CATALYTIC SUBUNIT OF MOLLUSCAN CYCLIC AMP-DEPENDENT PROTEIN KINASES

这项工作目的是通过cDNAs分离和鉴定 环磷酸腺苷依赖蛋白激酶(A)各亚基的克隆 在海洋软体动物海兔的神经系统中存在 并确定这些蛋白质如何参与神经元 调制。目前的工作主要集中在隔离和 四个相关的A蛋白催化(C)亚基的鉴定 通过选择性剪接从单个基因转录的mRNA而产生 与哺乳动物转录的亚型的比较 独立的基因。独特的氨基酸序列存在于 另一种表达的轴突使产生轴突特异性成为可能 区分核酸和蛋白质异构体的探针 级别。已经产生的抗体可以区分 具有交替N-末端的蛋白质。使用这些工具，我们打算 研究C亚基对神经调节剂的反应 A激酶级联反应。除了这些亚型外，还有五分之一，甚至更多 远缘异构体被克隆并被发现是独有的 在精子中表达。该蛋白的氨基酸含量约为58% 与其他C亚基同源，并通过EM定位 成熟精子轴丝底部的免疫细胞化学。 目前正在努力确定这种激酶是否有助于 获得精子的能动性或参与受精。

The aim of this endeavor was to isolate and characterize, through cDNA cloning, the various subunits of cyclic AMP-dependent protein kinase (A kinase) present in the nervous system of the marine mollusc Aplysia californica and to determine how these proteins participate in neuronal modulation. The current work focuses on the isolation and characterization of four related catalytic (C) subunits of A kinase that arise via alternative splicing of mRNA transcribed from a single gene by comparison to the mammalian isoforms which are transcribed from independent genes. Unique amino acid sequences present in the alternatively expressed axons make it possible to produce axon-specific probes to discriminate between isoforms at the nucleic acid and protein levels. Antibodies have already been generated that distinguish between proteins having alternate N-termini. Using these tools, we intend to investigate how C subunits respond to neuromodulators that act through the A kinase cascade. In addition to these isoforms, a fifth, more distantly related isoform was cloned and found to be exclusively expressed in sperm. This kinase has approximately 58% amino acid homology with the other C subunits, and localizes by EM immunocytochemistry to the base of the axoneme in mature spermatozoa. Efforts are currently underway to determine if this kinase contributes to the acquisition of sperm motility or participates in fertilization.