CDP phosphotransferase activity in spinach intact chloroplasts: Possible involvement of nucleoside diphosphate kinase II

Nucleotides formation after addition of [gamma(32)P]-ATP has been analysed in isolated chloroplasts in the presence of exogenous CDP, UDP and GDP. The highest level of phosphotransfer was observed on CDP and UDP after 10 min incubation. Interestingly, the phosphorylation increase of chloroplastic CD P in organello correlated with the time-dependent dephosphorylation of a 18 -kDa polypeptide, thereby indicating that CDP, the major endogenous phospho rylated NDP, is likely to be a potent in vivo substrate for this phosphopro tein. The 18-kDa polypeptide was immunoprecipitated with antibodies directe d against human nucleoside diphosphate kinase (NDPK) A/B and spinach NDPK-I I, both belonging to the ubiquitous family of NDPKs (EC 2.7.4.6). Using rec ombinant NDPK-II, we could not show a preference for CDP in vitro, suggesti ng either that CDP is the most available NDPK-II substrate in intact chloro plasts or a chloroplastic factor modulates the enzyme affinity for nucleosi de diphosphate substrates in vivo. (C) 1999 Editions scientifiques et medic ales Elsevier SAS.