Adenosine 5′-phosphosulfate sulfotransferase (APSSTase) was purified over 2700-fold to homogeneity from the thalli of the marine macroalga Porphyra yezoensis Ueda (Rhodophyta), using a combination of ammonium sulfate precipitation, hydrophobic chromatography, anion-exchange chromatography and gel-filtration. The native Mr measured by gel-filtration was 350000. The subunit Mr was estimated to be 43 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. In addition, APSSTase had a relatively broad pH optimum of pH 9.0-9.8 with a peak at pH 9.5. The apparent Km value for adenosine 5′-phosphosulfate (APS) was 2.1 μM, when dithiothreitol was acceptor substrate. 3′-Phosphoadenosine 5′-phosphosulfate and inosine 5′-phosphosulfate could not substitute for APS as a sulfate donor. The enzyme utilized several organic thiols as acceptor substrates (artificial substrates): dithiothreitol (apparent K m = 1.5 mM) and dithioerythritol (apparent Km = 1.5 mM) gave the highest activity, and appreciable activity was also obtained using L-glutathione (reduced form) which exhibited slight substrate inhibition (apparent Km = 0.6 mM; the initial velocity was maximal at 3.0-4.0 mM). While APSSTase was markedly unstable in vitro: the half-life for activity loss at 25 °C and pH 9.5 was about 8 min, the instability was decreased in the presence of a relatively high concentration of Na2SO4 or (NH4)2SO4, and in the presence of APS or its analogs (AMP and β-methylene-APS). Most of the thiols, with the sole exception of glutathione, were found to inactivate APSSTase irreversibly. The thiol-mediated inactivation was completely inhibited by the high concentration of Na2SO4, and by the analogs of APS.
- Adenosine 5′-phosphosulfate sulfotransferase
- Sulfate metabolism
- Sulfate reduction
ASJC Scopus subject areas
- Plant Science