Li G, Comte M, Wollheim C B, Cox J A
Institut de Biochimie Clinique, University of Geneva, Switzerland.
Biochem J. 1989 Jun 15;260(3):771-5. doi: 10.1042/bj2600771.
The effect of Ca2+ and calmodulin (CaM) on the activation of purified bovine brain Ins(1,4,5)P3 kinase was quantified and interpreted according to the model of sequential equilibria generally used for other calmodulin-stimulated systems. Two main conclusions can be drawn. (i) CaM.Ca3 and CaM.Ca4 together are the biologically active species in vitro, as is the case for the great majority of other calmodulin targets. (ii) These species bind in a non-co-operative way to the enzyme with an affinity constant of 8.23 x 10(9) M-1, i.e. approx 10-fold higher than for most calmodulin-activated target enzymes. The dose-response curve of the activation of Ins(1,4,5)P3 kinase by calmodulin is not significantly impaired by melittin and trifluoperazine, whereas under very similar assay conditions the half-maximal activation of bovine brain cyclic AMP phosphodiesterase requires over 30-50-fold higher concentrations of CaM when 1 microM melittin or 20 microM-trifluoperazine is present in the assay medium. Similarly, 1 microM of the anti-calmodulin peptides seminalplasmin and gramicidin S, as well as 20 microM of N-(6-aminohexyl)-5-chloro-1-naphthalene-sulphonamide (W7), do not inhibit the activation process. These data suggest that binding and activation of Ins(1,4,5)P3 kinase require surface sites of calmodulin which are different from those involved in the binding of most other target enzymes or of model peptides.
根据通常用于其他钙调蛋白刺激系统的顺序平衡模型，对Ca2+和钙调蛋白（CaM）对纯化的牛脑肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）激酶激活的影响进行了定量和解释。可以得出两个主要结论。（i）CaM·Ca3和CaM·Ca4共同构成体外的生物活性物种，这与绝大多数其他钙调蛋白靶点的情况相同。（ii）这些物种以非协同方式与酶结合，亲和力常数为8.23×10⁹ M⁻¹，即比大多数钙调蛋白激活的靶酶高约10倍。蜂毒肽和三氟拉嗪不会显著损害钙调蛋白对Ins(1,4,5)P3激酶激活的剂量反应曲线，而在非常相似的测定条件下，当测定介质中存在1 μM蜂毒肽或20 μM三氟拉嗪时，牛脑环磷酸腺苷磷酸二酯酶的半最大激活需要高30 - 50倍以上浓度的钙调蛋白。同样，1 μM的抗钙调蛋白肽精浆蛋白和短杆菌肽S，以及20 μM的N-(6-氨基己基)-5-氯-1-萘磺酰胺（W7），均不抑制激活过程。这些数据表明，Ins(1,4,5)P3激酶的结合和激活需要钙调蛋白的表面位点，这些位点与大多数其他靶酶或模型肽结合所涉及的位点不同。
