title: Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor.
abstract: The purpose of the present investigation was to test three calpain inhibitors (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpain in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super family of calcium-activated, cysteine proteases (EC 34.22.17). Lp82 and m-calpain proteolytic activities and protein levels were measured by casein zymography and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fractions with calcium. Most of the Lp82 activity could be inhibited by irreversible inhibitor E64 and reversible inhibitor SJA6017. However, a major finding of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domain I from the endogenous tissue inhibitor of ubiquitous calpains, calpastatin, than m-calpain. By using recombinant calpastatin to inhibit endogenous lens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82-induced proteolysis in rodent lenses may occur even in the presence of calpastatin.
authors.affiliation: Research Laboratories, Senju Pharmaceutical Co., Ltd., 1-5-4 Murotani, Nishi-ku, Kobe, 651-2241, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Nakamura
authors.forename: C
authors.initials: C
authors.lastname: Fukiage
authors.forename: H
authors.initials: H
authors.lastname: Ma
authors.forename: M
authors.initials: M
authors.lastname: Shih
authors.forename: M
authors.initials: M
authors.lastname: Azuma
authors.forename: T R
authors.initials: TR
authors.lastname: Shearer
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors*; D002154:Calpain / Q000502:physiology*; D003546:Cysteine Endopeptidases / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors; D015151:Immunoblotting; D007908:Lens, Crystalline / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
chemical_list: D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; C033668:calpastatin; D002154:Calpain; D003546:Cysteine Endopeptidases; C118624:calpain Lp82