Rates of carbamylation of specific lysyl residues in bovine alpha-crystallins.
Previous investigations indicate that some forms of cataract may be due to the reactions of isocyanate with lens proteins. The present investigation was directed toward identifying the products of these reactions and determining rate constants for their formation. Bovine alpha-crystallins were incubated with isocyanate and separated into alpha A- and alpha B-crystallins by reversed-phase HPLC (high-performance liquid chromatography). Products of the reaction of isocyanate with alpha-crystallins were analyzed by mass spectrometry and isoelectric focusing. Proteolytic digests of carbamylated alpha A were analyzed by HPLC and fast atom bombardment mass spectrometry to determine the extent of reaction of each of the 7 lysyl residues present in alpha A. These results demonstrate that incubation of alpha-crystallins in 0.1 M KNCO leads to partial carbamylation of all 7 lysines of alpha A-crystallin. The extent of modification after 24 h of incubation varied from 7% at Lys 88 to 61% at Lys 11. Rate constants for the reaction of specific lysyl residues with isocyanate ranged from 5 to 54 x 10(-2) M-1 h-1. The distribution of reaction products, as determined by isoelectric focusing, indicates that the physiologically relevant initial stages of carbamylation of the 7 lysyl residues of alpha A proceed in a noncooperative manner.
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Department of Medicinal Chemistry and Pharmacognosy, Purdue University, West Lafayette, Indiana 47907.
Smith
J B
JB
Smith
D L
DL
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D003459:Crystallins / Q000302:isolation & purification / Q000378:metabolism*; D003485:Cyanates / Q000032:analysis / Q000378:metabolism*; D010450:Endopeptidases; D007525:Isoelectric Focusing; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism; D008239:Lysine; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D016339:Spectrometry, Mass, Fast Atom Bombardment
D003459:Crystallins; D003485:Cyanates; D010446:Peptide Fragments; D010450:Endopeptidases; D008239:Lysine