Poster: Seed Biology

Abs # 539: Purification and cDNA cloning of ADP-glucose pyrophosphatase from rice seedlings

Presenter:	Mitsui, Toshiaki , t.mitsui@agr.niigata-u.ac.jp
Authors	Mitsui, Toshiaki  (A)   Nanjo, Yohei  (A)   Kurokawa, Shunsuke  (A)   Pozueta-Romero, Javier  (B)
Affiliations:	(A): Department of Applied Biological Chemistry, Niigata University (B): Instituto de Agrobiotecnologia y Recursos Naturales (UPNA/CSIC)

We found that there exists an activity of ADP-glucose pyrophosphatase in the shoot tissues of germinating rice seeds. We succeeded to purify the enzyme to homogeneity, and the K_m, V_max and k_cat/K_m values toward ADP-glucose were determined to be 0.8 mM, 183 x 10^-6 mol min^-1 (mg of protein)^-1 and 2.50 x 10⁵ M^-1 sec^-1, respectively.�@The enzyme exhibited a significant hydrolyzing activity against the other sugar nucleotides, such as ADP-ribose, UDP-glucose, CDP-glucose, TDP-glucose, GDP-mannose, and bis-p-nitrophenyl phosphate, indicating that the enzyme is nucleotide pyrophosphatase/phosphodiesterase (NPP). The optimal temperature and pH for enzyme reaction were 50^oC and 6.0. The molecular size of rice NPP was estimated by gel filtration column chromatography and SDS-polyscrylamide gel electrophoresis to be 290 and 70 kDa, respectively. The MALDI-TOF-MS analysis strongly suggested that the rice enzyme exists as a homotetramer. In addition, the enzyme tightly bound to the Con A-Sepharose 4B column but the binding was prevented by methyl-mannopyranoside, indicating that the enzyme bears an oligosaccharide chain. We also determined the N-terminal and internal amino acid sequences of enzyme by protein sequencer and nESI-MS/MS, and succeeded the cDNA cloning of rice NPP (ORYsa;npp). The cDNA is 2202 bp in length including a single open reading frame of 1872 bp that encodes 623 amino acid residues, forming a 69.9 kDa precursor protein. The deduced amino acid sequence of ORYsa;npp clearly indicated that rice ADP-glucose pyrophosphatase is a novel NPP distinct from the barley soluble ADP-glucose pyrophophatase 1 and 2 [FEBS Lett. 490 (2001) 44-48]. This research was supported in part by a grant of Rice Genome Project PR-1102 (to T.M.), MAFF, Japan.