ADENOSINE 5'-PHOSPHOSULFATE SULFOTRANSFERASE FROM THE MARINE MACROALGA PORPHYRA-YEZOENSIS UEDA (RHODOPHYTA) - STABILIZATION, PURIFICATION, AND PROPERTIES

Adenosine 5'-phosphosulfate sulfotransferase (APSSTase) was purified o ver 2700-fold to homogeneity from the thalli of the marine macroalga P orphyra yezoensis Ueda (Rhodophyta), using a combination of ammonium s ulfate precipitation, hydrophobic chromatography, anion-exchange chrom atography and gel-filtration. The native M(r) measured by gel-filtrati on was 350 000. The subunit M(r) was estimated to be 43 000 by polyacr ylamide 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 K-m value for adenosine 5'-phosph osulfate (APS) was 2.1 mu M, when dithiothreitol was acceptor substrat e. 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 K-m = 1.5 mM) gave the highest activity, and appreciable activity was also obtained using L-glutathione (reduced form) which exhibited slig ht substrate inhibition (apparent K-m = 0.6 mM; the initial velocity w as maximal at 3.0-4.0 mM). While APSSTase was markedly unstable in vit ro: the half-life for activity loss at 25 degrees C and pH 9.5 was abo ut 8 min, the instability was decreased in the presence of a relativel y high concentration of Na2SO4 or (NH4)(2)SO4, and in the presence of APS or its analogs (AMP and beta-methylene-APS). Most of the thiols, w ith the sole exception of glutathione, were found to inactivate APSSTa se irreversibly. The thiol-mediated inactivation was completely inhibi ted by the high concentration of Na2SO4, and by the analogs of APS.