title: The active site of antithrombin. Release of the same proteolytically cleaved form of the inhibitor from complexes with factor IXa, factor Xa, and thrombin.
abstract: Reactions between near equimolar amounts of antithrombin and Factors IXa or Xa resulted in the formation of a free proteolytically modified, two-chain form of the inhibitor, in addition to the inactive antithrombin-protease complexes. The modified inhibitor produced by either enzyme was electrophoretically identical with that formed in the reaction with thrombin. As in the latter reaction, the formation of the modified antithrombin by Factor Xa was increased in the presence of heparin, while only small amounts were produced by Factor IXa both in the absence and presence of the polysaccharide. NH2-terminal sequence analyses of the isolated modified inhibitor formed by Factor Xa showed that a single Arg-Ser bond in the COOH-terminal end of the inhibitor had been cleaved. This cleavage site is identical with that identified in free thrombin-modified antithrombin. The purified antithrombin-Factor IXa and antithrombin-Factor Xa complexes were dissociated by ammonia or hydroxylamine into free enzyme and a modified two-chain form of the inhibitor. Electrophoresis studies and NH2-terminal sequence analyses showed that the modified antithrombin obtained from either complex was identical with that produced in free form by the two enzymes and also with the modified inhibitor that is released from the antithrombin-thrombin complex. The fact that identical results were obtained for the reactions between antithrombin and three enzymes with different specificities strongly suggests that the observed Arg-Ser cleavage site is the active site of antithrombin.
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000991:Antithrombins / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D005164:Factor IX / Q000378:metabolism*; D015949:Factor IXa; D005170:Factor X / Q000378:metabolism*; D015951:Factor Xa; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D013917:Thrombin / Q000378:metabolism*
chemical_list: D000991:Antithrombins; D046911:Macromolecular Substances; D005164:Factor IX; D005170:Factor X; D015949:Factor IXa; D013917:Thrombin; D015951:Factor Xa