Preparation and high-performance liquid chromatography of 3'-phosphoadenosine-5'-phospho[35S]sulfate with a predetermined specific activity
| Autor: | Delfert Dm, H. E. Conrad |
|---|---|
| Rok vydání: | 1985 |
| Předmět: |
Sulfotransferase
Chromatography Paper Phosphoadenosine Phosphosulfate Biophysics Chick Embryo Sulfur Radioisotopes Biochemistry High-performance liquid chromatography Sulfation Animals Electrophoresis Paper Molecular Biology Chromatography High Pressure Liquid chemistry.chemical_classification Chromatography Chemistry Adenine Nucleotides Cell Biology Oligosaccharide Electrophoresis Enzyme Cartilage Isotope Labeling Specific activity Counts per minute |
| Zdroj: | Analytical biochemistry. 148(2) |
| ISSN: | 0003-2697 |
| Popis: | 3′-Phosphoadenosine-5′-phospho[35S]sulfate (PAP35S) was prepared by incubating ATP and carrier-free H235SO4 with a 100,000g supernatant fraction prepared from chick embryo chondrocytes. The product was partially purified by paper by paper electrophoresis and mixed with unlabeled PAPS to give a solution of PAP35S with a specific activity and a concentration approximating those required for the desired metabolic studies. The product was analyzed by high-performance liquid chromatography on an anion-exchange column to determine the proportion of the 35SO4 cpm and A260 material found in the PAPS and other contaminating nucleotides. The PAP35S was purified further by preparative high-performance liquid chromatography. The exact specific activity of the PAP35S was then determined by using this PAP35S preparation as the SO4 donor in a sulfotransferase reaction using a microsomal preparation from the chick embryo chondrocytes as the enzyme and an 3H-labeled oligosaccharide as the SO4 acceptor. The sulfated oligosaccharide was then isolated and the number of 3H and 35SO4 counts per minute in this product were used to calculate the specific activity of the donor. The features of this generally useful approach for preparing PAP35S of any desired specific activity and concentration are discussed. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|