SDS-polyacrylamide gel electrophoresis (PAGE) of polyP-GK and analysis of polyP-GK activity. (A) The purified polyP-GK was analyzed by SDS-10% PAGE. (B) [³²P]polyP (6) and [γ-³²P]ATP were used for the phosphorylation of 1 mM glucose by 0.1 μg of the purified polyP-GK. The reaction was performed in the presence of TMB buffer (0.1 M Tris, 0.1 M boric acid, 0.1 M maleic acid [pH 5.5])-50 mM KCl-4 mM MgCl₂ at 30°C. After 1 h of incubation, the reaction mixture was plotted onto a polyethyleneimine-cellulose plate and the plate was developed with 1 M formic acid and 0.4 M LiCl. The ³²P-labeled products were visualized using BAS1000 (Fujifilm).

Comparison of the deduced amino acid sequences of the M. phosphovorus polyP-GK, the M. tuberculosis polyP/ATP-GK, and the E. coli ATP-GK. The shaded amino acids were conserved among the GKs (CLUSTALW analysis; DDBJ). The Phosphate-1, Phosphate-2, Connect-1, and Connect-2 motifs are boxed. The underlined sequences (WRGPLGVTYPGV, KNDWTYPKWAKQ, and FIAGGG) are the proposed regions of glucose, polyP, and adenosine binding in the polyP/ATP-GK (9), respectively.