Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure.
This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (greater than 600 mumol/min/mg protein), and was obtained with 30-36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP.
D000818:Animals; D001853:Bone Marrow / Q000201:enzymology*; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008297:Male; D009703:Nucleoside-Phosphate Kinase / Q000302:isolation & purification*; D010770:Phosphotransferases / Q000302:isolation & purification*; D011479:Protamines; D051381:Rats; D011919:Rats, Inbred Strains; D012685:Sepharose / Q000031:analogs & derivatives
D011479:Protamines; C029483:Blue-Sepharose; D012685:Sepharose; D010770:Phosphotransferases; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase