Hwang S B
Merck Sharp & Dohme Research Laboratories, Department of Biochemical Regulation, Rahway, NJ 07065-0900.
Biochim Biophys Acta. 1991 May 7;1064(2):351-9. doi: 10.1016/0005-2736(91)90322-y.
Specific, saturable and reversible binding of tritium-labeled inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) to human platelet membranes is demonstrated. The Ins(1,4,5)P3-binding sites are abundant and display high selectivity for Ins(1,4,5)P3. Other inositol phosphates exhibit much lower affinity for this site. The specific [3H]Ins(1,4,5)P3 binding was found to be modulated by pH, monovalent and divalent cations, and GTP. A sharp increase in binding occurs at slightly alkaline pH. The monovalent cations, Na+, K+ and Li+ almost double the binding at 30 mM. Mg2+ inhibits the specific [3H]Ins(1,4,5)P3 binding. At low concentrations of Ca2+, the binding is inhibited, but at concentrations higher than 5 mM the binding is potentiated and increases by almost 5-fold at 100 mM. Similar pattern of the effects is also observed for Mn2+ and Sr2+. The specific [3H]Ins(1,4,5)P3 binding is specifically inhibited by GTP. Other nucleotides also inhibit the binding but at higher concentrations. From saturation binding studies, Ca2+ potentiation seems to be due to the conversion of the receptor from the low-affinity state to the high-affinity one. In the absence of Ca2+, the Scatchard plot is nonlinear and concave, and statistically can be fitted best with two equilibrium dissociation constants (Kd values), 0.19 +/- 0.11 and 13.2 +/- 18.1 nM, respectively, for high- and low-affinity binding sites. However, in the presence of 100 mM CaCl2, the Scatchard plot reveals only the high-affinity binding sites with a Kd value of 0.32 +/- 0.15 nM. The specific Ins(1,4,5)P3 receptor in human platelets could therefore exist in multiple conformational states to regulate the intracellular Ca2+ concentration.
已证实氚标记的肌醇1,4,5 - 三磷酸[（³H]Ins(1,4,5)P3）与人血小板膜存在特异性、可饱和且可逆的结合。Ins(1,4,5)P3结合位点丰富，且对Ins(1,4,5)P3表现出高选择性。其他肌醇磷酸对此位点的亲和力则低得多。发现特异性的[³H]Ins(1,4,5)P3结合受pH、单价和二价阳离子以及GTP的调节。在略碱性pH下结合急剧增加。单价阳离子Na⁺、K⁺和Li⁺在30 mM时使结合几乎增加一倍。Mg²⁺抑制特异性的[³H]Ins(1,4,5)P3结合。在低浓度Ca²⁺时，结合受到抑制，但在高于5 mM的浓度时，结合增强，在100 mM时增加近5倍。Mn²⁺和Sr²⁺也观察到类似的效应模式。特异性的[³H]Ins(1,4,5)P3结合受到GTP的特异性抑制。其他核苷酸也抑制结合，但浓度更高。从饱和结合研究来看，Ca²⁺增强作用似乎是由于受体从低亲和力状态转变为高亲和力状态。在没有Ca²⁺的情况下，Scatchard图是非线性且凹形的，统计学上分别用两个平衡解离常数（Kd值）0.19±0.11和13.2±18.1 nM来拟合高亲和力和低亲和力结合位点最为合适。然而，在存在100 mM CaCl₂的情况下，Scatchard图仅显示Kd值为0.32±0.15 nM的高亲和力结合位点。因此，人血小板中的特异性Ins(1,4,5)P3受体可能以多种构象状态存在，以调节细胞内Ca²⁺浓度。
