Open AccessIdentification of the Proteolytic Thrombin Fragments Formed after Cleavage with Rat Mast Cell Protease 1
Author(s) -
Pejler Gunnar,
Karlström Anders R.,
Berg Lotta
Publication year - 1995
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1995.tb20364.x
Subject(s) - thrombin , cleavage (geology) , protease , serine protease , chemistry , biochemistry , serine , chymotrypsin , microbiology and biotechnology , trypsin , enzyme , biology , platelet , paleontology , fracture (geology) , immunology
We have previously identified rat mast cell protease 1 (RMCP‐1), a chymotrypsin‐like secretory granule serine protease, as a potent inactivator of thrombin. The present study outlines the cleavage pattern obtained after degradation of thrombin by RMCP‐1. The cleavage sites in thrombin were identified by N‐terminal amino acid sequence analysis of recovered thrombin fragments. Incubation of thrombin with RMCP‐1 resulted in the rapid formation of a 37‐kDa fragment, due to cleavage of the Phe1G‐Gly1F bond in the thrombin A chain (numbering of amino acid residues according to topological equivalencies with chymotrypsinogen). Further incubation resulted in cleavage of the Trp148‐Thr149 bond in the B chain, along with the formation of fragments of 27 kDa and 15 kDa. When the RMCP‐1/thrombin mixtures were incubated further, successive degradation of the 37‐kDa, 27‐kDa and 15‐kDa fragments was observed, along with cleavage of the Tyr117‐Ile118 bond in the B chain and the formation of fragments of 12, 9 and 6 kDa. No residual thrombin activity was detected after the degradation process had proceeded to this stage. Heparin was shown to markedly enhance the rate of thrombin degradation by RMCP‐1.