Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.
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.
Bedell-Hogan
D
D
Department of Biochemistry, Boston University School of Medicine, Massachusetts 02118.
Trackman
P
P
Abrams
W
W
Rosenbloom
J
J
Kagan
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001011:Aorta / Q000201:enzymology; D002417:Cattle; D002478:Cells, Cultured; D002645:Chickens; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D004926:Escherichia coli / Q000235:genetics; D005091:Exons; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D010084:Oxidation-Reduction; D008249:Protein-Lysine 6-Oxidase / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D014333:Tropoelastin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011994:Recombinant Proteins; D014333:Tropoelastin; D008249:Protein-Lysine 6-Oxidase