Resistance of α-crystallin-glutathione mixed-disulfide to tryptic digestion

Publisher Website
Google Scholar
Abstract
Protein-mixed disulfides (PSSG) were formed by interaction of glutatione disulfide (GSSG) with lens crystallins. Total water-soluble crystallins and .alpha.-crystallin purified on a Sephacryl S-200 column were separately incubated with 0, 2, 4, and 8 mM (final concentrations) GSSG overnight and then dialyzed to remove unbound GSSG and GSH. Eight TPCK-treated trypsin or TLCK-treated .alpha.-chymotrypsin were added to about 200 .mu.g crystallin samples and incubated for 20 min at room temperature. Reactions were terminated by boiling in SDS-mercaptoethanol-Tris (pH 6.8) solution and subjected to electrophoresis on 10% polyacrylamide slab gels. Comparison of SDS-PAGE patterns of proteolysis with or without GSSG treatment showed that GSSG at a concentration of 2 mM or higher reduced or abolished proteolysis of .alpha.-crystallin by trypsin but not by .alpha.-chymotrypsin. The protective effect of GSSG was greater with .alpha.-crystallin than with .beta.-crystallins. Addition of .alpha.-crystallin-mixed-disulfide to an assay system in which trypsin was hydrolyzing N-.alpha.-benzoyl-DL-arginine-P-anilide (BAPNA) inhibited the tryptic activity. Direct addition of GSSG or native .alpha.-crystallin had no significant inhibitory effect on trypsin. Based on these results, it is speculated that .alpha.-crystallin glutathione mixed-disulfide appears to become resistant to trypsin probably by non-competitive inhibition of the enzyme.

This publication has 16 references indexed in Scilit: