Nucleoside Diphosphate Kinase from Bovine Retina: Purification, Subcellular Localization, Molecular Cloning, and Three-Dimensional Structure
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- 作者：Najmoutin G. Abdulaev ; Galina N. Karaschuk ; Jane E. Ladner ; Dmitri L. Kakuev ; Alexei V. Yakhyaev ; Maria Tordova ; Ibragim O. Gaidarov ; Viktor I. Popov ; John H. Fujiwara ; Diana Chinchilla ; Edward Eisens
- 刊名：Biochemistry
- 出版年：1998
- 出版时间：October 6, 1998
- 年：1998
- 卷：37
- 期：40
- 页码：13958 - 13967
- 全文大小：167K
- 年卷期：v.37,no.40(October 6, 1998)
- ISSN：1520-4995
文摘
The biochemical and structural properties of bovine retinal nucleoside diphosphate kinase wereinvestigated. The enzyme showed two polypeptides of ~17.5 and 18.5 kDa on SDS-PAGE, whileisoelectric focusing revealed seven to eight proteins with a pI range of 7.4-8.2. Sedimentation equilibriumyielded a molecular mass of 96 ± 2 kDa for the enzyme. Carbohydrate analysis revealed that bothpolypeptides contained Gal, Man, GlcNAc, Fuc, and GalNac saccharides. Like other nucleoside diphosphatekinases, the retinal enzyme showed substantial differences in the Km values for various di- and triphosphatenucleotides. Immunogold labeling of bovine retina revealed that the enzyme is localized on both themembranes and in the cytoplasm. Screening of a retinal cDNA library yielded full-length clones encodingtwo distinct isoforms (NBR-A and NBR-B). Both isoforms were overexpressed in Escherichia coli andtheir biochemical properties compared with retinal NDP-kinase. The structures of NBR-A and NBR-Bwere determined by X-ray crystallography in the presence of guanine nucleotide(s). Both isoforms arehexameric, and the fold of the monomer is similar to other nucleoside diphosphate kinase structures. TheNBR-A active site contained both a cGMP and a GDP molecule each bound at half occupancy while theNBR-B active site contained only cGMP.