caa a22 4500 477100759 CHVBK 20180405111540.0 cr unu---uuuuu 170330e19960901xx s 000 0 eng 10.1007/BF00620061 doi (NATIONALLICENCE)springer-10.1007/BF00620061 Adenosine 5′-phosphosulfate sulfotransferase from the marine macroalga Porphyra yezoensis Ueda (Rhodophyta): stabilization, purification, and properties [Elektronische Daten] [Nobuhiro Kanno, Eizou Nagahisa, Minoru Sato, Yoshikazu Sato] 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. Springer-Verlag, 1996 Adenosine 5′-phosphosulfate sulfotransferase nationallicence Porphyra nationallicence Sulfate metabolism nationallicence Sulfate reduction nationallicence APS : adenosine 5′-phosphosulfate nationallicence APSSTase : adenosine 5′-phosphosulfate sulfotransferase nationallicence β-m-APS : β-methylene-adenosine 5′-phosphosulfate nationallicence DTT : dithiothreitol nationallicence IPS : inosine 5′-phosphosulfate nationallicence PAPS : 3′-phosphoadenosine 5′-phosphosulfate nationallicence Kanno Nobuhiro School of Fisheries Sciences, Kitasato University, Sanriku, 022-01, Iwate, Japan aut Nagahisa Eizou School of Fisheries Sciences, Kitasato University, Sanriku, 022-01, Iwate, Japan aut Sato Minoru Tohoku University, Sendai, 981, Miyagi, Japan aut Sato Yoshikazu School of Fisheries Sciences, Kitasato University, Sanriku, 022-01, Iwate, Japan aut Planta Springer-Verlag 198/3(1996-09-01), 440-446 0032-0935 198:3<440 1996 198 425 https://doi.org/10.1007/BF00620061 text/html Onlinezugriff via DOI 1 research-article jats NATIONALLICENCE 856 40 https://doi.org/10.1007/BF00620061 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Kanno Nobuhiro School of Fisheries Sciences, Kitasato University, Sanriku, 022-01, Iwate, Japan aut NATIONALLICENCE 700 1- Nagahisa Eizou School of Fisheries Sciences, Kitasato University, Sanriku, 022-01, Iwate, Japan aut NATIONALLICENCE 700 1- Sato Minoru Tohoku University, Sendai, 981, Miyagi, Japan aut NATIONALLICENCE 700 1- Sato Yoshikazu School of Fisheries Sciences, Kitasato University, Sanriku, 022-01, Iwate, Japan aut NATIONALLICENCE 773 0- Planta Springer-Verlag 198/3(1996-09-01), 440-446 0032-0935 198:3<440 1996 198 425 Metadata rights reserved Springer special CC-BY-NC licence nationallicence BK010053 XK010053 XK010000 NATIONALLICENCE NATIONALLICENCE NL-springer