Differences in activities and substrate specificity of human and murine pyrimidine nucleoside phosphorylases: implications for chemotherapy with 5-fluoropyrimidines.
Enzyme inhibition studies on extracts from human liver, mouse liver, and human placenta indicate that there are considerable differences between human and murine hepatic uridine phosphorylases (UrdPase, EC 2.4.2.3) and thymidine phosphorylases (dThdPase, EC 2.4.2.4) with regard to their specificities and roles in the phosphorolysis of natural and 5-fluoropyrimidine nucleosides. To confirm further these differences between human and murine pyrimidine nucleoside phosphorylases, UrdPase and dThdPase were isolated from human liver, mouse liver, and human placenta using diethylaminoethyl-cellulose ion exchange chromatography. The pattern of elution from the column suggests that the hydrophobicity or charges on the human enzymes at pH 8 are different from those on their murine counterparts. The amount of each enzyme present differed between tissues and species. The apparent Km, Vmax, and efficiency of catalysis (Vmax/Km) values were determined for each enzyme using uridine, thymidine, deoxyuridine, 5-fluorouridine (FUrd), 5-fluoro-2'-deoxyuridine (FdUrd), and 5'-deoxy-5-fluorouridine (5'-dFUrd) as substrates. Kinetic parameters and inhibition studies were used to ascertain the binding affinity, substrate specificity, and contributions of UrdPase and dThdPase to the phosphorolysis of the various nucleosides in the 3 tissues. The roles of UrdPase and dThdPase in human liver were quite distinct from those of their counterparts from human placenta and mouse liver. In human liver, UrdPase appears to be highly specific to uridine. Human hepatic UrdPase contributes only 15% to the cleavage of FUrd and does not contribute to the cleavage of the deoxyribosides (thymidine, deoxyuridine, FdUrd, and 5'-dFUrd). In mouse liver, UrdPase has a broader specificity as it cleaves over 85% of FUrd, 15% of FdUrd, and 25% of 5'-dFUrd. On the other hand, human hepatic dThdPase has a broader specificity than murine hepatic dThdPase. Human hepatic dThdPase cleaves all nucleosides tested including the ribosides, uridine, and FUrd. Approximately 15% of uridine and 85% of FUrd phosphorolysis in human liver is carried out by dThdPase. This contrasts with the murine hepatic dThdPase, which is more specific to deoxyribosides, as it does not contribute to the phosphorolysis of uridine, and contributes only 15% toward the cleavage of FUrd. dThdPase is the principal enzyme responsible for the phosphorolysis of 5'-dFUrd in both human and murine livers. The specificities of UrdPase and dThdPase from human placenta resembled the enzymes from the murine liver more than those from human liver. Thus, it appears that the specificities of human hepatic pyrimidine nucleoside phosphorylases are distinct from those from extrahepatic tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
el Kouni
M H
MH
Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912.
el Kouni
M M
MM
Naguib
F N
FN
D000818:Animals; D005467:Floxuridine / Q000378:metabolism*; D005472:Fluorouracil / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D051379:Mice; D010920:Placenta / Q000378:metabolism; D013045:Species Specificity; D013379:Substrate Specificity; D013939:Thymidine Phosphorylase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014529:Uridine / Q000031:analogs & derivatives* / Q000378:metabolism; D014543:Uridine Phosphorylase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D005467:Floxuridine; C001943:5-fluorouridine; D014543:Uridine Phosphorylase; D013939:Thymidine Phosphorylase; D005472:Fluorouracil; C025034:doxifluridine; D014529:Uridine