[Biochemical characteristics of bovine retina nucleoside diphosphate kinase]. / Biokhimicheskie svoistva nukleoziddifosfatkinazy setchatki glaza byka.

Nucleoside diphosphate kinase (NDP kinase; ATP: NDP phosphotransferase; EC 2.7.4.6) was purified from bovine retina. The molecular mass of the native enzyme was found to be 72 kDa, and those of its subunits were 17.5 and 18.5 kDa. Kinetic characteristics of the enzyme were determined. It was shown that NDP kinase exists in retina in both soluble and membrane-bound forms.

Nucleoside diphosphate kinase from bovine retina: purification, subcellular localization, molecular cloning, and three-dimensional structure.

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.

[Guanylate kinase from bovine retina: isolation, primary structure, and expression in E. coli]. / Guanilatkinaza iz setchatki byka: vydelenie, pervichnaia struktura i ékspressiia v E. coli.

Guanylate kinase (EC 2.7.4.8), catalysing the reaction GMP+ATP = GDP+ADP, was purified to homogeneity from bovine retina. Primary structure of the enzyme was determined by parallel analyses of amino acid sequences of its peptides and nucleotide sequence of the corresponding cDNA. It is shown that the bovine retinal guanylate kinase like the analogous enzyme from yeast Saccharomyces cerevisiae contains a characteristic glycine-rich motif, involved in ATP binding. All of the amino acids, involved in GMP binding in the yeast enzyme, are conserved or conservatively substituted in the bovine retinal guanylate kinase. The bovine retinal enzyme was expressed in E. coli as a fusion protein. Data are presented on the purification of the fusion protein, its digestion by enteropeptidase, purification of the recombinant enzyme and its functional characteristics.

Enzymes of the cyclic GMP metabolism in bovine retina. I. Cloning and expression of the gene for guanylate kinase.

Guanylate kinase (EC 2.7.4.8) catalyzing the reaction GMP + ATP = GDP + ADP, was purified to homogeneity from bovine retina. Using oligonucleotides based on the amino acid sequence of this enzyme, the cDNA encoding guanylate kinase (GK) was isolated and its nucleotide sequence was determined. Expression of the GK cDNA in E. coli, and the purification and functional characterization of the expressed enzyme are presented. It is shown that bovine retinal GK, like its yeast counterpart, contains the characteristic glycine-rich motif and all the amino acids involved in GMP binding. Bovine retinal enzyme is extended for several amino acid residues both at the N- and C-termini, compared to the yeast enzyme.