The culture of rat lenses in high sugar media: effect on mixed disulfide levels.
Lens proteins are long lived proteins with those in the center of the lens predating the birth of the individual. As a result, they are subject to a host of modifications and damage through a variety of mechanisms. Two such modifications have been proposed as primary events which could cause conformational changes potentiating further modifications. These are non-enzymatic glycation and mixed disulfide formation. Human lenses accumulate protein-thiol mixed disulfides of three kinds throughout the lifespan. The presence of one of these, protein-glutathione (PSSG) mixed disulfide has been shown to be intimately involved in protein aggregation. We have utilized ex vivo lens culture and in vitro incubations of purified gamma-crystallin to evaluate the following hypotheses. A) Lenses cultured with a high sugar media will form higher mixed disulfide levels than controls; B) glycation of lens proteins will be dependent on initial mixed disulfide level. Xylose levels in the cultured lens rise rapidly (to 23 mM by 4 h), and the level of glycation after one week is elevated 6-7% over control values. Mixed disulfide levels are also substantially increased but not more than for lenses cultured in control media. gamma-Crystallin modified with 0, 1, or 5 equivalents of GSH was differentially glycated by radioactive fructose. The amount of fructose bound by the protein was found to be inversely related to the extent of mixed disulfide formation. These results indicate that 1) protein modification of one kind may influence further modifications of other types; 2) glycation of lens proteins has no effect on mixed disulfide formation in this system; 3) the sulfhydryl status of lens proteins can affect the potential for protein glycation.
Dickerson
J E
JE
Cataract Research Group, Alcon Laboratories, Inc., Fort Worth, TX 76134, USA.
Lou
M F
MF
Gracy
R W
RW
D000818:Animals; D002846:Chromatography, Affinity; D003459:Crystallins / Q000378:metabolism*; D003470:Culture Media; D046508:Culture Techniques; D004220:Disulfides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005632:Fructose / Q000494:pharmacology*; D005978:Glutathione / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D014994:Xylose / Q000494:pharmacology*
D003459:Crystallins; D003470:Culture Media; D004220:Disulfides; D005632:Fructose; D014994:Xylose; D005978:Glutathione