[Decreased chaperone activity of alpha-crystallin by carbamylation in vitro].
OBJECTIVE
To investigate the effort of carbamylation, an important post-translational modification of lens proteins, on chaperone activity of alpha-crystallin.


METHODS
The alphaL-crystallin and betaL-crystallin were isolated from bovine lens. Carbamylation was performed by different concentrations of potassium [14C] cyanate to alphaL-crystallin at 37 degrees C for 7 days. The binding of cyanate to alphaL-crystallin was determined by measuring the radioactivity incorporated into trichloroacetic acid precipitable protein from potassium [14C] cyanate. In addition, alphaL-crystallin was incubated with unlabelled potassium cyanate (50 and 100 mmol.L(-1)) for 3 and 7 days at 37 degrees C and then its chaperone activity against heat-induced betaL-crystallin aggregation was assayed. Analysis of the carbamylated alphaL-crystallin was further performed by high performance liquid chromatography (HPLC).


RESULTS
The rate of binding (cyanate bound /mol of alphaL-crystallin) and the decreased chaperone activity of alphaL-crystallin induced by carbamylation in vitro were dose-dependent and time-dependent. The decreased chaperone activity corresponded with the higher binding of cyanate. After 7 days incubation with 100 mmol.L(-1), almost all chaperone activity was lost compared with a simultaneously incubation control sample. HPLC analysis revealed that aggregation of alphaL-crystallin by potassium cyanate has occurred after 3 days incubation in dose-dependent fashion.


CONCLUSIONS
The alpha-crystallin can be modified in vitro by carbamylation through a high-molecular-weight aggregates formation. The loss of its chaperone activity may result from carbamylation-induced aggregation.
Yan
Hong
H
Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China.
Yao
Linong
L
Hui
Yannian
Y
D000215:Acylation; D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D018832:Molecular Chaperones / Q000378:metabolism; D011190:Potassium Cyanide / Q000008:administration & dosage* / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D038201:alpha-Crystallins / Q000737:chemistry / Q000378:metabolism*
D018832:Molecular Chaperones; D038201:alpha-Crystallins; D011190:Potassium Cyanide