Studies on the Action of Lysyl Oxidase on Soluble Elastin
We have previously demonstrated that incubation of soluble elastin with lysyl oxidase results in the formation of isodesmosine and desmosine. We now report studies characterizing this reaction further.
Lysine labeled soluble elastin was purified from pulse labeled embryonic chick aortas by coacervation followed by organic solvent extraction. Lysyl oxidase was obtained from embryonic chick cartilage by affinity chromatography and DEAE-cellulose chromatography. Routinely 40 – 50,000 cpm of soluble elastin were incubated with lysyl oxidase at 37° for 24 hours in the presence of non-radioactive aorta extract; then the incubation mixtures were lyophilized, hydrolyzed and the radioactive desmosines determined after separation on an amino acid analyzer.
The purified lysyl oxidase of chick cartilage catalyzed the formation of crosslinks in soluble elastin as well as collagen. Therefore a single lysyl oxidase, irrespective of the presence of another species in the tissue, appears to be capable of acting on soluble elastin and collagen.
Radioactive isodesmosine and desmosine were formed when a homogeneous preparation of soluble elastin was incubated with lysyl oxidase; no other cofactor was necessary. Therefore only lysyl oxidase and possibly no additional enzyme is necessary for the formation of the desmosines.
Soluble elastin with unhydroxylated proline residues (prepared by incubation of aortas in the presence of α, α′-dipyridyl) formed same amounts of desmosines as did the preparation with normal hydroxyproline content. Hydroxylation of proline was also not necessary for the secretion of soluble elastin as indicated by pulse-chase experiments in the presence and absence of α, α′-dipyridyl and autoradiography. Therefore hydroxyproline does not seem to be required for secretion as well as crosslinking of soluble elastin.
Prior heating of soluble elastin to 60° or 100° did not affect the quantity of desmosines formed. However, at 15°, a temperature not conducive to coacervation, no desmosines were synthesized even though lysyl oxidase activity was present. It is concluded that in order to be crosslinked, soluble elastin must be converted to an insolubilized form.
KeywordsEmbryonic Chick Lysyl Oxidase Organic Solvent Extraction Homogeneous Preparation Coacervate Phase
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