Identification of the site of glycation of gamma-II-crystallin by (14C)-fructose.
Cataract formation in diabetes may be via non-enzymic glycosylation (glycation) of lens proteins due to increased concentrations of sugars present in the lenses of diabetic patients. The objective of this project was to identify the site(s) of glycation of bovine gamma-II-crystallin by [14C]fructose. gamma-II-crystallin was isolated from soluble lens nucleus proteins by gel chromatography, followed by ion-exchange chromatography and was then glycated by incubation with [14C]fructose. Radioactively labelled gamma-II-crystallin was cleaved with trypsin. Affinity chromatography of the tryptic peptides gave a single main peak containing the majority of the radioactivity. This indicated that fructose had reacted at a single site on the protein. Amino acid analysis of this peptide showed it to contain only lysine and a trace amount of glycine. By relating the results of the amino acid analysis to the amino acid sequence of gamma-II-crystallin, it was concluded that the labelled peptide corresponded to the N-terminal dipeptide. The site of glycation of bovine gamma-II-crystallin by fructose was thereby identified as the alpha-NH2 group of the N-terminal glycine.
Pennington
J
J
Nuffield Laboratory of Ophthalmology, University of Oxford, UK.
Harding
J J
JJ
D000818:Animals; D001665:Binding Sites; D002250:Carbon Radioisotopes; D002386:Cataract / Q000209:etiology; D002417:Cattle; D003459:Crystallins / Q000737:chemistry*; D048909:Diabetes Complications; D005632:Fructose / Q000737:chemistry*; D006031:Glycosylation; D010446:Peptide Fragments / Q000302:isolation & purification; D014357:Trypsin
D002250:Carbon Radioisotopes; D003459:Crystallins; D010446:Peptide Fragments; D005632:Fructose; D014357:Trypsin