RAT CHONDROSARCOMA ATP SULFURYLASE AND ADENOSINE 5'-PHOSPHOSULFATE KINASE RESIDE ON A SINGLE BIFUNCTIONAL PROTEIN

The sulfate-activation pathway consists of the sequential action of AT P sulfurylase (ATP: sulfate adenylyltransferase, EC 2.7.7.4) and adeno sine 5'-phosphosulfate kinase (ATP:adenylylsulfate 3'-phosphotransfera se, EC 2.7.1.25). Both sulfurylase and kinase from rat chondrosarcoma were copurified through substrate affinity chromatography using stable analogs of APS (adenosine 5'-phosphosulfate) and PAPS (3'-phosphoaden osine 5'-phosphosulfate). A 56-kDa protein, containing both activities , was then purified to apparent homogeneity through reversed-phase chr omatography and observed on denaturing polyacrylamide gels. The molecu lar mass of the native active unit containing both activities in the p urified preparation corresponded to approximately 60 kDa by analytical gel filtration. Coincident binding and elution of ATP sulfurylase and APS kinase by immunoaffinity chromatography, using polyclonal serum g enerated against the 56-kDa protein, also demonstrated that the enzyme contains both activities. Lastly, a single N-terminal amino acid sequ ence was obtained from the 56-kDa band isolated by gel electrophoresis . These results all suggest that ATP sulfurylase and APS kinase from r at chondrosarcoma reside on a single bifunctional protein.