A
pyrimidine phosphoribosyltransferase, previously shown to utilize
5-fluorouracil and possibly also
uracil and orotate (Reyes, P. (1969) Biochemistry 8, 2057-2062), has been purified about 100-fold from murine
leukemia P1534J. Roughly 20% of the original activity was recovered to yield an
enzyme preparation with a specific activity of 7.4 mumol of
5-fluorouracil utilized/hour/mg of
protein. Disc gel electrophoresis of this preparation revealed the presence of a major band of
protein accompanied by several trace contaminants. Emphasis was placed on a study of the substrate specificity of this
enzyme.
5-Fluorouracil,
uracil, and
orotate phosphoribosyltransferase activities purified in parallel during fractionation with
ammonium sulfate and
protamine sulfate and eluted together from columns of
Sephadex tG-150 and
DEAE-cellulose. The three phosphoribosyltransferase activities eluted from the
Sephadex columns with an apparent molecular weight of 55,000 to 60,000. In spite of this coordinate fractionation, preferential losses of orotate activity were experienced during
DEAE-cellulose chromatography. Orotate activity continued to behave in a unique manner under other conditions, such as during proteolytic digestion. In the latter case, however, all three activities responded in parallel when digestion took place in the presence of 5mM
UMP. The following results provided additional evidence to support the view that all three phosphoribosyltransferase activities may be catalyzed by the same
enzyme: (a) the apparent Km for 5-phosphoribosyl 1-pyrophosphate (PP-
ribose-P) did not change significantly when
enzyme activity was measured with either
5-fluorouracil,
uracil, or orotate; (b)
5-fluorouracil and
uracil were found to be mutually competitive inhibitors; the effect of
5-fluorouracil on orotate activity was likewise competitive in nature; (c) in the absence of
UMP, orotate was a noncompetitive inhibitor of
5-fluorouracil and
uracil activities, but in the presence of 5mM
UMP it became a competitive inhibitor of both of these activities; (d)
5-fluorouracil and orotate activities co-sedimented in 5 to 20%
sucrose gradients (
uracil activity was not examined); and (e) a wide variety of normal mouse tissues displayed virtually the same
5-fluorouracil to
uracil to orotate activity ratio as found in P1534J
enzyme preparations. The apparent Km and Ki values reported in this study indicate that the preferred
pyrimidine substrate is orotate. It seems likely, therefore, that this
enzyme functions in vivo as an
orotate phosphoribosyltransferase.
Orotate phosphoribosyltransferase and
orotidine 5'-monophosphate (
OMP) decarboxylase activities (a) eluted together during gel filtration on
Sephadex G-150, (b) co-sedimented in 5 to 20%
sucrose gradients, (c) remained associated during fractionation with
ammonium sulfate and
protamine sulfate, and (d) separated into a phosphoribosyltransferase and
decarboxylase component when
enzyme preparations previously subjected to limited proteolysis by
elastase were sedimented in
sucrose gradients...