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：确定生物学的重要性， 非第二信使环核苷酸（CN）的作用机制 代谢系统，这里指定为“兴奋性”，表现出 显著加快周转率的显著特点， 响应细胞信号，细胞CN浓度无变化， 促进细胞内Ca 2+浓度增加的离子载体;和 利用基于测量的独特有效的分析方法， 代谢中间体磷酰基中18-0的出现率 以确定酶途径在完整细胞中的动态行为， 在高能磷酰基转移中起作用。 在计划高的研究中 能量磷酸盐代谢将在亚细胞中表征 嗜铬细胞的隔室，以确定需要和细胞 能源消耗事件的场所。 A的可能操作 磷酸肌酸穿梭和腺苷酸激酶磷酸转移 系统运行以保持高能磷酸盐的状态， 将定义嗜铬细胞中的代谢颗粒。 兴奋性CN代谢系统将以几种方式表征。 首先，关于它的细胞内运作，通过确定其代谢 与Ca 2+瞬变和兴奋/分泌偶联相关的行为 由不同的Ca 2+相关信号诱导;第二，通过评估 其调节机制涉及钙调蛋白（CM），通过确定其 与原位动力学相关的代谢行为 磷酸二酯酶（PDE）激活Ca 2 +/CM使用构象特异性 检测Ca 2 +/CM PDE复合物形成率的抗体;第三， 评估对“下游”G蛋白在调节或 通过确定肉毒杆菌是否能将兴奋性CN系统与分泌偶联， 毒素分泌抑制超过细胞内Ca 2+释放 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基化;以及第四，通过测定 这种兴奋性CN系统的功能性效用的机制是否 来源于CN PDE的固有特性，作为高浓度的效应物， 电导Ca 2+激活的K+通道“打开”密切对应 与腮腺兴奋性CN系统的Ca ~（2+）连锁刺激有关。 PDE的这一拟议行动将与其最近的 证明了对感光细胞阳离子通道的直接影响， CN水解速率决定通道开放时间。

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.