Bradford P G, Rubin R P
J Biol Chem. 1986 Nov 25;261(33):15644-7.
myo-Inositol 1,4,5-trisphosphate is an intracellular second messenger generated from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C. In the present study, we have used the abilities of inositol 1,4,5-trisphosphate to inhibit inositol 1,4,5-tris[32P]phosphate binding and to stimulate release of sequestered stores of 45Ca2+ to assay the mass of inositol 1,4,5-trisphosphate in extracts derived from [3H]inositol-prelabeled chemoattractant-stimulated neutrophils. These assays are specific for inositol 1,4,5-trisphosphate since the relative capacity of the extracts to compete with inositol 1,4,5-tris[32P]phosphate binding and to release 45Ca2+ correlated well with the [3H]inositol 1,4,5-trisphosphate content of the extract as determined by high pressure liquid chromatography. No correlation of these activities was observed with the content in the extract of either [3H]inositol 1,3,4-trisphosphate or [3H]inositol 1,3,4,5-tetrakisphosphate, whose formation exhibited kinetics distinct from [3H]inositol 1,4,5-trisphosphate. Thus, within 10 s of stimulation with 10 nM formyl-methionyl-leucyl-phenylalanine, the inositol 1,4,5-trisphosphate content of the extract increased from 0.05 to 0.55 pmol/10(6) cells, equivalent to a change in intracellular concentration from 100 nM to 1.1 microM. These studies demonstrate that neutrophils produce sufficient quantities of inositol 1,4,5-trisphosphate to mobilize Ca2+ from intracellular stores.
肌醇1,4,5 -三磷酸是一种细胞内第二信使，由磷脂酶C水解磷脂酰肌醇4,5 -二磷酸产生。在本研究中，我们利用肌醇1,4,5 -三磷酸抑制肌醇1,4,5 -三[32P]磷酸结合以及刺激释放螯合的45Ca2+储存的能力，来测定从[3H]肌醇预标记的趋化因子刺激的中性粒细胞提取物中肌醇1,4,5 -三磷酸的量。这些测定对肌醇1,4,5 -三磷酸具有特异性，因为提取物与肌醇1,4,5 -三[32P]磷酸结合以及释放45Ca2+的相对能力，与通过高压液相色谱法测定的提取物中[3H]肌醇1,4,5 -三磷酸含量密切相关。未观察到这些活性与提取物中[3H]肌醇1,3,4 -三磷酸或[3H]肌醇1,3,4,5 -四磷酸的含量相关，它们的形成表现出与[3H]肌醇1,4,5 -三磷酸不同的动力学。因此，在用10 nM甲酰甲硫氨酰 - 亮氨酰 - 苯丙氨酸刺激10秒内，提取物中肌醇1,4,5 -三磷酸含量从0.05增加到0.55 pmol/10(6)细胞，相当于细胞内浓度从100 nM变化到1.1 microM。这些研究表明，中性粒细胞产生足够量的肌醇1,4,5 -三磷酸以从细胞内储存中动员Ca2+。
