Liu B H, Yu F Y, Huang X, Chu F S
Department of Food Microbiology & Toxicology, University of Wisconsin-Madison 53706, USA.
Toxicon. 2000 May;38(5):619-32. doi: 10.1016/s0041-0101(99)00176-2.
Using anti-microcystin-LR monoclonal antibodies, an immunoblotting procedure was developed to monitor the formation of microcystin-protein phosphatase adducts in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 s after the addition of MCYST-LR into the reaction mixture. Reduction of the methyldehydroalanine (Mdha) residue of MCYST-LR with ethanethiol totally abolished the covalent binding of the toxin to PP1, but retained its inhibitory toxicity on PP1. Immunoblotting analyses and enzyme-linked immunosorbant assay showed that between 5 min to 16 h after i.p. injection of single dose (35 microg/kg) of MCYST-LR into mice, approximately 0-27% of the injected toxin was found covalently bound while 0.2-9.2% existed as free form in liver cytosol.
利用抗微囊藻毒素-LR单克隆抗体，开发了一种免疫印迹程序，用于监测微囊藻毒素-蛋白磷酸酶加合物在体外和体内的形成。发现MCYST-LR与重组蛋白磷酸酶1（PP1）和兔肝细胞溶质蛋白共价结合的检测限分别为每次测定0.1 ng和0.3 ng。在向反应混合物中加入MCYST-LR后30秒检测到MCYST-PP1加合物。用乙硫醇还原MCYST-LR的甲基脱氢丙氨酸（Mdha）残基完全消除了毒素与PP1的共价结合，但保留了其对PP1的抑制毒性。免疫印迹分析和酶联免疫吸附测定表明，在给小鼠腹腔注射单剂量（35μg/kg）的MCYST-LR后5分钟至16小时之间，发现约0-27%的注射毒素共价结合，而0.2-9.2%以游离形式存在于肝细胞溶质中。
