Analysis of alpha-crystallin chaperone function using restriction enzymes and citrate synthase

Purpose: To compare the abilities of alphaA-crystallin, alphaB-crystallin, and mini-alphaA-crystallin (a synthetic peptide chaperone representing the functional unit of alphaA-crystallin) to protect against heat-induced inact ivation of citrate synthase (CS) and restriction enzymes, SmaI and NdeI. Methods: Restriction enzymes, SmaI and NdeI were heated at different temper atures in the presence of various amounts of molecular chaperones and teste d for their ability to cleave plasmid DNA. The aggregation of CS was measur ed at 43 degreesC while the loss in activity was monitored at 37 degreesC i n the presence of various crystallins. Results: Restriction enzyme activities were protected by the crystallin sub units up to 37 degreesC for SmaI and 43 degreesC for NdeI. However, the min i-alphaA-crystallin was unable to protect endonuclease activity. The crysta llin subunits and the peptide chaperone were able to suppress thermal aggre gation of CS at 43 degreesC, but failed to stabilize its activity at 37 deg rees C. Conclusions: The ability of alpha -crystallin subunits to stabilize denatur ing proteins varies from enzyme to enzyme as evidenced by the inactivation of CS and protection of SmaI and NdeI activity in the presence of alpha -cr ystallin subunits. Additionally, our results show that there could be more than one site in alphaA-crystallin responsible for its chaperone-like actio n. By addition of crystallin subunits to restriction enzymes prior to or du ring storage, transport, or assay would maintain or improve their activity thereby decreasing their cost.