Purification and characterization of paralytic shellfish toxin transforming enzyme from Mactra chinensis.
A carbamoylase, which catalyzes hydrolysis of the carbamoyl (or N-sulfocarbamoyl) moiety of paralytic shellfish toxins, was purified from the digestive glands of the Japanese clam Mactra chinensis. Using five steps of column chromatography, 290 microg of Carbamoylase I showing homogeneity on SDS-PAGE was obtained. Carbamoylase I was revealed to be a glycoprotein, having estimated molecular weight of 190 kDa. Observation of single band equivalent to 94 kDa on SDS-PAGE under reducing conditions suggested it to be a homodimer. The optimal temperature and pH were 20 degrees C and 7.0. Carbamoylase I did not require a divalent cation and its activity was inhibited by the serine proteinase inhibitors, benzenesulfonyl fluoride and 4-(2-aminoethyl)-benzenesulfonyl fluoride. Carbamoylase I hydrolyzed both carbamate and N-sulfocarbamate toxins. The presence or absence of a hydroxyl moiety at the N-1 position of the substrate toxins did not significantly alter the reaction rate, but the stereochemistry of sulfate esters at C-11 greatly affected it. The K(m) was 3.02 microM for saxitoxin as a substrate. Nineteen amino acids of the N-terminal sequence were identified by the Edman method. MALDI-TOF-MS/MS spectra of (18)O-labeled tryptic peptides indicated the possible internal amino acid sequences of five peptides.
Lin
Hsi-Pin
HP
Laboratory of Bioorganic Chemistry, Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
Cho
Yuko
Y
Yashiro
Hitoshi
H
Yamada
Tomoko
T
Oshima
Yasukatsu
Y
D000595:Amino Acid Sequence; D000818:Animals; D049872:Bivalvia / Q000201:enzymology* / Q000378:metabolism; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007564:Japan; D008387:Marine Toxins / Q000737:chemistry*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds; D012530:Saxitoxin / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity; D013696:Temperature
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D008387:Marine Toxins; D009943:Organophosphorus Compounds; D012530:Saxitoxin; D002265:Carboxylic Ester Hydrolases; C092629:paralytic shellfish poisoning toxin carbamoylase