Department of Biotechnology, Dalian Medical University, Dalian, China.
Biochimie. 2010 Apr;92(4):343-9. doi: 10.1016/j.biochi.2010.01.013. Epub 2010 Jan 25.
An L-amino acid oxidase (Akbu-LAAO) was isolated from the venom of Agkistrodon blomhoffii ussurensis snake using DEAE Sephadex A-50 ion-exchange, Sephadex G-75 gel filtration, and high performance liquid chromatographies. The homogeneity and molecular mass of Akbu-LAAO were analyzed by SDS-PAGE and MALDI-TOF spectrometry. The sequences of ten peptides from Akbu-LAAO were established by HPLC-nESI-MS/MS analysis. Protein sequence alignment indicated that i) that Akbu-LAAO is a new snake venom LAAO, and ii) Akbu-LAAO shares homology with several LAAOs from the venoms of Calloselasma rhodost, Agkistrodon halys, Daboia russellii siamensis, and Trimeresurus stejnegeri. Akbu-LAAO is a homodimer with a molecular mass of approximately 124.4 kDa. It reacts optimally with its enzymatic substrate, Leu, at pH 4.7 with a K(m) of 2.1 mM. ICP-AES measurements showed that Akbu-LAAO contains four Zn(2+) per dimer that are unessential for the hydrolytic activity of the enzyme. The emission fluorescence intensity of Akbu-LAAO decreases by 61% on removal of Zn(2+) indicating that the zinc probably helps maintain the structural integrity of the enzyme. The addition of exogenous metal ions, including Mg(2+), Mn(2+), Ca(2+), Ce(3+), Nd(3+), Co(2+) and Tb(3+), increases the l-Leu hydrolytic activity of the enzyme. Akbu-LAAO shows apparent anti-aggregation effects on human and rabbit platelets. It exhibits a strong bacteriostasis effect on Staphylococcus aureus, eighteen fold that of cephalosporin C under the same conditions. Taken together, the biochemical, proteomic, structural and functional characterizations reveal that Akbu-LAAO is a novel LAAO with promise for biotechnological and medical applications.
从东北长白山亚种白眉蝮蛇的毒液中分离出一种 L-氨基酸氧化酶（Akbu-LAAO），采用 DEAE Sephadex A-50 离子交换、Sephadex G-75 凝胶过滤和高效液相色谱进行分离。Akbu-LAAO 的均一性和分子量通过 SDS-PAGE 和 MALDI-TOF 光谱分析。通过 HPLC-nESI-MS/MS 分析建立了 Akbu-LAAO 的十个肽序列。蛋白序列比对表明：i）Akbu-LAAO 是一种新的蛇毒 LAAO，ii）Akbu-LAAO 与来自尖吻蝮、银环蛇、原矛头蝮和圆斑蝰蛇毒液中的几种 LAAO 具有同源性。Akbu-LAAO 是一个约 124.4 kDa 的同源二聚体。它在 pH 4.7 时与酶的最佳底物 Leu 反应，Km 为 2.1 mM。ICP-AES 测量表明，Akbu-LAAO 每个二聚体含有四个 Zn(2+)，这些 Zn(2+)对于酶的水解活性不是必需的。Akbu-LAAO 中的 Zn(2+)被去除后，其发射荧光强度降低了 61%，表明锌可能有助于维持酶的结构完整性。添加外源金属离子，包括 Mg(2+)、Mn(2+)、Ca(2+)、Ce(3+)、Nd(3+)、Co(2+)和 Tb(3+)，可增加酶对 L-Leu 的水解活性。Akbu-LAAO 对人血小板和兔血小板具有明显的抗聚集作用。在相同条件下，它对金黄色葡萄球菌的抑菌作用比头孢菌素 C 强 18 倍。综上所述，Akbu-LAAO 的生化、蛋白质组学、结构和功能特性表明，它是一种具有生物技术和医学应用前景的新型 LAAO。
