NUCLEOSIDE DIPHOSPHATE KINASE FROM BOVINE RETINA - PURIFICATION, SUBCELLULAR-LOCALIZATION, MOLECULAR-CLONING, AND 3-DIMENSIONAL STRUCTURE

The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase were investigated. The enzyme showed two polypepti des of similar to 17.5 and 18.5 kDa on SDS-PAGE, while isoelectric foc using revealed seven to eight proteins with a pi range of 7.4-8.2. Sed imentation equilibrium yielded a molecular mass of 96 +/- 2 kDa for th e enzyme. Carbohydrate analysis revealed that both polypeptides contai ned Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleosi de diphosphate kinases, the retinal enzyme showed substantial differen ces in the K-m values for various di- and triphosphate nucleotides. Im munogold labeling of bovine retina revealed that the enzyme is localiz ed 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 co li and their biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-B were determined by X-ray crystallogr aphy in the presence of guanine nucleotide(s). Both isoforms are hexam eric, and the fold of the monomer is similar to other nucleoside dipho sphate 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.