title: Human pituitary tryptase: molecular forms, NH2-terminal sequence, immunocytochemical localization, and specificity with prohormone and fluorogenic substrates.
abstract: A human pituitary-derived serine protease, immunologically identical to human lung tryptase (Smith, T. J., Hougland, M.W., and Johnson, D.A. (1984) J. Biol. Chem. 259, 11046-11051), was found immunohistochemically to be associated with mast cells present in pituitary connective tissue. Western blotting combined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of multiple forms: a major Mr 36,300 form and three minor forms with Mr 32,400, 33,400, and 34,600. Two major forms with Mr 35,600 and 34,100 were detected by affinity labeling with 125I-D-Tyr-Glu-Phe-Lys-Arg-CH2Cl. Treatment of the pituitary tryptase preparation with N-glycosidase F indicated that some of the molecular weight heterogeneity results from N-linked glycosylation. The multiple molecular weight forms appear to have the same NH2-terminal sequence: Ile-Val-Gly-Gly-Gln-Glu-Ala-Pro. Pituitary tryptase has an apparent Mr = 110,000 by gel filtration on Sephadex G-200 in the presence of 0.3 M NaCl, indicating that the enzyme may be a tetramer of Mr = 32,400-36,300 subunits. However, this quaternary structure was not stable to gradient polyacrylamide gel electrophoresis. Human pituitary tryptase was so reactive toward synthetic tripeptide coumarin-containing substrates containing a pair of basic amino acids at the site of cleavage such as benzyloxylcarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide (k cat/Km = 2.38 X 10(8) M-1 s-1) that Briggs-Haldane kinetics may apply. The reversible inhibitor NaCl at a concentration of 1 M decreased the k cat/Km for benzyloxylcarbonyl-L-Ala-L-Lys-L-Arg-4-methylcoumarin-7-amide to 6.53 X 10(6) M-1 s-1, which reflected a 100-fold increase in apparent Km. Based on active site titration with fluorescein mono-p-guanidinobenzoate hydrochloride, NaCl had no effect on the number of accessible active sites. Substrate specificity studies with prohormones indicated that pituitary tryptase has a preference for cleaving COOH-terminal to arginine or lysine residues which are preceded by a proline residue 4 or 6 residues NH2-terminal to the site of cleavage.
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003374:Coumarins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes; D006651:Histocytochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007159:Immunologic Tests; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D046911:Macromolecular Substances; D008407:Mast Cells / Q000201:enzymology; D008970:Molecular Weight; D010446:Peptide Fragments; D010447:Peptide Hydrolases / Q000032:analysis / Q000378:metabolism*; D010902:Pituitary Gland / Q000201:enzymology*; D011498:Protein Precursors / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D003374:Coumarins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011498:Protein Precursors; D012965:Sodium Chloride; C050485:fluorescein mono-p-guanidinobenzoate; D010447:Peptide Hydrolases; C023073:tosylarginine methyl ester hydrolase; C028743:7-amino-4-methylcoumarin