Purification, properties and specificity of a NDP kinase from Alyssum murale grown under Ni2+ toxicity

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

“…Upon completion, the gel slab was removed from the apparatus and equilibrated for 15 min in a reaction buffer (25 mM TrisHCl (pH 7.4), 5 mM MgCl 2 ). Mg was included since this divalent metal is required for 100% transfer of the phosphate from the donor to the acceptor [15]. The in-gel activity of NDPK was detected by coupling the formation of ATP to PMS and iodonitrotetrazolium chloride (INT).…”

“…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.…”

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

“…It has been reported that the phosphatase activity of A. bertolonii is increased by high Ni 2 + concentrations (Gabbrielli et al, 1990). Further, the response of a new DNase in relation to Ni 2 + and Mn 2 + accumulation in A. murale has been also reported (Abou Auda et al, 2002), and recently, a new NDP-kinase from the root of A. murale (named R-NDPK-B) grown under Ni 2 + (0.5 mM) toxicity was purified and characterized (Yupsanis et al, 2007). The autophosphorylated catalytic subunit (16 kDa) of R-NDPK-B continued to be present in Ni 2 + exposed plants, while the autophosphorylated shoot peptides (16 and 18 kDa) disappeared on the 19th day of growth.…”

“…In plants, NDP-kinases are involved in responses to phytochrome and UV-B light signaling (Zimmermann et al, 1999;Hetmann and Kowalczyk, 2009), growth (Pan et al, 2000), heat stress (EscobarGalvis et al, 2001), reactive oxygen species (Moon et al, 2003), and Mn-and Ni-toxicity in Ni-accumulator plants (Yupsanis et al, 2007). Furthermore, a pea mitochondrial nucleoside diphosphate kinase also displays nuclease activity (Hammargren et al, 2007).…”

“…The autophosphorylated catalytic subunit (16 kDa) of R-NDPK-B continued to be present in Ni 2 + exposed plants, while the autophosphorylated shoot peptides (16 and 18 kDa) disappeared on the 19th day of growth. R-NDPK-B had a very low K m (8.4 nM) towards ADP and could perform a crucial function in the formation of the amount of ATP necessary for A. murale to survive Ni 2 + stress (Yupsanis et al, 2007).…”