NUCLEOTIDASE ACTIVITIES IN SOLUBLE FRACTION OF RAT LIVER HOMOGENATE - PARTIAL PURIFICATION AND PROPERTIES OF A 5'-NUCLEOTIDASE WITH PH OPTIMUM-6.3

(NH4)2SO4 fractionation of the nonsedimentable fraction of rat liver homogenates indicated that two 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) with pH optima around 6 were present. One of the enzymes had a broader specificity for 5′-nucleotides than the other, which acted mainly on dTMP and dUMP. A procedure is described for purifying the former enzyme to the stage where it is essentially free of nonspecific phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1 and EC 3.1.3.2). The method involves (NH4)2SO4 fractionation and precipitation by dialysis at pH 6.0. Chromatography on DEAE-cellulose and partial heat inactivation indicated that only one 5′-nucleotidase was present in the preparation. The enzyme had a pH optimum at 6.3, it was Mg2+ dependent, and had a lower Km value for IMP (Km = 0.2 mM) than for any of the other nucleotides tested. Evidence is presented indicating that the enzyme is identical with the 5′-nucleotidase isolated recently from rat liver acetone powder by Itoh, Mitsui and Tsushima (J. Biochem. Tokyo, 63 (1968) 165). The enzyme is localized in the soluble cell cytoplasm and has properties different from the 5′-nucleotidases detected previously in particulate structures. © 1969.