Rosenthal W, Koesling D, Rudolph U, Kleuss C, Pallast M, Yajima M, Schultz G
Eur J Biochem. 1986 Jul 15;158(2):255-63. doi: 10.1111/j.1432-1033.1986.tb09745.x.
Antisera were raised against the retinal guanine-nucleotide-binding protein (N-protein), transducin, purified from bovine rod outer segments. Sera obtained after repeated injections of antigen recognized all transducin subunits (alpha, beta and gamma). One antiserum, tested for cross-reactivity with non-retinal N-proteins, was found to cross-react with the beta subunits of the ubiquitously occurring N-proteins, Ns and Ni, but not with their respective alpha and gamma subunits. The antiserum also cross-reacted with the beta subunit of the recently identified N-protein, No, which has been found in high abundance in the central nervous system. These data support the similarity of the beta subunits of the N-proteins identified so far. Purification of N-proteins from porcine cerebral cortex without the use of activating ligands yielded fractions containing the isolated alpha subunit of No, free beta gamma complex, Ni, No and fractions containing both N-proteins in various proportions. The purity of the preparations was at least 80% as judged by Coomassie-blue-stained SDS gels. No pure Ns was obtained. Use of the transducin antibody during the course of the purification revealed that the beta subunits coeluted from a gel filtration column largely with the alpha subunits of Ni and No but were hardly detectable in fractions that were able to reconstitute Ns activity into membranes of an Ns-deficient cell line (S49 cyc- lymphoma cells). This indicates that in the central nervous system the concentrations of Ni and No are of magnitudes higher than that of Ns. Two-dimensional gel electrophoresis of N-proteins, purified from porcine cerebral cortex, resulted in the resolution of two major peptides in the 35-kDa region, which differed in their pI values and were identified as beta subunits by the use of the antiserum. Identical results were achieved using crude cholate extracts from membranes of the same tissue instead of purified proteins. The occurrence of different beta subunits may be explained by posttranslational N-protein modification.
制备了针对从牛视杆外段纯化的视网膜鸟嘌呤核苷酸结合蛋白（N蛋白）——转导素的抗血清。多次注射抗原后获得的血清识别所有转导素亚基（α、β和γ）。检测了一种抗血清与非视网膜N蛋白的交叉反应性，发现它与普遍存在的N蛋白Ns和Ni的β亚基发生交叉反应，但不与其各自的α和γ亚基发生交叉反应。该抗血清还与最近鉴定出的N蛋白No的β亚基发生交叉反应，No在中枢神经系统中含量很高。这些数据支持了目前已鉴定出的N蛋白β亚基的相似性。在不使用激活配体的情况下从猪大脑皮层纯化N蛋白，得到的组分包含分离出的No的α亚基、游离的βγ复合物、Ni、No以及含有不同比例两种N蛋白的组分。根据考马斯亮蓝染色的SDS凝胶判断，制剂的纯度至少为80%。未获得纯的Ns。在纯化过程中使用转导素抗体显示，β亚基从凝胶过滤柱上洗脱时主要与Ni和No的α亚基一起洗脱，但在能够将Ns活性重建到缺乏Ns的细胞系（S49 cyc -淋巴瘤细胞）膜中的组分中几乎检测不到。这表明在中枢神经系统中，Ni和No的浓度比Ns高几个数量级。对从猪大脑皮层纯化的N蛋白进行二维凝胶电泳，在35 kDa区域分离出两个主要肽段，它们的pI值不同，通过使用抗血清鉴定为β亚基。使用来自同一组织膜的粗胆酸盐提取物代替纯化蛋白也得到了相同的结果。不同β亚基的出现可能是由N蛋白的翻译后修饰解释的。
