Inactivation of thrombin by a complex between rat mast-cell protease 1 and heparin proteoglycan.
Rat peritoneal mast cells were shown to inactivate thrombin rapidly. The thrombin-inactivating activity was purified to homogeneity by a combination of anion-exchange chromatography and h.p.l.c. on a Superdex 75 column. The purified thrombin inactivator had an apparent molecular mass of 29 kDa and an N-terminal amino acid sequence identical to rat mast-cell protease 1 (RMCP-1). After labelling of the mast cells in vivo with 35SO4(2-), RMCP-1 was recovered in a macromolecular complex with [35S]heparin proteoglycans. Dissociation of RMCP-1 from the heparin proteoglycans by Superdex 75 chromatography in the presence of 2 M NaCl resulted in a marked loss of the thrombin-inactivating activity displayed by the enzyme. When RMCP-1 was reconstituted with either endogenous [35S]heparin proteoglycans or standard pig mucosal heparin, the enzyme regained its thrombin-inactivating properties. Affinity chromatography of endogenous [35S]heparin on matrix-linked RMCP-1 demonstrated that all of the heparin molecules contained high-affinity binding sites for the mast-cell protease. In contrast, the endogenous mast-cell heparin showed low affinity for antithrombin, a protease inhibitor involved in the regulation of coagulation enzymes.
Pejler
G
G
Department of Veterinary Medical Chemistry, Biomedical Center, Uppsala, Sweden.
Söderström
K
K
Karlström
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D053818:Chymases; D006493:Heparin / Q000031:analogs & derivatives* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008407:Mast Cells / Q000201:enzymology; D008970:Molecular Weight; D011509:Proteoglycans / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors*
D011509:Proteoglycans; C036090:heparin proteoglycan; D006493:Heparin; D012697:Serine Endopeptidases; D053818:Chymases; C509231:Mcpt1 protein, rat; D013917:Thrombin