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.