Abdulaev N G, Karaschuk G N, Ladner J E, Kakuev D L, Yakhyaev A V, Tordova M, Gaidarov I O, Popov V I, Fujiwara J H, Chinchilla D, Eisenstein E, Gilliland G L, Ridge K D
Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, University of Maryland Biotechnology Institute, Rockville 20850, USA.
Biochemistry. 1998 Oct 6;37(40):13958-67. doi: 10.1021/bi980853s.
The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypeptides of approximately 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for the enzyme. Carbohydrate analysis revealed that both polypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphate kinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphate nucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both the membranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encoding two distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms are hexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. The NBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while the NBR-B active site contained only cGMP.
对牛视网膜核苷二磷酸激酶的生化和结构特性进行了研究。该酶在SDS-PAGE上显示出两条分子量约为17.5 kDa和18.5 kDa的多肽，而等电聚焦显示有7至8种蛋白质，其pI范围为7.4 - 8.2。沉降平衡得出该酶的分子量为96±2 kDa。碳水化合物分析表明，两条多肽均含有半乳糖（Gal）、甘露糖（Man）、N-乙酰葡糖胺（GlcNAc）、岩藻糖（Fuc）和N-乙酰半乳糖胺（GalNac）糖类。与其他核苷二磷酸激酶一样，视网膜酶对各种二磷酸和三磷酸核苷酸的Km值存在显著差异。牛视网膜的免疫金标记显示该酶定位于膜和细胞质中。筛选视网膜cDNA文库得到了编码两种不同同工型（NBR-A和NBR-B）的全长克隆。两种同工型均在大肠杆菌中过表达，并将其生化特性与视网膜NDP-激酶进行了比较。在存在鸟嘌呤核苷酸的情况下，通过X射线晶体学确定了NBR-A和NBR-B的结构。两种同工型均为六聚体，单体的折叠与其他核苷二磷酸激酶结构相似。NBR-A活性位点分别以半占据状态结合了一个cGMP分子和一个GDP分子，而NBR-B活性位点仅含有cGMP。
