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A nucleotidase present in rat liver cytosol which hydrolyzes certain 3′- and 5′-nucleotides, is subject to allesteric activation by dIno, dGuo, dThd, dUrd, and the corresponding 5′-deoxyribonuelcotides. A systematic study of enzyme activation as a function of effector concentration was carried out with Urd-3′-P or [14C]dThd-3′-P as substrate. It was found that optimal effector concentration was 6—8 mM for the nucleosides, 4 nm for dThd-5′-P, and 1—2 mM for the other 5′-deoxyribonucleotides. Deoxyinosine was a stronger activator than any of the other effectors and afforded a 3.3-fold increase in the rate of Urd-3′-P hydrolysis at optimal concentration. At 1 mM concentration the enzyme was stimulated 2.3 times by dIno and, furthermore, by dGuo dGuo-5′-P, dThd-5′-P, dUrd-5′-P, and dIno-5′-P in decreasing order of effectiveness. The di- and triphosphates of dThd were more stimulatory than the monophosphate at concentrations above 3 mM. The p-nitrophenyl ester of dThd-5′-P showed stimulation similar to that of the unesterified compound. A kinetic enzyme model was developed which explains all the observed relations between enzyme activity and effector concentration. The parameters of the model were calculated by means of a programmed electronic computer and showed that the apparent dissociation constants for binding of dIno and the 5′-monophasphates of dIno, dGuo, and dUrd to the regulatory site were about 1.7 mM. The other effectors had higher dissociation constants. The apparent Km values of the 5′-deoxyribonucleotides, calculated by means of the computer, were about 0.2 mM. This value is similar to the Km values of the 3′-nucleotides. The stimulation of enzyme activity is due to an increase in F without a detectable change in the apparent Km, a mechanism which is less common for regulatory enzymes. [ABSTRACT FROM AUTHOR]

The activity of a highly purified nucleotidase from rat liver cytosol which splits certain 3'- anti 5'-nucleotides, Was studied in the presence of each of 36 (different nucleic acid nucleic acid constituents including14C, labeled and chemically modified nucleotides. It was found that the compounds either inhibited or had no effect, on dephosphorylation of thymidine 5'-phosphate, which was used as substrate for measuring 5'nucleotidase activity. On the other hand , the 3'-nucleotidase activity, which was measured with uridine 3'-phosphate as substrate, was stimulated 2.6 times by deoxy- guanosine and, furthermore, by dexyguanosine and, furthermore, by deoxyinosine, thymidine, deoxyuridine and inosine in decreasing order of effectiveness. Experiments with various phosphorylated derivatives of thymidine indicated that a 5'-phosphoryl group increase the stimulating effect of the nucleoside whereas a 3'-phosphoryl substitution reduces its ability to activate the enzyme. Di-and triphosphates were less stimulatory than the mono phosphate. The results are interpreted to indicate that the same catalytic site is responsible for the hydrolysis of the 3'-and 5'-nucleotides and that the enzyme possesses a regularly site, topographically different from the catalytic site, at which the deoxyribonucleic acid constituents act as stimulators of enzyme activity. [ABSTRACT FROM AUTHOR]