Calpain inhibition by peptide epoxides

Abstract

A Ca2+-activated cysteine proteinase (calpain II) was purified from chicken gizzard smooth muscle by use of isoelectric precipitation, (NH4)2SO4 fractionation, chromatography on DEAE-Sepharose CL-6B, Reactive-Red 120-agarose and Mono Q. The apparent second-order rate constants for the inactivation of calpain by a series of structural analogues of L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-guanidino)butane (E-64) were determined. The fastest rate of inactivation was observed with L-3-carboxy-trans-2, 3-epoxypropionyl-leucylamido-(4-benzyloxy-carbonylamino)buta ne. It was possible to determine the active-site molarity of solutions of calpain by titration with E-64. When incubated with Ca2+, calpain underwent several steps of intermolecular limited proteolysis, via multiple pathways, followed by a slower loss of enzymic activity. The proteolytic steps preceding the loss of activity did not affect the rates of reaction of calpain with E-64 analogues.

Involvement of a serine protease in tumour‐necrosis‐factor‐mediated cytotoxicity

“…o-Phenanthroline is a heavy metal chelator with high affinity for zinc [42] and strongly inhibited TNF action. E64 specifically inhibits thiol proteases, but not serine proteases [41,43] and had no effect in our assay. Neither did we find any inhibition with other sulfhydryl-blocking agents, such as iodoacetic acid 1431 or N-ethylmaleimide [42].…”

“…The latter could not be tcstcd at higher concentrations, as the solvent MezSO itself inhibited too strongly. The macromolecular protease inhibitors soybean trypsin inhibitor, inhibiting both trypsin-and chymotrypsinlike activity [41], and aprotinin (also called trasylol or bovine pancreatic trypsin inhibitor), had no detectable influence on the activity of TNF. o-Phenanthroline is a heavy metal chelator with high affinity for zinc [42] and strongly inhibited TNF action.…”

Calcium‐activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

“…EGTA). Leupeptin, antipain, and E64 (an epoxysuccinyl derivative) are potent inhibitors [7]. It should be noted that the active-site SH group is buried in the molecule and exposed to the surface by a conformational change induced upon binding of Ca 2÷ [21. The presence of Ca 2÷ is essential for the reaction of CANP with inhibitors as well as substrates.…”

Structural modifications associated with the change in Ca2+ sensitivity on activation of m‐calpain

“…For chicken m-calpain conversion from the 80 kDa polypeptide to a 76 kDa polypeptide can be observed on SDS-PAGE [3,11]; but for mammalian mcalpains no change in electrophoretic mobility is detectable [5,12,13]. For both forms of calpain these initial autolytic events are followed by the conversion of the large subunit to a polypeptide of approximately 50 kDa [7,11], and by a slow loss of enzyme activity.…”