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, a nd human placenta indicate that there are considerable differences bet ween 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 b etween human and murine pyrimidine nucleoside phosphorylases, UrdPase and dThdPase were isolated from human liver, mouse liver, and human pl acenta using diethylaminoethyl-cellulose ion exchange chromatography. The pattern of elution from the column suggests that the hydrophobicit y 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 K(m), V(max), and efficiency of catalysis (V(max)/K(m)) values were determined for each enzyme usi ng uridine, thymidine, deoxyuridine, 5-fluorouridine (FUrd), 5-fluoro- 2'-deoxyuridine (FdUrd), and 5'-deoxy-5-fluorouridine (5'-dFUrd) as su bstrates. Kinetic parameters and inhibition studies were used to ascer tain 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 we re quite distinct from those of their counterparts from human placenta and mouse liver. In human liver, UrdPase appears to be highly specifi c to uridine. Human hepatic UrdPase contributes only 15% to the cleava ge of FUrd and does not contribute to the cleavage of the deoxyribosid es (thymidine, deoxyuridine, FdUrd, and 5'-dFUrd). In mouse liver, Urd Pase 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, uridi ne, and FUrd. Approximately 15% of uridine and 85% of FUrd phosphoroly sis 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 contribu tes only 15% toward the cleavage of FUrd. dThdPase is the principal en zyme responsible for the phosphorolysis of 5'-dFUrd in both human and murine livers. The specificities of UrdPase and dThdPase from human pl acenta resembled the enzymes from the murine liver more than those fro m human liver. Thus, it appears that the specificities of human hepati c pyrimidine nucleoside phosphorylases are distinct from those from ex trahepatic tissues. This suggests the existence of tissue-specific iso zymes of pyrimidine nucleoside phosphorylases in humans. The inter- an d intraspecies differences in substrate specificities and activities b etween human and murine pyrimidine nucleoside phosphorylases may have an important impact on the validity of attempts to introduce inhibitor s of these enzymes into the clinic or on drawing conclusions about the metabolism and the chemotherapeutic use of pyrimidine analogues in hu mans based on studies in mice.