THE CROSS-LINKING OF COLLAGEN AND ELASTIN: ENZYMATIC CONVER&ION OF LYSINE IN PEPTIDE LINKAGE TO a-AMINOADIPIC-6-SEMIALDEHYDE (ALLYSINE) BY AN EXTRACT FROM BONE*

Current evidence indicates that the initial step in the cross-linking of both collagen' and elastin2 I is the conversion of peptide-bound lysine to a-aminoadipic-6-semialdehyde. For the sake of brevity, we have given the latter compound the trivial name allysine to indicate that it is an aldehyde derived from lysine. In the case of collagen, one lysyl residue per chain of -103 amino acids4 has been found to be converted to this aldehyde as compared to 5-16 lysyl residues per 103 amino acids in elastin.2' 5 The mechanism of formation of the actual cross-links is probably due both to the spontaneous condensation of aldehydes and to their reaction with still other groups on these proteins.3' 6 The enzyme converting lysyl residues to allysyl residues has not been previously identified. In this paper, we will describe assays for, and some of the properties of, an enzyme from bone that converts peptide-bound lysine to allysine. The enzyme is inhibited by physiologically active levels of the lathyrogen f3-aminopropionitrile, a compound previously shown in vivo and in tissue culture to inhibit the cross-linking of collagen7' I and elastin9 by blocking the lysine-to-allysine conversion.2' 4 Materials and Methods. -Preparation of substrate: Eighteen aortas from 16-day-old embryonic chicks were incubated in a 50-ml Erlenmeyer flask with 10 ml of Eagle's minimal essential medium lacking free lysine but containing 20 gg/ml #-aminopropionitrile fumarate (BAPN) and either D-lysine-6-3H (200 gc) or ilysine-U-14C (50 Muc). After being flushed with a mixture of 95% 02 and 5% C02, the stoppered flasks were incubated with shaking for 24 hr at 37°. Following incubation, the aortas were rinsed with distilled water and lyophilized. Lyophilized aortas were ground in a glass homogenizer at room temperature in 0.16 M saline, and the homogenate was centrifuged at 11,000 g. The supernatant was discarded and the pellet was rehomogenized in saline and reisolated by centrifugation twice more. In general, substrate equivalent to the pellet from two aortas containing approximately 500,000 cpm 'H or 100,000 cpm 14C was used per incubation. Preparation of bone extract (105S): Tibias and femurs from 16-day-old embryonic chicks were homogenized (6 bones/ml) in 0.16 M NaCl, 0.1 M phosphate buffer (pH 7.4) in a Polytron homogenizer. The supernatant (105S) obtained by centrifuging the homogenate (105,000 g for 2 hr at 5°) was used as the source of the enzyme and contained 5-8 mg protein/ml. In some experiments, 105S extracts were incubated at 230 with and without 50 ,g/ml of BAPN for 2 hr. These extracts were then dialyzed extensively against 0.16 M NaCi, 0.1 M phosphate buffer (pH 7.4) at 5°. Standard incubation mixture: The standard incubation mixture consisted of the labeled substrate and 1.5 ml of 105S. Since allysine may be an intermediate in the degradation of free lysine in mitochondria,'0 sufficient carrier lysine (1 mM) was added to the reaction mixture to dilute any free lysine-6-'H carried over in the preparation of the labeled substrate. BAPN (1-50 jug/ml) was added to some of the tubes, and the final volume was