title: 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.
mesh_terms: D000818:Animals; D001665:Binding Sites; D002154:Calpain / Q000302:isolation & purification; D002645:Chickens; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004852:Epoxy Compounds / Q000494:pharmacology*; D004988:Ethers, Cyclic / Q000494:pharmacology*; D006023:Glycoproteins / Q000494:pharmacology*; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D010455:Peptides; D011480:Protease Inhibitors / Q000494:pharmacology*
chemical_list: D004852:Epoxy Compounds; D004988:Ethers, Cyclic; D006023:Glycoproteins; D010455:Peptides; D011480:Protease Inhibitors; C039304:calpain inhibitors; D002154:Calpain; D007930:Leucine; C024974:E 64