Dudkin S M, Mikchaylova L I, Severin E S
Adv Enzyme Regul. 1983;21:333-52. doi: 10.1016/0065-2571(83)90022-5.
The mechanisms of regulation of cyclic AMP phosphodiesterases were studied using the cytoplasmic fraction of PC-12 cells sensitive to the action of nerve growth factor. The cells contain phosphodiesterases of two types. One of them possesses a high affinity for cyclic AMP (Km = 2.46 mM), whereas the other has the affinity by an order worse (Km = 37.1 mM). PC-12 cell differentiation under the action of nerve growth factor is connected with the cyclic nucleotide elevation; however, activities of both phosphodiesterases remain unchanged. This indicates that the regulation of activity of these enzymes in PC-12 cells is mediated by second messenger effects. The main object of cell regulation is phosphodiesterase with low affinity for the substrate. Its activity is modulated by the calmodulin-Ca2+ complex, cyclic GMP and NAD+ at micromolar concentrations. The effect on the phosphodiesterase system of both a "quick" messenger, Ca2+ and "slow" messengers, cyclic GMP and NAD+, has the same consequences: the turnover number of the enzymic reaction increases that is accompanied by a proportional decrease in the enzyme affinity for cyclic AMP so that the ratio Vmax/Km remains constant. A possible explanation of functional significance of such an activity modulation may be the necessity to maintain the conditions for phosphodiesterase functioning when Km much greater than [cyclic AMP] and the reaction rate are directly proportional to the substrate concentration: v = Vmax/Km [cyclic AMP]. Then the cells are transferred into such a mode when autoregulation of the cyclic nucleotide level takes place. Besides the transient effects causing changes in phosphodiesterase activity, studies of PC-12 cells revealed a chronic effect of phosphodiesterase activity change under the action of staphylococcal enterotoxin A. This protein which induces differentiation of PC-12 cells and possesses a NAD+-glycohydrolase activity is translocated into cytoplasm of cells in the presence of NAD+ and accomplishes ADP-ribosylation of phosphodiesterase. As a result, the enzyme activity falls, cyclic AMP level increases and cell differentiation starts. The activity of soluble phosphodiesterase of PC-12 cells also decreases under the effect of two neurotoxins from bee venom, melittin and tertiapin. Both the toxins at concentration of 10 microM completely block calcium regulation of the enzyme. The mechanism of tertiapin action was investigated on a model system of calmodulin-bovine brain phosphodiesterase. It appeared that inhibition of Ca2+ action is achieved as the result of binding of two toxin molecules with Kd = 2 mM to the activated calmodulin molecule.(ABSTRACT TRUNCATED AT 400 WORDS)
利用对神经生长因子作用敏感的PC-12细胞的细胞质部分，研究了环磷酸腺苷磷酸二酯酶的调节机制。这些细胞含有两种类型的磷酸二酯酶。其中一种对环磷酸腺苷具有高亲和力（Km = 2.46 mM），而另一种的亲和力则差一个数量级（Km = 37.1 mM）。在神经生长因子作用下PC-12细胞的分化与环核苷酸水平升高有关；然而，两种磷酸二酯酶的活性均保持不变。这表明这些酶在PC-12细胞中的活性调节是由第二信使效应介导的。细胞调节的主要对象是对底物亲和力低的磷酸二酯酶。其活性在微摩尔浓度下受到钙调蛋白-Ca2+复合物、环鸟苷酸和NAD+的调节。“快速”信使Ca2+以及“缓慢”信使环鸟苷酸和NAD+对磷酸二酯酶系统的作用具有相同的结果：酶促反应的转换数增加，同时酶对环磷酸腺苷的亲和力成比例降低，从而使Vmax/Km比值保持恒定。对这种活性调节功能意义的一种可能解释是，当Km远大于[环磷酸腺苷]且反应速率与底物浓度成正比时，即v = Vmax/Km [环磷酸腺苷]，有必要维持磷酸二酯酶发挥作用的条件。然后细胞进入环核苷酸水平自动调节的模式。除了引起磷酸二酯酶活性变化的瞬时效应外，对PC-12细胞的研究还揭示了葡萄球菌肠毒素A作用下磷酸二酯酶活性变化的慢性效应。这种诱导PC-12细胞分化并具有NAD+ - 糖水解酶活性的蛋白质，在有NAD+存在时会转位到细胞的细胞质中，并完成磷酸二酯酶的ADP - 核糖基化。结果，酶活性下降，环磷酸腺苷水平升高，细胞分化开始。在蜂毒的两种神经毒素蜂毒素和蜂毒肽的作用下，PC-12细胞可溶性磷酸二酯酶的活性也会降低。两种毒素在10 microM的浓度下完全阻断了该酶的钙调节。在钙调蛋白 - 牛脑磷酸二酯酶的模型系统上研究了蜂毒肽的作用机制。结果发现，两个毒素分子以Kd = 2 mM与活化的钙调蛋白分子结合，从而实现对Ca2+作用的抑制。（摘要截取自400字）
