Human cationic and anionic trypsins: differences of interaction with alpha 1-proteinase inhibitor

Area of Science:

• Biochemistry
• Enzymology
• Protease inhibitors

Background:

• Human cationic (trypsin 1) and anionic (trypsin 2) trypsins are serine proteases with distinct physiological roles.
• Alpha 1-proteinase inhibitor (alpha 1PI) is a primary inhibitor of serine proteases in circulation.
• Understanding the differential interactions between trypsin isozymes and alpha 1PI is crucial for comprehending protease regulation.

Purpose of the Study:

• To compare the in vitro interactions between human cationic trypsin 1, anionic trypsin 2, and human alpha 1-proteinase inhibitor (alpha 1PI).
• To elucidate the kinetic and stability differences in the inhibition of trypsin 1 and trypsin 2 by alpha 1PI.
• To assess the in vivo relevance of these interactions under physiological and pathological conditions.

Main Methods:

• Controlled activation of purified human trypsinogens 1 and 2 to obtain active trypsins.
• In vitro kinetic analysis of trypsin-alpha 1PI interactions using Glp-Gly-Arg-Nan substrate.
• Electrophoresis and immunoblotting to visualize and confirm equimolar complex formation.
• Calculation of inhibition delay times based on normal serum concentrations.

Main Results:

• The association rate constant for trypsin 2 inhibition by alpha 1PI was over 10 times faster than for trypsin 1.
• Both trypsin 1-alpha 1PI and trypsin 2-alpha 1PI complexes were formed but dissociated over time, yielding active enzyme.
• Trypsin 1-alpha 1PI complexes exhibited significantly greater stability than trypsin 2-alpha 1PI complexes.
• In vivo, alpha 1PI effectively inhibits trypsin 2, comparable to alpha 2-macroglobulin (alpha 2M) inhibition of trypsin 1.

Conclusions:

• Alpha 1-proteinase inhibitor plays a significant role in regulating anionic trypsin 2 activity under physiological conditions.
• Alpha 1PI contributes to the inhibition of cationic trypsin 1 primarily in pathological scenarios or when other inhibitors are saturated.
• The differential inhibition kinetics and complex stability highlight the specific roles of trypsin isozymes and their inhibitors in maintaining protease homeostasis.