HIGHER GLYCATION OF BETA(L)-CRYSTALLINS AND BETA(S)-CRYSTALLINS IN THE ANTERIOR LENS CORTEX AND MAXIMUM GLYCATION OF GAMMA-CRYSTALLINS IN THE BOVINE LENS NUCLEUS, DEMONSTRATED BY FROZEN SECTIONING, ISOELECTRIC-FOCUSING AND LECTIN STAINING

The aim of the current study was to demonstrate glycation of beta(L)-, beta(S)- and gamma-crystallins in the young bovine lens. To establish which of the crystallins are glycated and where they are located in t he lens, we carried out microsectioning of the lens, followed by isoel ectric focusing (IEF). Four bovine lenses of 1.183 +/- 0.070 years wer e frozen-sectioned into equator and 11 layers. Water-soluble crystalli ns were separated by IEF and stained: (l)with Coomassie brilliant blue for proteins; (2) with the lectin concanavalin A, followed by horsera dish peroxidase and diaminobenzidine, for glycated proteins. Experimen ts were performed with crystallins and proteins in native form, in the absence of denaturants. The crystallins were separated by IEF into al pha-crystallins of high molecular weight (HM), alpha(L)-, beta(H)-, be ta(L)-, beta(S)- and gamma-crystallins. In the lectin staining experim ents, only HM, beta(L)-, beta(S)- and gamma-crystallins were positive, whereas the alpha(L)- and beta(H)-crystallins were negative. Contrary to the glycated gamma-crystallins in the lens nucleus, the beta(S)- a nd gamma-crystallins were predominantly glycated in the anterior corte x and to a somewhat lower extent also in the posterior cortical region s. The degree of glycation (total densitometric readings of lectin-sta ined bands/Coomassie-blue-stained bands) is as follows: total gamma-cr ystallins 2.44, beta(S)-crystallins 0.77 and PL-crystallins 0.28. Thou gh glycation in the bovine lens is very low, lectin staining is suffic iently sensitive to detect the various glycated crystallins. The degre e of glycation of gamma-crystallins was 3 times higher than that of be ta(S)-crystallins and 9 times higher than that of beta(L)-crystallins.