A simple sensitive fluorimetric assay of APS-kinase activity.
Adenosine phosphosulphokinase (APS-kinase or ATP:adenylylsulphate 3'-phosphotransferase; EC 2.7.1.25) catalyses the formation of 3'-phosphoadenosine-5'-phosphosulfate (PAPS). Its activity in various tissues was measured by transferring the sulphate from PAPS, a product of APS-kinase reaction, to 4-methylumbelliferone (4-MU) to form 4-MU-sulphate (4-MUS) using phenolsulphotransferase (PST) extracted from rat liver. Desalting with Sephadex G-25, together with the addition of EDTA effectively removed the Mg2+ ions from the rat liver extract and thereby inhibited the APS-kinase activity therein in the subsequent PST reaction. 4-MUS formed was measured indirectly by a decrease in the fluorescence of 4-MU by a continuous fluorimetric assay. Kinetic data showed that the substrate, APS, at concentrations at and above 132 microM inhibited the APS kinase reaction. Pyrophosphate (PP) also inhibited the reaction. The apparent Km for APS was 14 microM. Two apparent Km values of 0.12 mM and 1.06 mM were obtained for ATP, while that for Mg2+ was 0.09 mM.
Wong
K P
KP
Department of Biochemistry, Faculty of Medicine, National University of Singapore, Kent Ridge.
D000818:Animals; D015239:Arylsulfotransferase / Q000378:metabolism; D006923:Hymecromone / Q000378:metabolism; D007421:Intestine, Small / Q000201:enzymology; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D051379:Mice; D010710:Phosphates / Q000494:pharmacology; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D010770:Phosphotransferases / Q000032:analysis* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D051381:Rats; D013050:Spectrometry, Fluorescence; D013431:Sulfates / Q000378:metabolism; D014018:Tissue Distribution
D010710:Phosphates; D013431:Sulfates; D006923:Hymecromone; D010724:Phosphoadenosine Phosphosulfate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C019617:adenylylsulfate kinase; D015239:Arylsulfotransferase