Radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) content of adult rat liver.
1. Rabbit antiserum was raised against purified carbamoyl-phosphate synthase (ammonia) from rat liver. 2. The antiserum proved to be specific in double-diffusion test and reacted in an in situ immunohistochemical test on rat liver proteins fractionated on a sodium dodecyl sulphate polyacrylamide gel only in the region where carbamoyl-phosphate synthase (ammonia) migrated. 3. This antiserum was used for setting up a radioimmunochemical determination of carbamoyl-phosphate synthase (ammonia) in cetyltrimethylammonium bromide extracts of rat liver. To obtain reproducible results in this assay it was necessary to treat the unlabelled ligand with sodium dodecyl sulphate and dithiothreitol. This treatment led to a large increase in the percentage of labelled ligand displaceable by added unlabelled ligand. 4. Radioimmunochemical determination showed that adult rat liver (3-month old) contains 5.5 mg carbamoyl-phosphate synthase (ammonia) protein per gram wet weight.
D000818:Animals; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000032:analysis* / Q000302:isolation & purification; D004229:Dithiothreitol; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005779:Immunodiffusion; D008099:Liver / Q000201:enzymology*; D010770:Phosphotransferases / Q000032:analysis*; D011863:Radioimmunoassay / Q000379:methods; D051381:Rats; D012967:Sodium Dodecyl Sulfate
D000939:Epitopes; D012967:Sodium Dodecyl Sulfate; D010770:Phosphotransferases; D002222:Carbamoyl-Phosphate Synthase (Ammonia); D004229:Dithiothreitol