Human pituitary tryptase: molecular forms, NH2-terminal sequence, immunocytochemical localization, and specificity...

Area of Science:

• Biochemistry
• Enzymology
• Protease Research

Background:

• A human pituitary-derived serine protease, immunologically identical to human lung tryptase, was investigated.
• This enzyme was found associated with mast cells in pituitary connective tissue.

Purpose of the Study:

• To characterize the molecular properties and enzymatic activity of the pituitary-derived serine protease.
• To investigate the enzyme's substrate specificity and potential quaternary structure.

Main Methods:

• Immunohistochemistry to localize the protease within pituitary tissue.
• Western blotting and SDS-PAGE to determine molecular weights and heterogeneity.
• Affinity labeling, N-glycosidase F treatment, and gel filtration (Sephadex G-200) for structural analysis.
• Kinetic analysis using synthetic substrates and active site titration.
• Substrate specificity studies with prohormones.

Main Results:

• Multiple molecular weight forms (major Mr 36,300; minor Mr 32,400-34,600) were identified, with heterogeneity attributed to N-linked glycosylation.
• The enzyme exhibited high catalytic efficiency (kcat/Km = 2.38 X 10^8 M-1 s-1) towards specific substrates, with activity modulated by NaCl.
• Pituitary tryptase demonstrated a preference for cleaving peptide bonds C-terminal to arginine or lysine residues preceding proline.

Conclusions:

• Human pituitary tryptase exists in multiple forms due to glycosylation and potentially forms a tetrameric structure.
• The enzyme displays high enzymatic activity and specific cleavage patterns, suggesting a functional role in the pituitary.
• Further research is warranted to elucidate the precise physiological function of pituitary tryptase.