CHARACTERIZATION OF GUANOSINE KINASE FROM BREVIBACTERIUM-ACETYLICUM ATCC-953

Guanosine kinase (GKase) activity was identified in a cell-free extrac t of Brevibacterium acetylicum ATCC 953, We have purified the enzyme 4 000-fold from the cell-free extract to apparent homogeneity. Molecular weight of 71 300 and 36 300 determined by gel filtration and sodium d odecyl sulfate gel electrophoresis. respectively, revealed that the en zyme is a dimer molecule. Maximum activity of the GKase was attained w hen the magnesium/ATP concentration ratio was close to 1. The GKase ac tivity was dependent on the presence of a divalent cation. The K-m val ues for guanosine, inosine, and 2'-deoxyguanosine as phosphate accepte rs were determined to be 0.022, 0.87, and 2.83 mM, respectively. In ad dition to ATP and dATP, GTP and dGTP were shown to be effective phosph ate donors for the GK;ise. The optimal pH seemed to be around pH 8.3, although relatively high activity was observed in the alkaline pH rang e of 7.5-9.8. The addition of 0.1 mM pyrimidine nucleotides, especiall y CMP and CTP, activated the GKase activity. On the other hand, the ad dition of I mM AMP, ADP, and GMP inhibited the GKase activity. It is t hus likely that the GKase activity might be regulated in viva by nucle otide concentrations and may control the nucleoside monophosphate leve l by efficiently recycling guanosine. (C) 1997 Elsevier Science B.V.