Lin Hsi-Pin, Cho Yuko, Yashiro Hitoshi, Yamada Tomoko, Oshima Yasukatsu
Laboratory of Bioorganic Chemistry, Graduate School of Life Sciences, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan.
Toxicon. 2004 Nov;44(6):657-68. doi: 10.1016/j.toxicon.2004.07.024.
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.
从中国蛤蜊的消化腺中纯化出一种氨甲酰酶，该酶催化麻痹性贝类毒素的氨甲酰基（或N - 磺基氨甲酰基）部分的水解。通过五步柱色谱法，获得了290微克在SDS - PAGE上显示均一性的氨甲酰酶I。氨甲酰酶I被证明是一种糖蛋白，估计分子量为190 kDa。在还原条件下，SDS - PAGE上观察到相当于94 kDa的单条带，表明它是同型二聚体。最佳温度和pH分别为20℃和7.0。氨甲酰酶I不需要二价阳离子，其活性受到丝氨酸蛋白酶抑制剂苯磺酰氟和4 - （2 - 氨基乙基） - 苯磺酰氟的抑制。氨甲酰酶I水解氨基甲酸酯和N - 磺基氨基甲酸酯毒素。底物毒素N - 1位羟基部分的存在与否对反应速率没有显著影响，但C - 11位硫酸酯的立体化学对其有很大影响。以石房蛤毒素为底物时，K(m)为3.02 microM。通过埃德曼方法鉴定了N端序列的19个氨基酸。(18)O标记的胰蛋白酶肽段的MALDI - TOF - MS/MS光谱表明了五个肽段可能的内部氨基酸序列。
