Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties
- 1 October 1983
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 156 (1), 198-204
- https://doi.org/10.1128/jb.156.1.198-204.1983
Abstract
Uridine phosphorylase was purified 1370-fold from sonicated extracts of A. laidlawii by (NH4)2SO4 precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography and Sephadex G-200 fractionation. The enzyme MW was .apprx. 65,000 by gel filtration. [U-14C]Ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-14C]inosine, was used in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a Km of 110 .mu.M for uracil and a Km of 17 .mu.M for Rib-1-P. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a Km of 170 .mu.M for uridine and a Km of 120 .mu.M for Pi. Thus, an intracellular flux from uracil to uridine is kinetically feasible. Such flux would be metabolically unproductive, since the low affinity of uridine kinase for uridine (Km = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. Evidently, uridine phosphorylase performs only a cataboic function in A. laidlawii.This publication has 28 references indexed in Scilit:
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