Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.
Oxidation, cross-linking, and insolubilization of recombinant tropoelastin by purified lysyl oxidase.
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DOI：
10.1016/s0021-9258(18)82207-4
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发表时间：
1993-05
期刊：
影响因子：
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通讯作者：
D. Bedell-Hogan;P. Trackman;William AbramsS;Joel RosenbloomS;Herbert Kagang
中科院分区：
文献类型：
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作者：
D. Bedell-Hogan;P. Trackman;William AbramsS;Joel RosenbloomS;Herbert Kagang
探索了重组人弹性蛋白原（rTE）及其所选变体作为体外赖氨酰氧化酶活性测定的底物的用途。还评估了不溶性弹性蛋白样产物可以在不存在与体内弹性蛋白相关的其他大分子的情况下从该弹性蛋白前体产生的可能性。赖氨酰氧化酶比常规使用的不溶性鸡主动脉弹性蛋白底物更有效地氧化rTE。阴离子两亲性弹性蛋白配体强烈抑制rTE氧化与静电酶-底物相互作用的重要性之前指出的不溶性弹性蛋白基板一致。一个rTE变体，rTE delta 26 A，缺乏由外显子26 A编码的亲水序列，是一种不如rTE有效的底物，主要是由于Km的增加，而rTE delta 36，缺乏由外显子36编码的C-末端多元序列的氧化动力学参数与rTE非常相似。rTE delta 26 A与赖氨酰氧化酶的孵育不仅导致肽基α-氨基己二酸-δ-半醛和赖氨酸衍生的交联的产生，而且产生不溶于热0.1 N NaOH的产物，与不溶性弹性蛋白的性质一致。因此，赖氨酰氧化酶对弹性蛋白底物的氧化、交联和不溶解可以在不存在涉及该体内过程的其他大分子的情况下发生，尽管这样的大分子对于获得用于特定放置的交联的原弹性蛋白单元和最佳功能性弹性纤维之间的适当对齐可能是必需的。
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.