The Nε-(γ-glutamic)lysine cross-link: Method of analysis, occurrence in extracellular and cellular proteins

Abstract

Publisher Summary The N ɛ -( γ-glutamic)lysine (Glu-Lys)cross-link is a covalent bond formed between protein chains. It is catalyzed by a Ca 2+ -dependent acyltransferase in which the γ- carboxamide groups of peptide-bound glutamine residues are the acyl donors and the ɛ-amino groups of peptide-bound lysine residues are the acyl acceptors. This chapter describes various procedures for the measurement of the Glu-Lys cross-link and its occurrence in extracellular and cellular proteins. The basic strategy for the analysis of the Glu-Lys bond involves the liberation of N ɛ -(y-glutamyl)lysine from a protein preparation by exhaustive enzyme digestion, followed by the chromatographic purification and measurement of the isodipeptide. Methods for exhaustive enzyme digestion of the cross-linked protein sample must be adjusted to the tractability of the material being studied. The choice of the analytic procedure depends on (1) the cross-link content of the protein being studied, (2) the sensitivity of the method of detection available, and (3) the suitability of the material for in vitro incorporation of [ 14 C]lysine into the protein being studied.