Complex U, which contains the last two
enzymes (
orotate phosphoribosyltransferase (EC 2.4.2.10) and
orotidylate decarboxylase (EC 4.1.23)) of the six
enzymes for the de novo biosynthesis of
UMP, has been purified 200-fold from mouse Ehrlich
ascites cells. The specific activity of the
orotate phosphoribosyltransferase and the
orotidylate decarboxylase activities of the complex were 0.115 and 0.290 mumol of product/mg of
protein/min; the recovery of the activities was high being 20 to 30%. The rate of the two activities remained similar to that of the homogenate. At the sixth step of the fractionation, one can obtain a fraction that has lost phosphoribosyltransferase activity but retains
decarboxylase activity. The apparent molecular weights, as determined by density gradient centrifugation, of the native complex and the fraction containing only
decarboxylase activity are identical, 55,700 +/- 4,000. Both activities of complex U are labile to very mild treatments such as dilution, dialysis, or storage at 3 degrees.
Dithiothreitol and 5-phosphoribosyl-1-pyrophosphate (PP-Rib-P), but not
orotic acid or
MgCl2, can stabilize either or both of the
enzyme activities. The degree of stabilization by three of these chemicals varies with the
reagent(s) used, with the nature of the treatment, and with the concentration of Complex U. When PP-Rib-P, Mg2+ and
dithiothreitol are present in the diluting
buffer the activity losses were slowed and then followed by a partial recovery of the phosphoribosyltransferase activity. Maximum activities of both
enzymes are observed by adding undiluted complex to a complete reaction mixture without preincubation. The complex cannot be exposed to pH values of 4 or below, or pH 9 or above. The stability studies have led to the development of conditions that permit one, for the first time, to subject the complex to electrophoresis and to recover a large percentage of both
enzyme activities, rather than only
decarboxylase activity as has occurred in the past. The electrophoretic studies indicate that PP-Rib-P produces a complex whose conformation and/or net charge differ significantly from that of the complex in the absence of PP-Rib-P. Kinetic characteristics of the
transferase are a pH optimum between 6.5 and 7.5, apparent Km values for orotate, PP-Rib-P, and Mg2+ of 1.9 muM, 16 muM, and 2.9 mM, respectively; for the
decarboxylase, a sharp pH optimum of 7.0 is observed, and a Km value for
orotidine 5'-phosphate of 0.8 muM.