Purification of an autophosphorylating protein from imbibing soybean (Glycine max L.) seed exudate and its identification as a nucleoside diphosphate kinase

A calcium-independent autophosphorylating protein was identified in the exudate of imbibed soybean (Glycine max L.) seeds. The inclusion of histone I or calmodulin in protein phosphorylation assays had no observable affect on the extent of phosphorylation of this protein. The protein was purified to apparent homogeneity, bad a native molecular mass of 72 Ku, a subunit molecular mass of 18 Ku, and was capable of in situ phosphorylation. The phosphorylated residue was identified as serine by two-dimensional TLE analysis. Polyclonal antibodies were raised against the purified protein and were used to isolate a cDNA clone from a lambda-ZAP soybean cDNA expression library. The nucleotide sequence of the clone and its deduced amino acid sequence had very high homology to those of nucleoside diphosphate kinases (NDP kinase, EC 2.7.4.6) from plants and other eukaryotic tissue. In addition, the purified autophosphorylating protein exhibited NDP kinase activity. The purified enzyme preferentially transferred the terminal phosphate from ATP to a purine, but no transfer to the pyrimidine, CDP was observed.