De Raad S, Comte M, Nef P, Lenz S E, Gundelfinger E D, Cox J A
Department of Pharmacology, University Medical Center, Geneva, Switzerland.
Histochem J. 1995 Jul;27(7):524-35. doi: 10.1007/BF02388752.
Neural Ca(2+)-binding proteins (NCaPs) constitute a subfamily of 4-EF-hand proteins, and display a histological and structural dichotomy: the A-type NCaPs are selectively expressed by the retina and pineal organ and display two canonical EF-hands, whereas the B-type NCaPs are found in the entire brain and present three regular EF-hands. In this study, antisera were raised against the A-type NCaP recoverin (26 kDa) and the B-type NCaPs VILIP and NCS-1 (22 kDa). Since the sequence identity among NCaPs is high, specific polyclonal antibodies were purified by double cross-immunoaffinity chromatography; both ELISA and immunoblot analyses determined that the resulting antibodies showed selectivity ratios inferior to 1/363 for the two other related NCaPs. Besides, the anti-VILIP antibodies displayed some affinity toward neurocalcin delta, and the antirecoverin antibodies recognized a 24 kDa protein, which is most likely visinin. Thus, immunohistochemical studies on the chicken, rat and cow retina revealed that anti-recoverin antibodies recognized the vertebrate photoreceptors and a small number of mammalian bipolar cells. Anti-VILIP antibodies exclusively labelled the inner retina, i.e. the amacrine and ganglion cells. NCS-1 was mainly present in the photoreceptor inner segments, the inner plexiform layer and the ganglion cells. NCS-1 showed the highest species disparity. The retinal localization of NCS-1 and VILIP offered an important morphological basis for the understanding of their function. Furthermore, specific antibodies against the NCaPs may enable the identification of cell populations in more complex neural tissues, such as the brain.
神经钙结合蛋白（NCaPs）构成了4-EF手蛋白的一个亚家族，并呈现出组织学和结构上的二分法：A型NCaPs在视网膜和松果体中选择性表达，并具有两个典型的EF手结构，而B型NCaPs则存在于整个大脑中，具有三个规则的EF手结构。在本研究中，制备了针对A型NCaP恢复蛋白（26 kDa）以及B型NCaPs维利蛋白（VILIP）和神经元钙传感器-1（NCS-1，22 kDa）的抗血清。由于NCaPs之间的序列同一性很高，因此通过双交叉免疫亲和层析纯化了特异性多克隆抗体；酶联免疫吸附测定（ELISA）和免疫印迹分析均确定，所得抗体对另外两种相关NCaPs的选择性比率低于1/363。此外，抗VILIP抗体对神经钙蛋白δ表现出一定亲和力，抗恢复蛋白抗体识别一种24 kDa的蛋白，很可能是视锥视蛋白。因此，对鸡、大鼠和牛视网膜进行的免疫组织化学研究表明，抗恢复蛋白抗体识别脊椎动物的光感受器和少数哺乳动物双极细胞。抗VILIP抗体专门标记视网膜内层，即无长突细胞和神经节细胞。NCS-1主要存在于光感受器内段、内网层和神经节细胞中。NCS-1表现出最高的物种差异。NCS-1和VILIP在视网膜中的定位为理解它们的功能提供了重要的形态学基础。此外，针对NCaPs的特异性抗体可能有助于识别更复杂神经组织（如大脑）中的细胞群体。
