Analysis of alpha-crystallin chaperone function using restriction enzymes and citrate synthase.
OBJECTIVE
To compare the abilities of [alpha]A-crystallin, [alpha]B-crystallin, and mini-[alpha]A-crystallin (a synthetic peptide chaperone representing the functional unit of [alpha]A-crystallin) to protect against heat-induced inactivation of citrate synthase (CS) and restriction enzymes, SmaI and NdeI.


METHODS
Restriction enzymes, SmaI and NdeI were heated at different temperatures in the presence of various amounts of molecular chaperones and tested for their ability to cleave plasmid DNA. The aggregation of CS was measured at 43 degrees C while the loss in activity was monitored at 37 degrees C in the presence of various crystallins.


RESULTS
Restriction enzyme activities were protected by the crystallin subunits up to 37 degrees C for SmaI and 43 degrees C for NdeI. However, the mini-[alpha]A-crystallin was unable to protect endonuclease activity. The crystallin subunits and the peptide chaperone were able to suppress thermal aggregation of CS at 43 degrees C, but failed to stabilize its activity at 37 degrees C.


CONCLUSIONS
The ability of [alpha]-crystallin subunits to stabilize denaturing 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]-crystallin subunits. Additionally, our results show that there could be more than one site in [alpha]A-crystallin responsible for its chaperone-like action. By addition of crystallin subunits to restriction enzymes prior to or during storage, transport, or assay would maintain or improve their activity thereby decreasing their cost.
Santhoshkumar
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Department of Ophthalmology, University of Missouri, Columbia, MO 65212, USA.
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D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D002950:Citrate (si)-Synthase / Q000378:metabolism*; D003459:Crystallins / Q000302:isolation & purification / Q000502:physiology*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006358:Hot Temperature; D007904:Lens Cortex, Crystalline / Q000737:chemistry; D018832:Molecular Chaperones / Q000302:isolation & purification / Q000502:physiology*; D010446:Peptide Fragments / Q000494:pharmacology; D011489:Protein Denaturation
D003459:Crystallins; D018832:Molecular Chaperones; D010446:Peptide Fragments; D002950:Citrate (si)-Synthase; C037106:endodeoxyribonuclease NdeI; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific