Phosphodiesterase I in human urine: purification and characterization of the enzyme.
Phosphodiesterase I [EC 3.1.4.1] was purified from normal human urine in a highly purified state free from phosphodiesterase II, RNase, DNase I, DNase II, and phosphatase by column chromatographies of DEAE-Toyopearl, butyl-Toyopearl, Affi-Gel blue, and Sephadex G-150. The molecular weight of the enzyme was 1.9 x 10(5) and the pH optimum around 9.0 with p-nitrophenyl deoxythymidine 5'-phosphate as the substrate. The enzyme hydrolyzed the 3'-5' linkage of various dinucleoside monophosphates at approximately the same rate and the phosphodiester bonds of cyclic 3',5'-mononucleotides to produce mononucleoside 5'-phosphate. The enzyme also hydrolyzed ADP to 5'-AMP and Pi, ATP to 5'-AMP and PPi, and NAD+ to 5'-AMP and NMN. The enzyme activity was abolished by removal of metal ions with EDTA, and the metal-free enzyme was reactivated on the addition of Zn2+. The enzyme activity was also abolished by some reducing agents and the inhibition was reversed by Zn2+. The metal-free enzyme was less stable than the native enzyme, and Zn2+ and Co2+ restored the stability of the metal-free enzyme to the level of the native enzyme. The enzyme degraded oligonucleotides and high molecular nucleotides stepwise from the 3'-termini to give 5'-mononucleotides. The enzyme hydrolyzed single-stranded DNA more preferentially than double-stranded DNA. The enzyme also nicked superhelical covalently closed circular phi X174 DNA to yield first open circular DNA and then linear DNA.
Ito
K
K
Faculty of Science, Osaka City University.
Yamamoto
T
T
Minamiura
N
N
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006801:Humans; D007202:Indicators and Reagents; D007700:Kinetics; D008970:Molecular Weight; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000302:isolation & purification / Q000652:urine*; D013379:Substrate Specificity; D013816:Thermodynamics
D007202:Indicators and Reagents; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I