Prorok M, Castellino F J
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
J Biol Chem. 1998 Jul 31;273(31):19573-8. doi: 10.1074/jbc.273.31.19573.
The binding isotherms of the divalent metal cations, Ca2+, Mg2+, and Zn2+, to the synthetic gamma-carboxyglutamic acid-containing neuroactive peptides, conantokin-G (con-G) and conantokin-T (con-T), have been determined by isothermal titration calorimetry (ITC) at 25 degreesC and pH 6.5. We have previously shown by potentiometric measurements that con-G contains 2-3 equivalent Ca2+ sites with an average Kd value of 2800 microM. With Mg2+ as the ligand, two separate exothermic sites are obtained by ITC, one of Kd = 46 microM and another of Kd = 311 microM. Much tighter binding of Zn2+ is observed for these latter two sites (Kd values = 0.2 microM and 1.1 microM), and a third considerably weaker binding site is observed, characterized by a Kd value of 286 microM and an endothermic enthalpy of binding. con-T possesses a single exothermic tight binding site for Ca2+, Mg2+, and Zn2+, with Kd values of 428 microM, 10.2 microM, and 0.5 microM, respectively. Again, in the case of con-T, a weak (Kd = 410 microM) endothermic binding site is observed for Zn2+. The binding of these cations to con-G and con-T result in an increase in the alpha-helical content of the peptides. However, this helix is somewhat destabilized in both cases by binding of Zn2+ to its weakest site. Since the differences observed in binding affinities of these three cations to the peptides are substantially greater than their comparative Kd values to malonate, we conclude that the structure of the peptide and, most likely, the steric and geometric properties imposed on the cation site as a result of peptide folding greatly influence the strength of the interaction of cations with con-G and con-T. Further, since the Zn2+ concentrations released in the synaptic cleft during excitatory synaptic activity are sufficiently high relative to the Kd of Zn2+ for con-G and con-T, this cation along with Mg2+, are most likely the most significant metal ion ligands of these peptides in neuronal cells.
通过等温滴定量热法（ITC）在25℃和pH 6.5条件下测定了二价金属阳离子Ca2+、Mg2+和Zn2+与合成的含γ-羧基谷氨酸的神经活性肽芋螺毒素G（con-G）和芋螺毒素T（con-T）的结合等温线。我们之前通过电位测量表明，con-G含有2 - 3个等效的Ca2+位点，平均解离常数（Kd）值为2800微摩尔。以Mg2+作为配体时，通过ITC得到两个独立的放热位点，一个Kd值为46微摩尔，另一个Kd值为311微摩尔。对于后两个位点，观察到Zn2+的结合紧密得多（Kd值分别为0.2微摩尔和1.1微摩尔），并且观察到第三个结合位点明显较弱，其特征为Kd值为286微摩尔且结合焓为吸热。con-T对Ca2+、Mg2+和Zn2+具有单个放热紧密结合位点，Kd值分别为428微摩尔、10.2微摩尔和0.5微摩尔。同样，就con-T而言，观察到Zn2+有一个弱的（Kd = 410微摩尔）吸热结合位点。这些阳离子与con-G和con-T的结合导致肽的α-螺旋含量增加。然而，在这两种情况下，由于Zn2+与其最弱位点的结合，这种螺旋会有些不稳定。由于观察到这三种阳离子与肽的结合亲和力差异远大于它们与丙二酸的比较Kd值，我们得出结论，肽的结构以及很可能由于肽折叠而施加在阳离子位点上的空间和几何性质极大地影响阳离子与con-G和con-T相互作用的强度。此外，由于在兴奋性突触活动期间突触间隙中释放的Zn2+浓度相对于con-G和con-T的Zn2+ Kd值足够高，这种阳离子连同Mg2+很可能是这些肽在神经元细胞中最重要的金属离子配体。
