PENTAVALENT IONS DEPENDENCY OF MAMMALIAN ADENOSINE KINASE

The enzyme adenosine kinase (AK) has been purified to homogeneity from Syrian hamster and bovine livers. The purified enzymes from both thes e sources have a Mr of approximately 38 kDa, as determined by gel-filt ration and SDS-polyacrylamide gel electrophoresis. A novel characteris tic of AK observed here is that its catalytic activity shows a nearly complete dependence upon the presence of pentavalent ions such as phos phate (P-i), arsenate or vanadate. Maximal AK activity was observed in the presence of either 2-3 mM P-i, or 5-10 mM arsenate, or 10-20 mM v anadate. A low basal level of AK activity (similar to 1-5% of maximal) observed in the absence of these ions is attributed to P-i contaminat ion in the adenine nucleotides preparations. The presence of P-i had n o effect on the K-m for ATP (0.4 mM), but it markedly increased the af finity of the enzyme for adenosine. The K-m of AK for adenosine in pre sence of 0, 0.1 mM and 2 mM P-i was estimated to be 1.4 mu M, 0.77 mu M and 0.095 mu M, respectively. Free P-i showed no exchange with any o f the reactants during the assay conditions, and its presence had no e ffect on the thermostability of the enzyme. These observations suggest that the pentavalent ions such as phosphate may be playing an importa nt role in the enzyme's catalytic mechanism by facilitating either bin ding of adenosine to the enzyme or in the formation of an enzyme-ATP-a denosine complex.