KREBS CYCLE & CYCLIC NUCLEOTIDE CONTROL

描述(改编自申请人的摘要)：计划 实验有两个主要目标：确定生物重要性和 非第二信使环核苷酸(CN)的作用机制 代谢系统，在这里被指定为“兴奋性的”，它表现出 显著加快周转速度的显著特征 细胞内CN浓度不随细胞信号变化而变化 促进细胞内钙浓度增加的离子载体；以及 利用基于测量的独特有效的分析方法 18-0在代谢中间体磷酸化中的出现率 为了定义完整细胞中酶途径的动态行为， 在高能磷酰化转移中的作用。在计划很高的研究中 能量磷酸盐代谢将在亚细胞中表现出来 用于确定嗜铬细胞和细胞需求的隔室 高耗能事件的地点。的可能的操作 磷酸化肌酸穿梭和腺苷酸激酶的磷酸化转移 系统运行以保持高能磷酸盐的活跃状态 将定义嗜铬细胞中的代谢颗粒。 兴奋性CN代谢系统将从几个方面进行描述。 首先，关于它的细胞内运作，通过确定它的代谢 与钙瞬变和兴奋/分泌耦合有关的行为 由不同的钙信号诱导；第二，通过评估是否 其调控机制涉及钙调蛋白(CM)。 药物代谢行为与其原位动力学的关系 构象特异性钙/CM激活磷酸二酯酶(PDE) 检测钙/CM PDE复合体形成速率的抗体；第三，通过 评估“下游”G-蛋白在调节或 通过确定肉毒杆菌是否存在将兴奋性CN系统与分泌物偶联 毒素抑制细胞内钙释放以外的分泌 通过ADP干扰CN代谢系统的激活 22 kDa G蛋白毒素靶标的核糖化；第四，通过确定 这种兴奋性CN系统的功能效用是否有机制 派生自CN PDE充当High的效应器的固有属性 电导钙激活的K通道紧密对应地“开放” 与兴奋性CN系统在腮腺的钙结合刺激有关。 PDE的这一拟议行动将与其最近的 显示对光感受器阳离子通道的直接影响 CN水解率决定了通道的开放时间。

DESCRIPTION (Adapted from the applicant's abstract): The planned experimentation has 2 major goals: to define the biological importance and operational mechanism of a non-second messenger, cyclic nucleotide (CN) metabolic system, designated here as "excitatory," that exhibits the distinctive characteristic of markedly accelerated rates of turnover with no change in cellular CN concentration in response to cell signals and ionophores that promote increases in intracellular Ca2+ concentration; and to utilize a uniquely effective analytical procedure based on measuring rates of appearance of 18-0 in the phosphoryls of metabolic intermediates to define the dynamic behavior in intact cells of the enzymic pathways that function in high energy phosphoryl transfer. In the studies planned high energy phosphate metabolism will be characterized in subcellular compartments of chromaffin cells to determine requirements for and cellular locales of energy-consuming events. The possible operation of a phosphorylcreatine shuttle and an adenylate kinase phosphoryl transfer system operating to maintain high energy phosphate status of actively metabolizing granules in chromaffin cells will be defined. The excitatory CN metabolic system will be characterized in several ways. First, regarding its intracellular operation by determining its metabolic behavior in relation to Ca2+ transients and excitation/secretion coupling induced by dissimilar Ca2+-linked signals; second, by assessing if the mechanism of its regulation involves calmodulin (CM by determining its metabolic behavior in relation to the in situ kinetics of phospho-diesterase (PDE) activation by Ca2+/CM using conformation-specific antibodies to detect rates of Ca2+/CM PDE complex formation; third, by assessing the requirement for a "down-stream" G-protein in regulating or coupling the excitatory CN system to secretion by determining if botulinum toxin inhibition of secretion beyond release of intracellular Ca2+ interferes with activation of this CN metabolic system through the ADP ribosylation of a 22 kDa G-protein toxin target; and fourth, by determining whether a mechanism for the functional utility of this excitatory CN system derives from an intrinsic property of CN PDE to act as an effector of high conductance Ca2+-activated K+ channels "opened" in close correspondence with the Ca2+-linked stimulation of the excitatory CN system in parotid. This proposed action of PDE would coincide with its very recently demonstrated direct effect on photoreceptor cation channels which predicts that rates of CN hydrolysis determine channel open time.