Characteristics of a pyrimidine-specific 5'-nucleotidase in human erythrocytes.
A 5'-nucleotidase with unique specificity has been identified in the soluble fraction of normal human erythrocytes. It mediates the hydrolytic dephosphorylation of pyrimidine 5'-ribosemonophosphates but is catalytically ineffective with purine nucleotides or with the 2'-, 3'-, or cyclic isomers of pyrimidine nucleotides. Activities at 37 degrees in dialyzed hemolysates of nromal human erythrocytes averaged 7.3 and 6.2 mumol of Pi liberated per hour per g of hemoglobin for the substrates UMP and CMP, respectively. Activity with TMP as substrate was approximately one-half as much as with UMP or CMP. Apparent Michaelis constants were 0.33 mM UMP, 0.15 mM CMP, and 1.0 mM TMP. Magnesium was required for optimal activity, and this cation could not be replaced by Mn2+. Maximum activity was obtained between pH 7.0 and 7.5 with rapid decreases in more alkaline media and moderate decreases with acidification. The enzyme was quite sensitive to heat and was strongly inhibited by AMP, by some purine bases, and by both purine and pyrimidine nucleosides. Divalent cations of heavy metals were also strongly inhibitory, as were agents active against sulfhydryl groups. The presence of substrates and/or 2-mercaptoethanol provided considerable protection against some of these deleterious agents and conditions. Pyrimidine 5'-nucleotidase activity in hemolysates was clearly distinguishable from erythrocyte acid phosphatase and from leukocyte and serum alkaline phosphatases and nucleotidases.
D002413:Cations, Divalent; D002857:Chromium / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009708:Nucleotidases / Q000097:blood*; D011743:Pyrimidines; D013329:Structure-Activity Relationship; D013696:Temperature
D002413:Cations, Divalent; D011743:Pyrimidines; D002857:Chromium; D009708:Nucleotidases; D008274:Magnesium