Christie D J, Alter G M, Magnuson A J
Biochemistry. 1978 Oct 17;17(21):4425-30. doi: 10.1021/bi00614a011.
Saccharide binding has been observed with demetallized concanavalin A in the presence of Ca(2+) only, using the fluorescent sugar 4-methylumbelliferyl alpha-D-mannopyranoside. At pH 7.2 both the nicked and intact forms of concanavalin A bound 4-methylumbelliferyl alpha-D-mannopyranoside with similar affinities. Competitive binding with methyl alpha-D-mannopyranoside was demonstrated. The association constants at 5 degrees C were 9.6 +/- 0.6 X 10(4) M(-1) for 4-methylumbelliferyl alpha-D-mannopyranoside and 1.1 +/- 0.3 X 10(4) M(-1) for methyl alpha-D-mannopyranoside. 4-Methylumbelliferyl alpha-D-mannopyranoside binding was also observed if demetallized concanavalin A was remetallized with less than stoichiometric amounts of Ca(2+). The association constants with low Ca(2+) concentrations were similar to those determined with saturating Ca(2+). With less than stoichiometric levels of Ca(2+), the number of sugar molecules bound per protein subunit was a reflection of the fraction of activated lectin subunits. These results show that saccharide binding activity of concanavalin A does not require a transition metal ion at pH 7.2; only Ca(2+) is required. At pH values near 5, where most previous studies have been carried out, both a transition metal ion and Ca(2+) are necessary.
仅在存在Ca(2+)的情况下，使用荧光糖4-甲基伞形酮基α-D-甘露吡喃糖苷，观察到脱金属伴刀豆球蛋白A与糖类的结合。在pH 7.2时，切口形式和完整形式的伴刀豆球蛋白A以相似的亲和力结合4-甲基伞形酮基α-D-甘露吡喃糖苷。证明了其与甲基α-D-甘露吡喃糖苷的竞争性结合。在5℃时，4-甲基伞形酮基α-D-甘露吡喃糖苷的缔合常数为9.6±0.6×10(4) M(-1)，甲基α-D-甘露吡喃糖苷的缔合常数为1.1±0.3×10(4) M(-1)。如果用化学计量以下的Ca(2+)对脱金属伴刀豆球蛋白A进行再金属化，也可观察到4-甲基伞形酮基α-D-甘露吡喃糖苷的结合。低Ca(2+)浓度下的缔合常数与用饱和Ca(2+)测定的缔合常数相似。在Ca(2+)化学计量以下时，每个蛋白质亚基结合的糖分子数反映了活化凝集素亚基的比例。这些结果表明，在pH 7.2时，伴刀豆球蛋白A的糖类结合活性不需要过渡金属离子；仅需要Ca(2+)。在pH值接近5（大多数先前研究在此进行）时，过渡金属离子和Ca(2+)都是必需的。
