Characterization of a cytosolic nucleoside diphosphate kinase associated with cell division and growth in potato

Abstract

A cDNA encoding Solanum chacoense cytosolic NDPK (NDPK1, EC 2.7.4.6) was isolated. The open reading frame encoded a 148 amino acid protein that shares homology with other cytosolic NDPKs including a conserved N-terminal domain. S. chacoense NDPK1 was expressed in Escherichia coli as a 6xHis-tagged protein and purified by affinity chromatography. The recombinant protein exhibited a pattern of abortive complex formation suggesting that the enzyme is strongly regulated by the NTP/NDP ratio. A polyclonal antibody generated against recombinant NDPK1 was specific for the cytosolic isoform in Solanum tuberosum as shown from immunoprecipitation experiments and immunoblot analysis of chloroplasts and mitochondria preparations. NDPK activity and NDPK1 protein were found at different levels in various vegetative and reproductive tissues. DEAE fractogel analyses of NDPK activity in root tips, leaves, tubers and cell cultures suggest that NDPK1 constitutes the bulk of extractable NDPK activity in all these organs. NDPK activity and NDPK1 protein levels raised during the exponential growth phase of potato cell cultures whereas no rise in activity or NDPK1 protein was observed when sucrose concentration in the culture was manipulated to limit growth. Activity measurements, immunoblot analysis as well as immunolocalization experiments performed on potato root tips and shoot apical buds demonstrated that NDPK1 was predominantly localized in the meristematic zones and provascular tissues of the apical regions. These data suggest that NDPK1 plays a specific role in the supply of UTP during early growth of plant meristematic and provascular tissues.

The monitoring of nucleotide diphosphate kinase activity by blue native polyacrylamide gel electrophoresis

“…Thus, GTP is preferentially used in the phosphotransferase reaction. These data are consistent with the previously observed Km values for NDPK isolated from Solanum chacoense [18]. The NDPK in this case had a calculated Km value of 75 mM for GTP in comparison to a value of 1030 mM for CTP.…”

“…However, UTP and CTP were not as effective as GTP in generating activity bands. It has been previously reported that NDPK preferentially utilizes GTP [18]. To evaluate the concept that NDPK has a higher affinity for GTP than CTP we performed reactions with concentrations of GTP and CTP ranging from 0.3 to 1.5 mM.…”

“…NDPK is generally monitored using enzyme-coupled spectrophotometric assays [18]. In these assays, the ATPgenerating capabilities of NDPK are coupled to exogenously added hexokinase (HK) and glucose-6-phosphate dehydrogenase (G6PDH) allowing the quantification of NADPH production.…”

“…Furthermore, high amounts of sample are required for the spectrophotometric measurements since the sensitivity of these assays are relatively low. In order to compensate for the shortcomings of spectrophotometric assays, techniques employing sensitive technologies such as HPLC or radiolabeled NTP have been developed [15,18]. Although sensitive, these techniques are quite costly and, in the case of using radiolabeled substrate, extreme care needs to be taken.…”

Research Article A proteomic approach to guarana seed and pericarp maturation.

“…NDPK is a ubiquitous enzyme found in all organisms and cell types that catalyzes the transfer of the γ-phosphoryl group from adenosine triphosphate (ATP) to nucleotide diphosphate (NDP) (Lascu and Gonin, 2000), maintaining the intracellular levels of all (d)NTPs required for nucleic acid biosynthesis (Moon et al, 2003). This protein appears to be involved in signal transduction processes associated with the regulation of cellular activities, such as proliferation, development and tissue differentiation (Otero, 2000;Dorion et al, 2006), which explains its occurrence mainly in the green condition. Variation in NDPK expression among different tissues and developmental stages has been reported (Escobar et al, 1984).…”