USE OF A COLORIMETRIC PROTEIN PHOSPHATASE INHIBITION ASSAY AND ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE STUDY OF MICROCYSTINS AND NODULARINS
USE OF A COLORIMETRIC PROTEIN PHOSPHATASE INHIBITION ASSAY AND ENZYME-LINKED-IMMUNOSORBENT-ASSAY FOR THE STUDY OF MICROCYSTINS AND NODULARINS
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DOI：
10.1016/0041-0101(94)90308-5
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发表时间：
1994-12-01
期刊：
影响因子：
2.8
通讯作者：
CARMICHAEL, WW
中科院分区：
文献类型：
--
作者：
AN, JS;CARMICHAEL, WW
微囊藻毒素和结核素是由几种蓝藻细菌产生的环肽肝毒素和肿瘤促进剂。制备兔抗微囊藻毒素lr多克隆抗体，检测其与18种微囊藻毒素和结节素变体的交叉反应性。一种疏水氨基酸，3-氨基-9-甲氧基-10-苯基-2,6,8-三甲基-十-4(E),6(E)-二烯酸（Adda），在微囊藻毒素和瘤毒素的C-6双键上具有(E)形式，被发现是这些毒素表达抗体特异性所必需的。微囊藻毒素和结节素谷氨酸中-COOH的修饰没有改变它们的抗原性。比较了这些毒素的抗体交叉反应性及其抑制1型蛋白磷酸酶（PP1）的能力。通过测定毒素对对硝基酚磷酸对PP1活性的抑制作用来检测对PP1的抑制作用。得到了在大肠杆菌中表达的重组PP1。用微孔板仪测定了5种微囊藻毒素和2种结核素对重组PP1对磷酸硝基酚活性的抑制作用。微囊藻毒素- lr对重组PP1活性抑制50%的浓度（IC50）约为0.3 nM，而两种修饰的微囊藻毒素的IC50均显著高于前者。微囊藻毒素- lr和结节素在C-6双键上具有(z)形式的Adda或具有谷氨酸单酯，对PP1没有抑制作用。这三种毒素在小鼠生物实验中也是无毒的。这些结果表明，氨基酸和谷氨酸在这些环肽的毒性中起重要作用，而PPI的抑制作用与毒素的作用机制有关。
Microcystins and nodularins are cyclic peptide hepatotoxins and tumor promoters produced by several genera of cyanobacteria. Using a rabbit anti-microcystin-LR polyclonal antibody preparation, the cross-reactivity with 18 microcystin and nodularin variants was tested. A hydrophobic amino acid, 3-amino-9-methoxy-10-phenyl-2,6,8-trimethyl-deca-4(E),6(E)-dienoic acid (Adda), which has the (E) form at the C-6 double bond in both microcystin and nodularin, was found essential for these toxins to express antibody specificity. Modification of -COOH in glutamic acid of microcystin and nodularin did not alter their antigenicity. Antibody cross-reactivity of these toxins was compared with their ability to inhibit protein phosphatase type 1 (PP1). Detection of PP1 inhibition was done by measuring the inhibition effect of the toxins on p-nitrophenol phosphate activity toward PP1. PP1 was obtained as recombinant PP1 expressed in E. coli. The inhibition effect of five microcystins and two nodularins on recombinant PP1 activity toward p-nitrophenol phosphate was measured in a microwell plate reader. The concentration of microcystin-LR causing 50% inhibition of recombinant PP1 activity (IC50) was about 0.3 nM, while that of two modified microcystins had a significantly higher IC50. Microcystin-LR and nodularin with the (z) form of Adda at the C-6 double bond or having the monoester of glutamic acid did not inhibit PP1. These three toxins were also nontoxic in the mouse bioassay. These results show the importance of Adda and glutamic acid in toxicity of these cyclic peptides and that PPI inhibition is related to the toxins' mechanism of action.