Abstract
The use of recombinant human tropoelastin (rTE) and selected variants thereof as substrates for the assay of lysyl oxidase activity in vitro was explored. The possibility was also assessed that an insoluble elastin-like product could be generated from this elastin precursor in the absence of other macromolecules found associated with elastin in vivo. rTE was more efficiently oxidized by lysyl oxidase than the insoluble chick aorta elastin substrate conventionally used. Anionic amphiphilic elastin ligands strongly inhibited rTE oxidation consistent with the importance of electrostatic enzyme-substrate interactions previously noted with the insoluble elastin substrate. An rTE variant, rTE delta 26A, lacking the hydrophilic sequence coded by exon 26A, was a less effective substrate than rTE, largely due to an increase in Km, while the kinetic parameters for the oxidation of rTE delta 36, lacking the C-terminal polybasic sequence coded by exon 36, were quite similar to those for rTE. Incubation of rTE delta 26A with lysyl oxidase not only resulted in the generation of peptidyl alpha-aminoadipic-delta-semialdehyde and lysine-derived cross-linkages, but also yielded a product insoluble in hot 0.1 N NaOH, consistent with the properties of insoluble elastin. Thus, oxidation, cross-linking and insolubilization of elastin substrates by lysyl oxidase can occur in the absence of other macromolecules implicated as being involved in this process in vivo, although such macromolecules may be essential to obtain the proper alignment between tropoelastin units for specifically placed cross-linkages and optimally functional elastic fibers.
Highlights
The use of recombinant human tropoelastin glycosaminoglycans [3]
Anionicamphiphilicelastinligands stronglyinhibited recombinant human tropoelastin (rTE) oxidation consistent with the importanceof electrostatiecnzyme-substratienteractionpsreviously noted with theinsoluble elastin substrate.An rTE variant, rTEA26A, lackingthe hydrophilicsequence matrix derived fromchickembryoaortae[5].Whilethis illustrates the potential for tropoelastin insolubilization in vitro, the complexity of the aortic preparation used in this instance did not permit the characterizatioonf lysyl oxidase/ substrate interactionsor the role of matrix components other coded by exon 26A, was a less effective substrate than than lysyl oxidase in the insolubilization of tropoelastin
Comparative Efficiencies of PHIElastin and rH]rTE Substrates-Equal aliquots of smooth muscle cell fractions or of purified bovine aorta lysyl oxidase were assayed for 2 h using
Summary
Comparative Efficiencies of PHIElastin and rH]rTE Substrates-Equal aliquots of smooth muscle cell fractions or of purified bovine aorta lysyl oxidase were assayed for 2 h using. As shown (Fig. l ) , considerably more tritium was released from the soluble rTE than theinsoluble elastin substrate with the crude enzyme obtained from the culture media (5-7-fold) and cell layer (5-fold),as well as by the purified bovine aorta enzyme (2.8-fold). Kinetic Characterization of rTE Substrates-Three isoforms of recombinant tropoelastin were kinetically compared as substrates for lysyl oxidase. Pg) lacked the C-terminal polybasic amino acid sequence encoded by exon 36(4), i.e. GGACLGKACGRKRK,normally present in human tropoelastin [4]A. s shown (Fig. 2), the time dependency of tritium release from these substratescatalyzed by purified bovine lysyl oxidase is approximately linear for at least the first 60 min of assay at 37 "C, with the rTEA36 showing the greatest deviation from linearity with longer incubation. Thecatalytic efficiency of these isoforms as substratesfor lysyl oxidase is greatest for rTE and rTEA36, as indicated by the V/K values (Table I)
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