Kayestha R, Hajela K
School of Life Sciences, Devi Ahilya Vishwavidyalaya, Vigyan Bhawan, India.
Biochim Biophys Acta. 1996 Feb 9;1289(1):51-6. doi: 10.1016/0304-4165(95)00135-2.
A Ca2+ -dependent, glucose-specific lectin was isolated from goat peripheral blood lymphocytes by affinity chromatography on N-acetyl D-glucosamine agarose gel. Since the lectin binding to carbohydrate ligands was metal dependent, it was important to study the divalent metal ion-induced conformational changes in the lectin. The conformational changes were studied by absorption, fluorescence and circular dichroism spectroscopy techniques. Binding of Ca2+, Mn2+ or Mg2+ results in shift in ultraviolet absorption maxima from 281 to 298 nm (red shift). A major increase in absorbance at 245 nm is also exhibited. Binding of Ca2+ and Mn2+ resulted in decrease in intrinsic fluorescence emission maxima with shift from 355.2 nm to 342.4 nm (blue shift). These shifts could be reversed on addition of EDTA. A double reciprocal analysis of fluorescence quenching data suggest that Ca2+ and Mn2+ interact with a single class of binding site with an apparent kd of 1.50 +/- 0.37 microM and 1.25 +/- 0.25 microM, respectively. These data clearly indicate that occupancy of metal binding sites on goat peripheral blood lymphocyte lectin induces a gross conformational change sequestering aromatic amino acids into a hydrophobic environment. These findings were further supported by circular dichroism spectrum which showed a massive alteration in the presence of Ca2+.
通过在N-乙酰-D-葡萄糖胺琼脂糖凝胶上进行亲和层析，从山羊外周血淋巴细胞中分离出一种钙依赖性、葡萄糖特异性凝集素。由于凝集素与碳水化合物配体的结合依赖于金属，因此研究二价金属离子诱导的凝集素构象变化很重要。通过吸收光谱、荧光光谱和圆二色光谱技术研究了构象变化。Ca2+、Mn2+或Mg2+的结合导致紫外吸收最大值从281nm移至298nm（红移）。在245nm处吸光度也有显著增加。Ca2+和Mn2+的结合导致内在荧光发射最大值降低，从355.2nm移至342.4nm（蓝移）。加入EDTA后，这些位移可以逆转。荧光猝灭数据的双倒数分析表明，Ca2+和Mn2+分别与一类结合位点相互作用，表观解离常数分别为1.50±0.37μM和1.25±0.25μM。这些数据清楚地表明，山羊外周血淋巴细胞凝集素上金属结合位点的占据诱导了一种总体构象变化，将芳香族氨基酸隔离到疏水环境中。圆二色光谱进一步支持了这些发现，该光谱显示在Ca2+存在下有大量改变。
