Decreased sensitivity of lens-specific calpain Lp82 to calpastatin inhibitor

The purpose of the present investigation was to test three calpain inhibito rs (recombinant calpastatin domain I, E64, and SJA6017) against Lp82 calpai n in rat lenses. Lp82 is a lens-specific isoenzyme from the calpain super f amily of calcium-activated, cysteine proteases (EC34.22.17). Lp82 and m-cal pain proteolytic activities and protein levels were measured by casein zymo graphy and immunoblotting. Activity of endogenous Lp82 against vimentin was also tested by in vitro incubation of rat lens soluble and insoluble fract ions with calcium. Most of the Lp82 activity could be inhibited by irrevers ible inhibitor E64 and reversible inhibitor SJA6017. However, a major findi ng of the present investigation was that Lp82 in the soluble and insoluble fractions of the lens was less sensitive to inhibition by recombinant domai n I from the endogenous tissue inhibitor of ubiquitous calpains, calpastati n, than m-calpain. By using recombinant calpastatin to inhibit endogenous l ens m-calpain, we were able to demonstrate the first example of a substrate for Lp82, vimentin. These data suggest that Lp82-induced proteolysis in ro dent lenses may occur even in the presence of calpastatin. (C) 1999 Academi c Press.