The role of choline on the activity-temperature relationship of brush-border alkaline phosphatase.
The role of choline on the activity-temperature relationship of brush-border alkaline phosphatase.
复制标题
胆碱对刷状缘碱性磷酸酶活性-温度关系的作用。
DOI：
10.1016/0005-2736(87)90068-x
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Seetharam,B
中科院分区：
文献类型：
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作者：
Tiruppathi,C;Alpers,DH;Seetharam,B
我们研究了胆碱对来自大鼠肠道（组织特异性类型）以及肾脏和胎盘（组织非特异性类型）的刷状缘碱性磷酸酶同工酶的活性和温度依赖性的影响。用磷脂酶 D 去除胆碱导致所有膜中酶活性的丧失，而阿伦尼乌斯图不连续性的原位丧失发生在肾脏和胎盘膜中，但不在肠膜中。通过添加游离胆碱或磷脂酰胆碱或通过从膜表面去除酶来恢复失去的活性。木瓜蛋白酶去除肠道酶，而枯草杆菌蛋白酶和磷脂酰肌醇特异性磷脂酶 C 释放组织非特异性酶。去除胆碱后，来自肾和胎盘膜的酶聚集 (ρ = 1.13)，添加胆碱导致解聚 (ρ = 1.03)。肾脏和胎盘膜中碱性磷酸酶的不连续线性图转变为连续线性图与膜磷脂酸含量的增加和总磷脂酰胆碱的减少平行。肠酶在所有磷脂酸/磷脂酰胆碱比率下产生具有断点的图。磷脂酶 D 处理带来的变化并不是由于底物半饱和动力学 (Km) 的变化所致。基于这些研究，我们得出结论：（1）组织非特异性磷酸酶的活性位点近似于外膜胆碱，如肠道同工酶的情况[7]； (2) 尽管对磷脂膜含量有类似的影响，但磷脂酶 D 处理对组织非特异性酶产生更大的影响，如阿伦尼乌斯图和密度离心所评估的； （3）这些效应是由于不同的蛋白质结构而不是每个刷状缘膜独特的脂质环境造成的。
We have studied the effect of choline on the activity and temperature dependency of the brush-border alkaline phosphatase isoenzymes from rat intestine (tissue-specific type), and from kidney and placenta (tissue-nonspecific type). The removal of choline with phospholipase D resulted in the loss of enzyme activity in all the membranes, whereas in situ loss in the discontinuity of Arrhenius plots occurred in the kidney and the placental membranes, but not in the intestinal membranes. The lost activity was restored either by addition of free choline or phosphatidylcholine or by the removal of the enzyme from the membrane surface. Intestinal enzyme was removed by papain, while the tissue-nonspecific enzyme was released by subtilisin and by phosphatidylinositol-specific phospholipase C. The enzyme from kidney and placental membranes aggregated (ρ= 1.13) upon removal of choline, and addition of choline resulted in disaggregation (ρ= 1.03). Conversion of discontinuous to continuous linear plots of alkaline phosphatase in the kidney and placental membranes paralleled the increase in membrane phosphatidic acid content, and the decrease in total phosphatidylcholines. The intestinal enzyme produced plots with break points at all phosphatidic acid/phosphatidylcholine ratios. The change brought about by treatment with phospholipidase D was not due to changes in the half-saturation kinetics (Km) for the substrate. Based on these studies we conclude (1) that the active site of the tissue-nonspecific phosphatase is approximated to exterior membrane cholines, as in the case of the intestinal isoenzyme [7]; (2) that despite similar effects on the membrane content of phospholipids, phospholipase D treatment caused much greater effects on the tissue-nonspecific enzyme, as assessed by Arrhenius plots and density centrifugation; (3) that these effects are due to different protein structures rather than to a lipid milieu unique to each brush-border membrane.