The glycation and cross-linking of isolated lens crystallins by ascorbic acid.
Individual lens crystallins were isolated from calf lens extracts and incubated in the presence of ascorbic acid for 3 weeks under aerobic conditions. Both alpha-crystallin and beta H-crystallin rapidly cross-linked to form high molecular weight proteins, which did not enter the resolving gel on SDS-PAGE. Beta L-crystallin was somewhat less reactive, but gamma-crystallin showed little or no crosslinking. Gamma-crystallin, however, was almost equivalent to the other crystallins as a substrate for glycation. This was measured by: (a) the binding of protein to a boronate affinity column; (b) the incorporation of 3H from NaB3H4 into protein; (c) amino acid analysis of the modified proteins to estimate the extent of lysine modification; and (d) the incorporation of [1-14C]ASA into individual crystallins. When the separated crystallins were combined with [125I]gamma-crystallin and incubated with ascorbic acid, radioactivity was readily incorporated into the cross-linked products with other crystallins, but again not with gamma-crystallin itself. Gel filtration chromatography of a mixture of [125I]gamma-crystallin and alpha-crystallin showed the formation of a complex between gamma- and alpha-crystallins. These data suggest that all crystallins are glycated, but that cross-linking occurs preferentially between proteins, which are already bound together non-covalently.
Prabhakaram
M
M
Mason Institute of Ophthalmology, University of Missouri, Columbia 65212.
Ortwerth
B J
BJ
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology*; D002417:Cattle; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D003459:Crystallins / Q000187:drug effects*; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D066298:In Vitro Techniques; D008970:Molecular Weight; D011485:Protein Binding; D013997:Time Factors
D000596:Amino Acids; D003432:Cross-Linking Reagents; D003459:Crystallins; D001205:Ascorbic Acid