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.
Wang
A Y
AY
Department of Agricultural Chemistry, National Taiwan University, Taipei.
Juang
R H
RH
Chang
C T
CT
Sung
H Y
HY
D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004247:DNA / Q000378:metabolism; D003854:Deoxyribonucleotides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D001467:Hordeum / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D015394:Molecular Structure; D008970:Molecular Weight; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology
D003854:Deoxyribonucleotides; D008670:Metals; D012265:Ribonucleotides; D012313:RNA; D004247:DNA; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I