Dog mastocytoma tryptase: affinity purification, characterization, and amino-terminal sequence.
A tryptic protease with the characteristics of a mast cell tryptase was purified from dog mastocytoma cells propagated in nude mice. Partial amino acid sequence of the mastocytoma tryptase revealed unexpected differences in comparison with other mast cell and leukocyte granule protease sequences. Extraction from mastocytoma homogenates at high ionic strength, followed by gel filtration and benzamidine affinity chromatography yielded a product with several closely spaced bands (Mr 30,000-32,000) on gel electrophoresis and a single N-terminal sequence. Nondenaturing analytical gel filtration revealed an apparent Mr of 132,000, suggesting noncovalent association as a tetramer. Studies with peptide p-nitroanilides indicated pronounced substrate preferences, with P1 arginine preferred to lysine. Benzoyl-L-Lys-Gly-Arg-p-nitroanilide was the best of the substrates screened. Inhibition by diisopropyl fluorophosphate and tosyllysine chloromethyl ketone indicated that the enzyme is a serine protease. Like the tryptases of human mast cells, mastocytoma tryptic protease was inhibited by NaCl, resistant to inactivation by alpha 1-proteinase inhibitor and plasma, and stabilized by heparin. Comparison of the N-terminal 24 residues of mastocytoma tryptase revealed 80% identity with the more limited sequence reported for human lung tryptase, and surprisingly, closer homology to serine proteases of digestion and clotting than to other leukocyte granule proteases sequenced to date, including mast cell chymase. The N-terminal isoleucine is the homolog of trypsinogen Ile-16 which becomes the new N-terminus upon cleavage of the activation peptide. Thus, the tryptase N-terminus is related to the catalytic domain of activated serine proteases, and lacks the N-terminal regulatory domains found in most clotting and complement serine proteases. These findings provide further evidence that tryptases are unique serine proteases and that they may be less closely related in evolution and function than are other leukocyte granule proteases described to date.
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004283:Dog Diseases / Q000201:enzymology*; D004285:Dogs; D004795:Enzyme Stability; D006493:Heparin / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D012515:Mast-Cell Sarcoma / Q000201:enzymology / Q000662:veterinary*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity
D006493:Heparin; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase