Co-purification and characterization of ATP-sulfurylase and adenosine-5'-phosphosulfate kinase from rat chondrosarcoma.
The two sulfate-activating enzymes, ATP-sulfurylase (EC 2.7.7.4) and adenosine-5'-phosphosulfate kinase (adenylylsulfate kinase, EC 2.7.1.25), were each purified about 2000-fold from crude rat chondrosarcoma homogenate. Throughout a purification protocol which included Sephacryl S-300 gel filtration, DEAE-Sephadex ion exchange, hydroxylapatite, and ATP-agarose affinity chromatography, these two activities consistently co-purified. ATP-sulfurylase and adenosine-5'-phosphosulfate kinase each showed a pH optima of 7.0-7.4 and a bimodal temperature optima of 46 and 52-54 degrees C. Both activities preferred Mg2+ as their divalent cation source over Mn2+, Co2+, or Zn2+. The apparent Km values determined for adenosine 5'-phosphosulfate in both assays was 1-5 microM; the Km for pyrophosphate in the sulfurylase reaction was 40 microM and for ATP in the kinase reaction was 5 mM. Gel electrophoresis indicated major bands at Mr = 160,000 in nondenaturing systems and 35,000-37,000 and 60,000 under dissociative conditions, whereas gel filtration of the most highly purified fractions yielded a coincident peak in the molecular weight range 260,000.
D000818:Animals; D002413:Cations, Divalent; D002813:Chondrosarcoma / Q000201:enzymology*; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009713:Nucleotidyltransferases / Q000378:metabolism*; D010770:Phosphotransferases / Q000302:isolation & purification / Q000378:metabolism*; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D013430:Sulfate Adenylyltransferase / Q000302:isolation & purification / Q000378:metabolism*; D013696:Temperature
D002413:Cations, Divalent; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D009713:Nucleotidyltransferases; D013430:Sulfate Adenylyltransferase; D008274:Magnesium