Laboratório de Moléculas Biologicamente Ativas (Biomol-Lab), Department of Biochemistry and Molecular Biology, Federal University of Ceará, Av. Humberto Monte s/n, Bloco 907, Lab. 1075, Campus do Pici, Zip code 60.400-970, Fortaleza, CE, Brazil.
J Mol Recognit. 2012 Dec;25(12):657-64. doi: 10.1002/jmr.2222.
A lectin from seeds of Dioclea lasiocarpa (DLL) was purified in a single step by affinity chromatography in a Sephadex G-50 column. DLL haemagglutinated rabbit erythrocytes showing stability even after 1 h of exposure to a different pH values (optimal between pH 6.0 and 8.0) but was inhibited after incubation with D-mannose and D-glucose. The pure protein possessed a molecular weight of 25 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 25,410Da by mass spectrometry. The results analyzed by the software SELCON 3 indicate that β-sheet secondary structures are predominant in DLL (approximately 40.2% antiparallel β-sheet, 4.6% parallel β-sheet, 7.2% α-helices, 17.3% turns, and 28.7% unordered structures). Mechanical activity of isolated aorta from rat measured by cumulative concentration curves of DLL, performed at the contraction plateau induced by phenylephrine in either endothelium-intact or denuded aorta. DLL (IC(50) = 34.12 ± 3.46 µg/ml) relaxed precontracted endothelized aortic rings by 34.61 ± 9.06%, 55.19 ± 11.9%, and 81.33 ± 14.35%, respectively, at 10 µg/ml (initial concentration), 30 µg/ml, and 100 µg/ml (maximum effect). All effects occurred via interaction with lectin domains and participation of nitric oxide.
从 Dioclea lasiocarpa(DLL)种子中纯化的凝集素通过在 Sephadex G-50 柱上进行亲和层析,可在单个步骤中进行纯化。DLL 可使兔红细胞凝集,即使在不同 pH 值(最佳 pH 值为 6.0 至 8.0)下暴露 1 小时后也具有稳定性,但在与 D-甘露糖和 D-葡萄糖孵育后会被抑制。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳,该纯蛋白的分子量为 25 kDa,通过质谱法为 25,410Da。通过软件 SELCON 3 分析的结果表明,DLL 中的β-折叠二级结构占主导地位(约 40.2%反平行β-折叠,4.6%平行β-折叠,7.2%α-螺旋,17.3%转角和 28.7%无规卷曲)。通过 DLL 的累积浓度曲线,在苯肾上腺素诱导的收缩平台上,分别在完整内皮或去内皮的大鼠离体主动脉中测量 DLL 的机械活性。DLL(IC50=34.12±3.46μg/ml)使预收缩的内皮化主动脉环分别舒张 34.61±9.06%、55.19±11.9%和 81.33±14.35%,在 10μg/ml(初始浓度)、30μg/ml 和 100μg/ml(最大效应)时。所有作用均通过与凝集素结构域的相互作用以及一氧化氮的参与而发生。