Abstract
Adenosine 5′-phosphosulfate sulfotransferase (APSSTase) was purified over 2700-fold to homogeneity from the thalli of the marine macroalgaPorphyra 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 350 000. 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 apparentK m 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 (apparentK m = 1.5 mM) and dithioerythritol (apparentK m = 1.5 mM) gave the highest activity, and appreciable activity was also obtained usingl-glutathione (reduced form) which exhibited slight substrate inhibition (apparentK m = 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.
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Abbreviations
- APS:
-
adenosine 5′-phosphosulfate
- APSSTase:
-
adenosine 5′-phosphosulfate sulfotransferase
- β-m-APS:
-
β-methylene-adenosine 5′-phosphosulfate
- DTT:
-
dithiothreitol
- IPS:
-
inosine 5′-phosphosulfate
- PAPS:
-
3′-phosphoadenosine 5′-phosphosulfate
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We wish to thank Mr. I. Kashiwase, Mr. Y. Endo and Mr. Y. Mimura, School of Fisheries Sciences, Kitasato University, for their technical assistance in this study. The research described in this paper was partly supported by the Kitasato Research Grant (H5-9 and H6-13 to N.K.).
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Kanno, N., Nagahisa, E., Sato, M. et al. Adenosine 5′-phosphosulfate sulfotransferase from the marine macroalgaPorphyra yezoensis Ueda (Rhodophyta): stabilization, purification, and properties. Planta 198, 440–446 (1996). https://doi.org/10.1007/BF00620061
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DOI: https://doi.org/10.1007/BF00620061