Degradation of native and oxidized beta- and gamma-crystallin using bovine lens epithelial cell and rabbit reticulocyte extracts.
In many types of cells, modified proteins are selectively and rapidly removed by various proteolytic systems. In eye lens, as in most cells, there appears to be a multiplicity of proteolytic pathways, including ubiquitin-dependent, ATP-dependent and ATP-independent pathways. Each of these appears to be involved in the degradation of alpha-crystallins. The objective of this study was to determine if oxidized beta- and gamma-crystallins would be selectively degraded and which proteolytic systems might be involved. beta- and gamma-crystallins were oxidized by exposure to 137Cs radiation under N2O. This system generates *OH primarily. Oxidation of beta- and gamma-crystallins was indicated by decreased protein sulfhydryl content and tryptophan fluorescence, as well as by increased levels of carbonyl and high molecular weight aggregates with increasing radiation dose. gamma-crystallin was more susceptible than beta-crystallin to oxidation based on loss of native crystallin, increase in aggregates and fragmentation products, and loss of tryptophan. Low molecular mass polymers (dimers) appear to be the precursors of high mass aggregates induced upon oxidation. At a specific level of oxidative insult interchain covalent bonds in addition to disulfides were more extensive in the polymers of gamma-crystallin as compared to beta-crystallin. Except for beta-crystallin irradiated with 1 krad, the degradation rate of crystallins using both reticulocyte and bovine lens epithelial cells (BLEC) proteolytic systems increased in proportion to the extent of oxidation. Proteolysis of oxidized gamma-crystallins increased 1775% and 900%, respectively, using reticulocytes and BLEC supernatants as the source of proteases.(ABSTRACT TRUNCATED AT 250 WORDS)
Shang
F
F
Laboratory for Nutrition and Vision Research, USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111.
Huang
L
L
Taylor
A
A
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002588:Cesium Radioisotopes; D003459:Crystallins / Q000378:metabolism* / Q000528:radiation effects; D004591:Electrophoresis, Polyacrylamide Gel; D004848:Epithelium / Q000201:enzymology; D007908:Lens, Crystalline / Q000201:enzymology*; D010084:Oxidation-Reduction; D010447:Peptide Hydrolases / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000201:enzymology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D002588:Cesium Radioisotopes; D003459:Crystallins; D013438:Sulfhydryl Compounds; D000255:Adenosine Triphosphate; D010447:Peptide Hydrolases