Hughes P J, Hughes A R, Putney J W, Shears S B
Inositol Lipid Section, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
J Biol Chem. 1989 Nov 25;264(33):19871-8.
High performance liquid chromatography analysis of supernatants from acid-quenched [3H]inositol-labeled parotid acinar cells revealed an inositol pentakisphosphate and three inositol tetrakisphosphates. Two of the latter were identified as the 1,3,4,5 and 1,3,4,6 isomers, whereas the third was probably a mixture of unknown proportions of the 3,4,5,6/1,4,5,6 enantiomeric pair. Methacholine (100 microM) produced a 40-50-fold increase in the levels of inositol trisphosphate (mainly the 1,3,4 isomer) and inositol 1,3,4,5-tetrakisphosphate, but inositol 1,3,4,6-tetrakisphosphate only increased 5-fold. Levels of inositol 3,4,5,6/1,4,5,6-tetrakisphosphate and inositol pentakisphosphate were unaffected by agonist stimulation. Thus, in parotid cells, an agonist-induced increase in both inositol trisphosphate and inositol 1,3,4,6-tetrakisphosphate formation does not result in an increase in the rate of formation of inositol pentakisphosphate. Following the addition of 100 microM atropine to methacholine-stimulated parotid cells, the levels of [3H]inositol 1,3,4,5-tetrakisphosphate fell rapidly, returning to basal levels within 5 min. Inositol trisphosphate was metabolized more slowly and was still elevated 20-fold above basal 5 min after the addition of atropine. Inositol 1,3,4,6-tetrakisphosphate was metabolized much more slowly (t1/2 approximately 15 min). Inositol 1,3,4-trisphosphate metabolism was examined in parotid homogenates as well as in 100,000 x g cytosolic and particulate fractions. Inositol 1,3,4-trisphosphate was both dephosphorylated and phosphorylated. Two inositol tetrakisphosphate products were formed, namely the 1,3,4,6 and 1,3,4,5 isomers. Over 90% of both kinase and phosphatase activities were found in the cytosolic fractions. The ratio of activities of kinase to phosphatase decreased as the levels of inositol 1,3,4-trisphosphate substrate were increased from 1 nM to 10 microM. These data led to the conclusion that the kinetic parameters of the inositol 1,3,4-trisphosphate kinases and phosphatases are such that in stimulated cells, dephosphorylation of inositol 1,3,4-trisphosphate is greatly favored. Inositol 1,3,4-trisphosphate kinase activity was potently inhibited by inositol 3,4,5,6-tetrakisphosphate (IC50 = 0.1-0.2 microM), which leads us to propose that inositol 3,4,5,6-tetrakisphosphate is an endogenous inhibitor of the kinase.
对酸淬灭的[³H]肌醇标记的腮腺腺泡细胞的上清液进行高效液相色谱分析，结果显示存在一种肌醇五磷酸和三种肌醇四磷酸。后两者中的两种被鉴定为1,3,4,5和1,3,4,6异构体，而第三种可能是3,4,5,6/1,4,5,6对映体对中比例未知的混合物。乙酰甲胆碱（100微摩尔）使肌醇三磷酸（主要是1,3,4异构体）和肌醇1,3,4,5 - 四磷酸水平增加了40 - 50倍，但肌醇1,3,4,6 - 四磷酸仅增加了5倍。肌醇3,4,5,6/1,4,5,6 - 四磷酸和肌醇五磷酸水平不受激动剂刺激的影响。因此，在腮腺细胞中，激动剂诱导的肌醇三磷酸和肌醇1,3,4,6 - 四磷酸生成增加并不会导致肌醇五磷酸生成速率增加。在向乙酰甲胆碱刺激的腮腺细胞中加入100微摩尔阿托品后，[³H]肌醇1,3,4,5 - 四磷酸水平迅速下降，在5分钟内恢复到基础水平。肌醇三磷酸代谢较慢，在加入阿托品5分钟后仍比基础水平高20倍。肌醇1,3,4,6 - 四磷酸代谢更慢（半衰期约15分钟）。在腮腺匀浆以及100,000×g的胞质和微粒体组分中研究了肌醇1,3,4 - 三磷酸的代谢。肌醇1,3,4 - 三磷酸既发生了去磷酸化也发生了磷酸化。形成了两种肌醇四磷酸产物，即1,3,4,6和1,3,4,5异构体。超过90%的激酶和磷酸酶活性存在于胞质组分中。随着肌醇1,3,4 - 三磷酸底物水平从1纳摩尔增加到10微摩尔，激酶与磷酸酶的活性比值降低。这些数据得出结论，肌醇1,3,4 - 三磷酸激酶和磷酸酶的动力学参数使得在受刺激的细胞中，肌醇1,3,4 - 三磷酸的去磷酸化极为有利。肌醇1,3,4 - 三磷酸激酶活性受到肌醇3,4,5,6 - 四磷酸的强烈抑制（IC50 = 0.1 - 0.2微摩尔），这使我们提出肌醇3,4,5,6 - 四磷酸是该激酶的内源性抑制剂。
