Purification and Characterization of Adenylate Kinase as an Apparent Adenosine Triphosphate-dependent Inhibitor of Ribonuclease II in Escherichia coli

Highly purified preparations of Escherichia coli RNase II are not inhibited by ATP alone. The reported inhibition of RNase II by ATP has been shown to require a soluble protein from E. coli. This putative ATP-dependent inhibitor of RNase II has been purified approximately 400-fold and identified as adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3). A trivial explanation for the apparent inhibition of RNase II by ATP is thus provided. The 5′-mononucleotide products released from RNA by RNase II are routinely detected by their solubility in alcohol. In the presence of ATP and appropriate nucleoside monophosphate kinases, the 5′-mononucleotides are converted to nucleoside diphosphates which are precipitated to a significant extent in alcohol. These observations contradict both the hypothesis that ATP is an important physiological regulator of RNase II activity in E. coli and the assumption that ATP can function as a specific inhibitor of RNase II in routine RNase V assays performed with crude extracts of E. coli. Our data support the suggestion that the putative RNase V activity in crude extracts of E. coli is a manifestation of previously characterized ribonucleases. Purified adenylate kinase from E. coli appears to be a single polypeptide chain with a molecular weight of approximately 27,000. The enzyme can utilize both ribo- and deoxyribonucleotides as substrates. E. coli adenylate kinase is highly specific for AMP and dAMP, but detectable activity is observed when ATP or dATP is replaced by the variety of ribonucleoside triphosphates tested.