Preparation and high-performance liquid chromatography of 3'-phosphoadenosine-5'-phospho[35S]sulfate with a predetermined specific activity.
3'-Phosphoadenosine-5'-phospho[35S]sulfate (PAP35S) was prepared by incubating ATP and carrier-free H2(35)SO4 with a 100,000g supernatant fraction prepared from chick embryo chondrocytes. The product was partially purified 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.
D000227:Adenine Nucleotides / Q000096:biosynthesis*; D000818:Animals; D002356:Cartilage / Q000378:metabolism; D002642:Chick Embryo; D002851:Chromatography, High Pressure Liquid; D002854:Chromatography, Paper; D004590:Electrophoresis, Paper; D007553:Isotope Labeling / Q000379:methods; D010724:Phosphoadenosine Phosphosulfate / Q000096:biosynthesis* / Q000302:isolation & purification; D013462:Sulfur Radioisotopes
D000227:Adenine Nucleotides; D013462:Sulfur Radioisotopes; D010724:Phosphoadenosine Phosphosulfate