THE USE OF POTENT INHIBITORS OF ALKALINE-PHOSPHATASE TO INVESTIGATE THE ROLE OF THE ENZYME IN INTESTINAL TRANSPORT OF INORGANIC-PHOSPHATE
THE USE OF POTENT INHIBITORS OF ALKALINE-PHOSPHATASE TO INVESTIGATE THE ROLE OF THE ENZYME IN INTESTINAL TRANSPORT OF INORGANIC-PHOSPHATE
复制标题
DOI：
10.1042/bj1940803
复制
发表时间：
1981-01-01
影响因子：
4.1
通讯作者：
BUTTERWORTH, PJ
中科院分区：
文献类型：
--
作者：
SHIRAZI, SP;BEECHEY, RB;BUTTERWORTH, PJ
为了研究哺乳动物小肠中Pi转运与碱性磷酸酶之间的联系，将大鼠小肠刷状缘膜囊泡摄取Pi的特性与组织碱性磷酸酶的特性进行了比较。NaCl依赖的Pi的吸收在pH 7.5时具有0.1 mM的Km，并且被1 mM-砷酸盐和1 mM-钒酸盐完全抑制。这些化合物也是囊泡碱性磷酸酶活性的有效竞争性抑制剂，在pH 7.5时Ki值< 5 μ M。当对碱性磷酸酶的其他几种有效抑制剂（包括膦酸盐和磷酸盐类似物）对Pi摄取的影响进行测试时，发现在磷酸酶活性完全抑制的条件下，对运输的抑制作用很小（如果有的话）。用氧化腺苷5“-[β]孵育囊泡20分钟。伽马亚氨基]三磷酸，然后快速凝胶过滤以除去抑制剂，导致磷酸酶活性的不可逆损失，但使Pi转运未受损。相反地，与腺苷5“-[β-腺苷]类似的延长孵育也是有效的。硫代]二磷酸或腺苷5“-[γ-硫代三磷酸对碱性磷酸酶活性没有影响，但导致运输能力的永久性部分丧失。由于未能证明碱性磷酸酶的抑制导致了Pi转运的抑制，以及酶活性和Pi转运对不可逆抑制的不同反应，使得该酶不太可能直接参与转运系统。
In an investigation of the link between Pi transport and alkaline phosphatase in mammalian small intestine, the characteristics of Pi uptake by brush-border membrane vesicles prepared from rat intestine were compared with the properties of the tissue alkaline phosphatase. The NaCl-dependent Pi uptake had a Km of 0.1 mM at pH 7.5 and was inhibited totally by 1 mM-arsenate and by 1 mM-vanadate. These compounds are also potent competitive inhibitors of the alkaline phosphatase activity of the vesicles, with Ki values < 5 .mu.M at pH 7.5. When the effect on Pi uptake of several other potent inhibitors of alkaline phosphatase, including phosphonates and phosphate analogs, was tested, it was found that there was little, if any, inhibition of transport under conditions in which the inhibition of phosphatase activity was total. Incubation of the vesicles for 20 min with oxidized adenosine 5''-[.beta..gamma.-imido]triphosphate followed by rapid gel filtration to remove the inhibitor resulted in an irreversible loss of phosphatase activity but left Pi transport unimpaired. Conversely, a similar prolonged incubation with adenosine 5''-[.beta.-thio]diphosphate or adenosine 5''-[.gamma.-thio]triphosphate had no effect on alkaline phosphatase activity but resulted in a permanent partial loss of transport capability. The failure to demonstrate an inhibition of Pi transport resulting from inhibition of alkaline phosphatase and the different responses of enzymic activity and Pi transport to irreversible inhibition make it very unlikely that the enzyme is directly involved in the transport system.