ISOELECTRIC-FOCUSING OF CRYSTALLINS IN MICROSECTIONS OF CALF AND ADULT BOVINE LENS - IDENTIFICATION OF WATER-INSOLUBLE CRYSTALLINS COMPLEXING UNDER NONDENATURING CONDITIONS - DEMONSTRATION OF CHAPERONE ACTIVITY OF ALPHA-CRYSTALLIN

Topographic studies of crystallin fractions from the young adult bovin e lens revealed that lenses do not have a homogeneous distribution of crystallins. There are, however, gradual differences between the corti ces and the nucleus. The isolated lenses were separated mechanically i nto lens equator and inner cylinder. The latter was then sectioned in a special sectioning machine into 11-12 morphological layers (from ant erior cortex through nucleus to posterior cortex). Matters of the lens sections were separated into water-soluble (WS) and water-insoluble ( WI) crystallins. The WI fractions were solubilized with 100% formamide , or dissolved into 7 M urea. Crystallin profiles were obtained for ea ch lens layer, using thin-layer isoelectric focusing in polyacrylamide gel. WS crystallins from the lens equator revealed a separation into HM-, alpha(L)-, beta(H)-, beta(L)-, beta(S)- and gamma-crystallins. Th e WI fractions of the layers dissolved in urea gave a separation into the individual HM- (3 components), alpha(L)- (4 components), beta- (6 component groups), beta(S)- (2 components) and gamma- (11 components) crystallins in the different morphological layers. The results confirm that a significant age-related increase in several beta- and gamma-cr ystallins incorporated into alpha-crystallins exists in the patterns o f WI fractions of the different layers from lenses of 2.2 and 5.9 year s. The WI crystallins solubilized in formamide showed only the presenc e of HM weight and alpha-crystallin moieties, due to the action of cha perone activity of alpha-crystallin. The nature of the WI protein frac tion in the separated lens layers reflected to the aggregated state of : alpha(L)-, beta(L)-, beta(S)- and gamma-crystallins in the different regions of the lens, concealed in the central cavity of the alpha-cry stallin chaperone model.