Purification of hamster dihydroorotate synthetase using Procion blue-Sepharose.
Dihydroorotate (DHO) synthetase is a trifunctional protein that catalyzes the first three reactions of de novo pyrimidine biosynthesis. A single-step procedure for purification of DHO synthetase from mutant hamster cells that overproduce this protein has been developed. The synthetase is adsorbed from a postmitochondrial supernatant to a column of Procion blue-Sepharose 4B and, after the column is washed, the synthetase is eluted as a single peak with 0.4 M KCl. Pooled fractions from the trailing side of this peak yield DHO synthetase with a specific activity for aspartate transcarbamylase of 14 mumol/min/mg protein, representing a purification factor of 8.5-fold and a recovery of 28% from the postmitochondrial supernatant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the DHO synthetase was of high purity. A further 34% of the DHO synthetase from the leading side of the eluted peak contained a minor proportion of a proteolytic fragment. Similar results were obtained with an established four-step purification procedure.
Crofts
L
L
Department of Biochemistry, University of Sydney, New South Wales, Australia.
Peide
Y
Y
Woodhouse
A
A
Algar
E M
EM
Christopherson
R I
RI
D000818:Animals; D001221:Aspartate Carbamoyltransferase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002460:Cell Line; D002847:Chromatography, Agarose / Q000379:methods*; D004396:Coloring Agents; D006224:Cricetinae; D004080:Dihydroorotase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D009154:Mutation; D014227:Triazines
C033626:CAD trifunctional enzyme; D004396:Coloring Agents; D009097:Multienzyme Complexes; D014227:Triazines; D001221:Aspartate Carbamoyltransferase; D004080:Dihydroorotase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)