EFFECTS OF OKADAIC ACID ON ISOMETRIC TENSION AND MYOSIN PHOSPHORYLATION OF CHEMICALLY SKINNED GUINEA-PIG TAENIA-COLI
EFFECTS OF OKADAIC ACID ON ISOMETRIC TENSION AND MYOSIN PHOSPHORYLATION OF CHEMICALLY SKINNED GUINEA-PIG TAENIA-COLI
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DOI：
10.1113/jphysiol.1988.sp017030
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发表时间：
1988-04-01
影响因子：
5.5
通讯作者：
TAKAI, A
中科院分区：
文献类型：
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作者：
BIALOJAN, C;RUEGG, JC;TAKAI, A
1. 在Triton X-100剥皮豚鼠大肠带绦虫中，海洋海绵毒素冈田酸(OA; 0.1-10 .mu。M)在低浓度（0.1-1 .mu）存在时产生剂量依赖性的等长张力增强。M)的Ca2+。2. 在0.2 μ m -钙调素存在下，带皮绦虫Ca2+-张力关系表现出高度的协同性（Hill系数，h = 5）。获得半最大张力（ED50）所需的Ca2+浓度为1.8 μ m。5 .亩。M)降低了协同性（h = 2.3），增加了Ca2+敏感性（ED50 = 0.92 .mu.M-Ca2+）。OA进一步增加了30 .mu产生的张力。M-Ca2+，而在无ca2溶液中不能产生任何机械效应。当钙调素浓度增加时，Ca2+敏感性也增加，但在OA存在和不存在的情况下，协同性不受影响。3. 二维凝胶电泳分析肌球蛋白磷酸化水平。OA使磷酸化轻链增加，同时使未磷酸化轻链减少。当OA被洗掉时，效果完全相反。4. 溶液中含有超过1。M-Ca2+，第三个蛋白带出现在光链带旁边的凝胶上。OA显著增加了第三条带，同时去除OA和Ca2+后，第三条带消失。5. 在30 μ m -Ca2+激活后，OA可逆地减缓了Ca2+去除引起的松弛和去磷酸化。大于0.1 μ m - oa时未发生完全松弛。使弛豫率降低50%所需的OA浓度为78 nM。6. OA （1 ~ 10 .mu）对带绦虫提取物的磷酸酶活性有抑制作用。M)剂量依赖性。这种抑制是一种混合的非竞争性抑制，并且随着底物（磷酸化轻链）浓度的增加，剂量-抑制关系向右偏移。改变底物浓度产生的ID50变化的下限和上限分别为10和165 nM-OA。7. 这些结果强烈提示，OA对肌球蛋白磷酸酶活性的抑制与张力增强和松弛减慢均有因果关系。
1. In guinea-pig taenia coli skinned with Triton X-100, the marine sponge toxin okadaic acid (OA; 0.1-10 .mu.M) produced a dose-dependent enhancement of isometric tension in the presence of low concentrations (0.1-1 .mu.M) of Ca2+. 2. The Ca2+-tension relation of the skinned taenia showed a high co-operativity (Hill coefficient, h = 5) in the presence of 0.2 .mu.M-calmodulin. The concentration of Ca2+ required to obtain half-maximal tension (ED50) was 1.8 .mu.M. OA (5 .mu.M) reduced the co-operativity (h = 2.3) and increased the Ca2+ sensitivity (ED50 = 0.92 .mu.M-Ca2+). OA further increased the tension produced with 30 .mu.M-Ca2+, while it failed to produce any mechanical effect in Ca2-free solution. When the calmodulin concentration was increased the Ca2+ sensitivity increased as well, but the co-operativity was not affected both in the absence and in the presence of OA. 3. The level of myosin phosphorylation was analysed by two-dimensional gel electrophoresis. OA produced an increase in phosphorylated light chains and a concomitant decrease in unphosphorylated light chains. The effect was completely reversed when OA was washed out. 4. In solutions containing more than 1 .mu.M-Ca2+, a third protein band appeared on the gels next to the bands of light chains. OA markedly increased the third band which disappeared when OA and Ca2+ were simultaneously removed. 5. OA reversibly slowed down both relaxation and dephosphorylation induced by Ca2+ removal following activation with 30 .mu.M-Ca2+. Complete relaxation did not occur in the present of more than 1 .mu.M-OA. The concentration of OA required to produce a 50% reduction (ID50) of the relaxation rate was 78 nM. 6. The phosphatase activity in the taenia extract was inhibited by OA (1-10 .mu.M) in a dose-dependent manner. The inhibition was well described as a mixed non-competitive inhibition, and the dose-inhibition relation was shifted to the right when the concentration of substrate (phosphorylated light chains) was increased. The lower and upper limits of the change of ID50 produced by changing the substrate concentration were estimated to be 10 and 165 nM-OA, respectively. 7. These results strongly suggest that the tension enhancement and the slow-down of relaxation are both causally related to inhibition of myosin phosphatase activity by OA.