Purification and characterization of two different thymidine-5'-triphosphosphate-hydrolysing enzymes in human serum.
Two different enzymes capable of hydrolysing dTTP to the corresponding diphosphate were purified from human serum in order to investigate their enzymatic properties. A specific dTTPase was purified to apparent homogeneity with a purification factor of ca. 10,000 and showed a molecular mass of 46,000 Da, consisting of two identical subunits. This enzyme revealed an isoelectric point of 5.8 and a Km value of 38 microM. The other enzyme showed substrate specificity for dTTP and dCTP and was purified with a factor of ca. 5,000. It seems to be a multifunctional enzyme of one subunit (96,000 Da) with two different catalytic sites for dTTP and dCTP. The isoelectric point was 5.2, the Km values were 20 microM for dTTP and 17 microM for dCTP, respectively. Both enzymes were sensitive to inorganic phosphate, but the dTTPase to a minor extent. In contrast to the dCTPase-dTTPase, the dTTPase was strongly inhibited by ZnSO4. Physico-chemical and biochemical data suggest the purification of two different enzymes.
Schultes
B C
BC
Institut für Klinische Biochemie, Universität Bonn, Germany.
Fischbach
E
E
Dahlmann
N
N
D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000097:blood*; D011087:Polycythemia Vera / Q000201:enzymology; D011755:Pyrophosphatases; D013379:Substrate Specificity; D013431:Sulfates / Q000494:pharmacology; D013942:Thymine Nucleotides / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology; D019287:Zinc Sulfate
D013431:Sulfates; D013942:Thymine Nucleotides; D019287:Zinc Sulfate; D010744:Phosphoric Monoester Hydrolases; D011755:Pyrophosphatases; C020007:dCTP pyrophosphatase; C023117:thymidine-triphosphatase; D015032:Zinc; C024157:thymidine 5'-triphosphate