Purification, properties and specificity of a NDP kinase from Alyssum murale grown under Ni(2+) toxicity.
During the growth of Alyssum murale, a nickel accumulator plant, three root peptides chains of 55, 18 and 16kDa undergo phosphorylation. The intensity of the phosphorylated bands decreased in the course of growth in nutrient solution supplied with 0.5mM Ni(2+). In the shoot only two phosphorylated peptide chains with a size of 18 and 16kDa were detected. These two shoot peptides disappeared on the 19th day of growth in Ni(2+)-exposed plants, while the root peptide of 16kDa continued to be present in less intensity. This peptide was identified as the catalytic subunit of nucleoside diphosphate kinase (NDP kinase: E.C. 2.7.4.6) and was named NDPK-B. The enzyme was purified by means of ammonium sulphate precipitation, DEAE-sepharose and hydroxyapatite column chromatography. NDPK-B was thermostable, displayed a molecular mass of 103,000 and was comprised of six catalytic subunits. The autophosphorylated enzyme displayed an isoelectric point (pI) of 6.5. The NDPK-B autophosphorylation activity was metal-dependent. With regard to the transfer reaction, NDPK-B exhibited the following properties: (a) the enzyme had an optimum pH of 7.6; (b) it was capable of using both (gamma-(32)P) ATP and (gamma-(32)P) GTP as phosphate donors and of using all the available NDPs except dCDP as phosphate acceptors; (c) its activity using NDPs as substrates was metal dependent; (d) in the presence of (gamma-(32)P) GTP as the phosphate donor, it phosphorylated exclusively ADP when a mixture of NDPs was added in the reaction mixture; and, (e) ADP had a very low K(m) value towards 8.4nM. This high affinity towards ADP suggests that the enzyme may play a crucial function in the formation of the amount of ATP necessary for Alyssum murale to survive Ni(2+) stress.
Yupsanis
Traianos
T
Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Greece. yupsanis@chem.auth.gr
Symeonidis
Lazaros
L
Vergidou
Cornilia
C
Siskos
Argirios
A
Michailidou
Sofia
S
Yupsani
Anastasia
A
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D019607:Brassicaceae / Q000187:drug effects* / Q000201:enzymology* / Q000254:growth & development; D018506:Gene Expression Regulation, Plant; D009532:Nickel / Q000633:toxicity*; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology; D011485:Protein Binding; D020033:Protein Isoforms; D013379:Substrate Specificity
D010940:Plant Proteins; D020033:Protein Isoforms; D009532:Nickel; D000255:Adenosine Triphosphate; D009701:Nucleoside-Diphosphate Kinase