Purification and characterization of adenosine nucleosidase from barley leaves.

Adenosine nucleosidase (adenosine ribohydrolase, EC 3.2.2.7) has been purified to a nearly homogeneous state from barley leaves. The enzyme is soluble in concentrated salt solution while it aggregates and precipitates at low ionic strength, factors which enabled a simple purification procedure to be carried out. A molecular weight of 66 000 +/- 3000 was estimated for the native enzyme by gel filtration. In sodium dodecyl sulphate polyacrylamide gel electrophoresis of the most purified fraction a single major band of polypeptide chains, with molecular weight of 33 000, was observed. Thus, the native enzyme seems to be dimer of alpha2 type. The pH optima are 4.7 and 5.4 for citrate and (N-morpholino)ethanesulphonic acid buffers, respectively. Adenine and adenosine protect the enzyme against heat inactivation. The enzyme is resistant to -SH reagents, dithiothreitol inhibits it. The Km for adenosine varied from 0.8 to 2.3 micronM depending on temperature and buffer system. The Km for deoxyadenosine was 120 micronM. Besides adenosine, of several nucleosides tested only adenosine N1-oxide, deoxyadenosine and purine riboside acted as substrates. Adenine as well as its derivatives, including plant hormones (cytokinins), have an inhibitory effect on the enzyme. The Ki values of some modified nucleosides and free bases were determined. The physiological role of adenosine nucleosidase in plants is discussed.