Purification and characterization of 5'-phosphodiesterase from barley rootlets

A phosphodiesterase was purified from barley rootlets by polyethylene glycol fractionation, calcium phosphate gel-cellulose adsorption, Sepharose CL-4B gel filtration, DEAE-Sepharose CL-6B ion exchange and preparative polyacrylamide gel electrophoresis (PAGE). The enzyme was purified by 103.6 folds and 11% of the enzyme activity was recovered. The purified enzyme was apparently homogeneous when examined on PAGE. It had a molecular weight of 100 kD, an optimum pH of 9.5, and a Km of 0.28 mM on the hydrolysis of p-nitrophenyl thymidine 5'-phosphate. SDS-PAGE revealed that the enzyme molecule might be composed of two or three subunits. Reducing agents, CuSO4, EDTA and 5'-nucleotides were inhibitory to the enzyme activity. This enzyme was able to hydrolyze RNA and denatured DNA efficiently, whereas native DNA was not a good substrate.