Purification, properties and substrate specificity of adenosine triphosphate sulphurylase from spinach leaf tissue.
1. ATP sulphurylase was purified up to 1000-fold from spinach leaf tissue. Activity was measured by sulphate-dependent [(32)P]PP(i)-ATP exchange. The enzyme was separated from Mg(2+)-requiring alkaline pyrophosphatase (which interferes with the PP(i)-ATP-exchange assay) and from other PP(i)-ATP-exchange activities. No ADP sulphurylase activity was detected. 2. Sulphate was the only form of inorganic sulphur that catalysed PP(i)-ATP exchange; K(m) (sulphate) was 3.1mm, K(m) (ATP) was 0.35mm and the pH optimum was 7.5-9.0. The enzyme was insensitive to thiol-group reagents and required either Mg(2+) or Co(2+) for activity. 3. The enzyme catalysed [(32)P]PP(i)-dATP exchange; K(m) (dATP) was 0.84mm and V (dATP) was 30% of V (ATP). Competition between ATP and dATP was demonstrated. 4. Selenate catalysed [(32)P]PP(i)-ATP exchange and competed with sulphate; K(m) (selenate) was 1.0mm and V (selenate) was 30% of V (sulphate). No AMP was formed with selenate as substrate. Molybdate did not catalyse PP(i)-ATP exchange, but AMP was formed. 5. Synthesis of adenosine 5'-[(35)S]sulphatophosphate was demonstrated by coupling purified ATP sulphurylase and Mg(2+)-dependent alkaline pyrophosphatase (also prepared from spinach) with [(35)S]sulphate and ATP as substrates; adenosine 5'-sulphatophosphate was not synthesized in the absence of pyrophosphatase. Some parameters of the coupled system are reported.
D000249:Adenosine Monophosphate; D002845:Chromatography; D005459:Fluorides; D006863:Hydrogen-Ion Concentration; D008274:Magnesium; D008982:Molybdenum; D009713:Nucleotidyltransferases / Q000302:isolation & purification*; D010759:Phosphorus Isotopes; D010944:Plants / Q000201:enzymology*; D011755:Pyrophosphatases; D012643:Selenium; D013431:Sulfates
D010759:Phosphorus Isotopes; D013431:Sulfates; D000249:Adenosine Monophosphate; D008982:Molybdenum; D009713:Nucleotidyltransferases; D011755:Pyrophosphatases; D012643:Selenium; D008274:Magnesium; D005459:Fluorides