Similarities and differences in the properties of multiple NDP-kinase isoforms of Alyssum murale, Ni2+-accumulator...

Two isoforms of NDPKs (diphosphonucleoside kinases: E.C. 2.7.4.6.) named S-NDPK-A and S-NDPK-B were separated and purified from shoots of Alyssum murale (19th day of growth), a nickel accumulator plant, by a four-step procedure involving ammonium sulphate precipitation and DEAE-sepharose and hydroxyapatite column chromatography. Shoot NDPKs underwent autophosphorylation, proved thermostable, displayed similar molecular mass of 105,000, and consisted of six catalytic subunits. The size of subunits of S-NDPK-A and S-NDPK-B were 18 and 16kDa, respectively. The autophosphorylated S-NDPK-A and S-NDPK-B displayed isoelectric points (pI) of 5.8 and 6.6, respectively. The shoot NDPKs using NDPs (diphosphonucleosides) as substrates were metal dependent, while these underwent autophosphorylation in the absence of metal. The specificity of S-NDPK-A, S-NDPK-B and root NDPK-B (R-NDPK-B); towards mixtures of purino- and pyrimidino-NDPs were tested by TLC (thin layer chromatography). UDP and CDP (pyrimidino-NDPs) and GDP (purino-NDP) were exclusively phosphorylated, using (gamma-(32)P) ATP as phosphate donor, by S-NDPK-A and R-NDPK-B, respectively. Both purino- and pyrimidino-NDPs were phosphorylated by S-NDPK-B. The above isoforms also displayed differences in preference towards a mixture of ADP, GDP, dGDP, TDP, dCDP, CDP and UDP in the presence of Cu(2+), Zn(2+), Mg(2+), Mn(2+), Ni(2+), Ca(2+), Hg(2+) or Co(2+). For example, GDP was mainly phosphorylated by R-NDPK-B independently of the metal used, TDP was mainly phosphorylated by S-NDPK-A in the presence of Mg(2+), Mn(2+), Ca(2+) or Co(2+) and S-NDPK-B was capable of phosphorylating more or less independently of the metal used. The purified and characterized NDPK isoforms may play different biological roles according to their preference towards NDPs.