Van der Kaay J, Draijer R, Van Haastert P J
Department of Biochemistry, University of Groningen, The Netherlands.
Proc Natl Acad Sci U S A. 1990 Dec;87(23):9197-201. doi: 10.1073/pnas.87.23.9197.
Dictyostelium discoideum cells that overexpress a ras gene with a Gly12----Thr12 mutation (Dd-ras-Thr12) have an altered phenotype. These cells were labeled with [3H]inositol and the incorporation of radioactivity into inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] was analyzed and found to be higher than in control cells. In contrast, the total mass of Ins(1,4,5)P3, as assessed with an assay using a specific Ins(1,4,5)P3-binding protein, was not significantly different between control and Dd-ras-Thr12 cells. Cells were labeled with [3H]inositol and the incorporation of radioactivity in all inositol metabolites was analyzed. Increased levels of radioactivity were observed for phosphatidylinositol phosphate (PtdInsP), phosphatidylinositol bisphosphate (PtdInsP2), Ins(1,4,5)P3, inositol 1,4-bisphosphate, inositol 4,5-bisphosphate, and inositol 4-monophosphate in Dd-ras-Thr12 cells relative to control cells. Decreased levels were found for phosphatidylinositol (PtdIns) and inositol 1-monophosphate. Calculations on the substrate/product relationships [i.e., Ins(1,4,5)P3/PtdInsP2] demonstrate that the observed differences are due only to the increased conversion of PtdIns to PtdInsP; other enzyme reactions, including phospholipase C, are not significantly different between the cell lines. The activity of PtdIns kinase in vitro is not different between Dd-ras-Thr12 and control cells, suggesting that either the regulation of this enzyme is altered or that the translocation of substrate from the endoplasmic reticulum to the kinase in the plasma membrane is modified. The results suggest multiple metabolic compartments of Ins(1,4,5)P3 in Dictyostelium cells. In Dd-ras-Thr12 transformants the increased conversion of PtdIns to PtdInsP leads to increased levels of Ins(1,4,5)P3 in the compartment with a high metabolic turnover. This Ins(1,4,5)P3 compartment is suggested to be involved in the regulation of cytosolic Ca2+ levels.
过表达具有甘氨酸12至苏氨酸12突变的ras基因（Dd-ras-Thr12）的盘基网柄菌细胞具有改变的表型。这些细胞用[3H]肌醇标记，分析放射性掺入肌醇1,4,5-三磷酸[Ins(1,4,5)P3]的情况，发现其高于对照细胞。相比之下，用使用特异性Ins(1,4,5)P3结合蛋白的测定法评估，对照细胞和Dd-ras-Thr12细胞之间Ins(1,4,5)P3的总质量没有显著差异。细胞用[3H]肌醇标记，并分析所有肌醇代谢物中放射性的掺入情况。相对于对照细胞，Dd-ras-Thr12细胞中磷脂酰肌醇磷酸（PtdInsP）、磷脂酰肌醇二磷酸（PtdInsP2）、Ins(1,4,5)P3、肌醇1,4-二磷酸、肌醇4,5-二磷酸和肌醇4-单磷酸的放射性水平增加。磷脂酰肌醇（PtdIns）和肌醇1-单磷酸的水平降低。对底物/产物关系[即Ins(1,4,5)P3/PtdInsP2]的计算表明，观察到的差异仅归因于PtdIns向PtdInsP转化的增加；其他酶反应，包括磷脂酶C，在细胞系之间没有显著差异。Dd-ras-Thr12细胞和对照细胞在体外PtdIns激酶的活性没有差异，这表明要么该酶的调节发生了改变，要么底物从内质网向质膜中激酶的转运发生了改变。结果表明盘基网柄菌细胞中Ins(1,4,5)P3存在多个代谢区室。在Dd-ras-Thr12转化体中，PtdIns向PtdInsP转化的增加导致高代谢周转率区室中Ins(1,4,5)P3水平升高。这个Ins(1,4,5)P3区室被认为参与了胞质Ca2+水平的调节。
