Tiruppathi C, Alpers D H, Seetharam B
Biochim Biophys Acta. 1987 Apr 23;898(3):283-92. doi: 10.1016/0005-2736(87)90068-x.
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 (rho = 1.13) upon removal of choline, and addition of choline resulted in disaggregation (rho = 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 that the active site of the tissue-nonspecific phosphatase is approximated to exterior membrane cholines, as in the case of the intestinal isoenzyme; 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; that these effects are due to different protein structures rather than to a lipid milieu unique to each brush-border membrane.
我们研究了胆碱对大鼠肠道（组织特异性类型）、肾脏和胎盘（组织非特异性类型）刷状缘碱性磷酸酶同工酶活性及温度依赖性的影响。用磷脂酶D去除胆碱会导致所有膜中的酶活性丧失，而肾脏和胎盘膜中阿累尼乌斯曲线的连续性出现原位丧失，但肠道膜中未出现。通过添加游离胆碱或磷脂酰胆碱，或从膜表面去除酶，可恢复丧失的活性。用木瓜蛋白酶可去除肠道酶，而枯草杆菌蛋白酶和磷脂酰肌醇特异性磷脂酶C可释放组织非特异性酶。去除胆碱后，肾脏和胎盘膜中的酶会聚集（ρ = 1.13），添加胆碱则会导致解聚（ρ = 1.03）。肾脏和胎盘膜中碱性磷酸酶从不连续线性图转变为连续线性图与膜磷脂酸含量的增加以及总磷脂酰胆碱的减少平行。肠道酶在所有磷脂酸/磷脂酰胆碱比例下都会产生有断点的图。用磷脂酶D处理所带来的变化并非由于底物的半饱和动力学（Km）发生改变。基于这些研究，我们得出结论：与肠道同工酶的情况一样，组织非特异性磷酸酶的活性位点靠近膜外胆碱；尽管对膜磷脂含量有相似影响，但通过阿累尼乌斯曲线和密度离心评估，磷脂酶D处理对组织非特异性酶的影响要大得多；这些影响是由于不同的蛋白质结构，而非每种刷状缘膜特有的脂质环境。
