Molecular cloning and characterization of nucleoside diphosphate kinase in cultured sugarcane cells

Abstract

A low molecular weight autophosphorylating protein (pp18) in cultured sugarcane cells was identified and characterized as nucleoside diphosphate (NDP) kinase. The purified NDP kinase separated into five isoforms of 16.5kD (NDP kinase I) and two isoforms of 18kD (NDP kinase IT) on two dimensional IEF/SDS-PAGE. The apparent K, values of the purified enzyme containing both size classes were 2.3mM and 0.2mM for ATP and GDP, respectively. Twenty one positive clones were isolated by screening a λgt11 cDNA library derived from cultured sugarcane cells with spinach NDP kinase I cDNA probe, and four were sequenced. The cDNA pSCNDK8 contained a 447 bp coding region (149 amino acids), and 58 bp and 238 bp 5' and 3' flanking sequences respectively. This cDNA hybridized to a 0.91kb mRNA. The deduced amino acid sequence of sugarcane NDP kinase cDNA clone showed over 60% sequence identity to many eukaryotic NDP kinases. The cDNA pSCNDK8 showed a frame shift resulting in 55 amino acid residues at the carboxyl terminus with no homology to NDP kinases. The sugarcane NDP kinase showed approximately a 3 to 4-fold enhancement in in situ autophosphorylation and 1.5 to 2 fold increase in NDP kinase activity in response to HS at 40-42°C for 2h. However, NDP kinase protein or mRNA levels did not show an increase during HS. The synthesis and phosphorylation of NDP kinase appears to be developmentally and heat shock regulated. NDP kinase levels were highest and showed a greater enhancement in autophosphorylation in response to HS in cells in fresh culture. In contrast to cultured sugarcane cells, mRNAs for NDP kinase I enhanced at least 2-fold in response to a 2 h HS at 40°C in young sugarcane shoots.