Phosphodiesterase I in Human Urine: Purification and Characterization of the Enzyme

Abstract

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 1.9×10⁵ 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 P₁, ATP to 5′-AMP and PP₁, 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 φX174 DNA to yield first open circular DNA and then linear DNA.