Measurement of glycinamide ribonucleotide synthetase activity in intact human fibroblasts and Chinese hamster ovary cells.
An intact cell assay system based on Tween 80 permeabilization was used to investigate glycinamide ribonucleotide (GAR) synthetase activity in human fibroblasts and Chinese hamster ovary cells. Optimal conditions for the assay of the enzyme were determined with regards to ATP, MgCl2, NH4Cl and ribose-5'-phosphate concentrations as well as pH. Using the optimal assay conditions, the Vmax values as determined by Lineweaver-Burke double reciprocal plots were found to be 5.19 nmol GAR formed/5 X 10(5) cells/30 min for the fibroblasts and 13.4 nmol GAR formed/5 X 10(5) cells/30 min for the Chinese hamster ovary cells.
D000255:Adenosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D006224:Cricetinae; D004789:Enzyme Activation / Q000187:drug effects; D005260:Female; D005347:Fibroblasts / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008025:Ligases / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D015636:Magnesium Chloride; D010053:Ovary / Q000201:enzymology*; D012265:Ribonucleotides / Q000096:biosynthesis
D012265:Ribonucleotides; D015636:Magnesium Chloride; C014978:glycineamide ribonucleotide; D000255:Adenosine Triphosphate; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C022387:phosphoribosylamine-glycine ligase; D008274:Magnesium; D005998:Glycine