Teng D H, Chen C K, Hurley J B
Howard Hughes Medical Institute, University of Washington, Seattle 98195.
J Biol Chem. 1994 Dec 16;269(50):31900-7.
Polymerase chain reaction was used to search for genes encoding recoverin-like proteins in Drosophila melanogaster. We identified a gene that codes for a cognate of bovine neurocalcin; hence, we have named it neurocalcin (nca). A cDNA of nca was isolated and sequenced. The deduced polypeptide product of the cDNA is 22 kDa in size, and its amino acid sequence is 88% identical to that of bovine neurocalcin. This deduced Drosophila neurocalcin (DrosNCa) protein has three putative EF-hands and has a sequence in its NH2 terminus required for fatty acylation. DrosNCa was expressed in Escherichia coli and subsequently purified by phenyl-Sepharose chromatography and Mono Q anion exchange fast protein liquid chromatography. This recombinant protein was capable of binding 45Ca2+ and exhibited Ca(2+)-dependent mobility shifts in both SDS-polyacrylamide gel electrophoresis and native gel electrophoresis. DrosNCa was tritiated when it was coexpressed in E. coli with N-myristoyl transferase in the presence of [3H]myristic acid. The nca transcript was approximately 1 kilobase long, and tissue in situ hybridization showed that this message was present in the brain of adult flies. Antibodies raised against recombinant DrosNCa cross-reacted with rat hippocalcin on an immunoblot but not with bovine recoverin. When immunohistochemical analysis was performed, staining was observed throughout the central nervous system of adult flies, particularly in the neuropil, where neurons synapse. The nca locus maps to or near 76F on the Drosophila third chromosome.
利用聚合酶链反应在黑腹果蝇中寻找编码类恢复蛋白的基因。我们鉴定出一个编码牛神经钙蛋白同源物的基因；因此，我们将其命名为神经钙蛋白（nca）。分离并测定了nca的cDNA序列。该cDNA推导的多肽产物大小为22 kDa，其氨基酸序列与牛神经钙蛋白的氨基酸序列有88%的同一性。这个推导的果蝇神经钙蛋白（DrosNCa）蛋白有三个假定的EF手结构，并且在其NH2末端有一个脂肪酰化所需的序列。DrosNCa在大肠杆菌中表达，随后通过苯基琼脂糖凝胶层析和Mono Q阴离子交换快速蛋白质液相层析进行纯化。这种重组蛋白能够结合45Ca2+，并且在SDS聚丙烯酰胺凝胶电泳和天然凝胶电泳中均表现出钙依赖性迁移率变化。当DrosNCa在大肠杆菌中与N-肉豆蔻酰转移酶在[3H]肉豆蔻酸存在下共表达时，它被氚化。nca转录本约1千碱基长，组织原位杂交显示该信息存在于成年果蝇的脑中。针对重组DrosNCa产生的抗体在免疫印迹上与大鼠海马钙蛋白发生交叉反应，但与牛恢复蛋白不发生交叉反应。当进行免疫组织化学分析时，在成年果蝇的整个中枢神经系统中均观察到染色，特别是在神经元形成突触的神经毡中。nca基因座定位于果蝇第三染色体上的76F或其附近。
