with the large chain of thrombin-modified AT, i.e. the form of AT which dissociates from the AT-thrombin complex and which is cleaved at the reactive Arg-Ser bond of the inhibitor. Control experiments showed that the smaller of the two chains was formed by tryptic cleavage of the larger chain. Antisera specific for thrombin-modified AT reacted with purified AT-trypsin complex, demonstrating that the inhibitor was present in the complex in a form immunologically identical to thrombin-modified AT. An analogous finding has been reported earlier for the anti thrombin- thrombin complex. Together, these results suggest the same general mechanism for inhibition of trypsin and thrombin by AT.

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