DIFFERENCES IN ACTIVITIES AND SUBSTRATE-SPECIFICITY OF HUMAN AND MURINE PYRIMIDINE NUCLEOSIDE PHOSPHORYLASES - IMPLICATIONS FOR CHEMOTHERAPY WITH 5-FLUOROPYRIMIDINES
Mh. Elkouni et al., DIFFERENCES IN ACTIVITIES AND SUBSTRATE-SPECIFICITY OF HUMAN AND MURINE PYRIMIDINE NUCLEOSIDE PHOSPHORYLASES - IMPLICATIONS FOR CHEMOTHERAPY WITH 5-FLUOROPYRIMIDINES, Cancer research, 53(16), 1993, pp. 3687-3693
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.