Calpain inhibition by peptide epoxides

Area of Science:

• Biochemistry
• Enzymology
• Muscle Physiology

Background:

• Calpains are calcium-activated cysteine proteases crucial in muscle function.
• Understanding calpain II regulation is key to muscle physiology and pathology.

Purpose of the Study:

• To purify and characterize chicken gizzard calpain II.
• To investigate the kinetics of calpain II inactivation by E-64 analogues.
• To determine the active-site molarity of purified calpain II.

Main Methods:

• Purification using isoelectric precipitation, ammonium sulfate fractionation, and multiple chromatography techniques (DEAE-Sepharose, Reactive-Red 120-agarose, Mono Q).
• Determination of second-order rate constants for calpain II inactivation by E-64 analogues.
• Active-site titration of calpain II using E-64.

Main Results:

• Calpain II was successfully purified from chicken gizzard smooth muscle.
• The apparent second-order rate constants for inactivation by E-64 analogues were determined.
• L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-benzyloxy-carbonylamino)butane showed the fastest inactivation rate.
• Active-site molarity was determined via E-64 titration.
• Calpain II underwent Ca2+-dependent intermolecular limited proteolysis preceding activity loss, without affecting inactivation rates by E-64 analogues.

Conclusions:

• Chicken gizzard calpain II can be purified using established biochemical methods.
• Specific E-64 analogues are potent inhibitors of calpain II.
• The study provides a method for determining calpain II active-site molarity.
• Autoproteolysis of calpain II occurs independently of its reaction kinetics with E-64 analogues.