Irvine R F, Anggård E E, Letcher A J, Downes C P
Biochem J. 1985 Jul 15;229(2):505-11. doi: 10.1042/bj2290505.
A complete separation of myo-inositol 1,4,5-[4,5-(32)P]trisphosphate prepared from human erythrocytes, and myo-[2-3H]inositol 1,3,4-trisphosphate prepared from carbachol-stimulated rat parotid glands [Irvine, Letcher, Lander & Downes (1984) Biochem. J. 223, 237-243], was achieved by anion-exchange high-performance liquid chromatography. This separation technique was then used to study the metabolism of these two isomers of inositol trisphosphate in carbachol-stimulated rat parotid glands. Fragments of glands were pre-labelled with myo-[2-3H]inositol, washed, and then stimulated with carbachol. At 5s after stimulation a clear increase in inositol 1,4,5-trisphosphate was detected, with no significant increase in inositol 1,3,4-trisphosphate. After this initial lag however, inositol 1,3,4-phosphate rose rapidly; by 15s it predominated over inositol 1,4,5-trisphosphate, and continued to rise so that after 15 min it was at 10-20 times the radiolabelling level of the 1,4,5-isomer. In contrast, after the initial rapid rise (maximal within 15s), inositol 1,4,5-trisphosphate levels declined to near control levels after 1 min and then rose again very gradually over the next 15 min. When a muscarinic blocker (atropine) was added after 15 min of carbachol stimulation, inositol 1,4,5-trisphosphate levels dropped to control levels within 2-3 min, whereas inositol 1,3,4-trisphosphate levels took at least 15 min to fall, consistent with the kinetics observed earlier for total parotid inositol trisphosphates [Downes & Wusteman (1983) Biochem. J. 216, 633-640]. Phosphatidylinositol bisphosphate (PtdInsP2) from stimulated and control cells were degraded chemically to inositol trisphosphate to seek evidence for 3H-labelled PtdIns(3,4)P2. No evidence could be obtained that a significant proportion of PtdInsP2 was this isomer; in control tissues it must be less than 5% of the total PtdInsP2 radiolabelled by myo-[2-3H]inositol. These data indicate that, provided that inositol 1,4,5-trisphosphate is studied independently of inositol 1,3,4-trisphosphate, the former shows metabolic characteristics consistent with its proposed role as a second messenger for calcium mobilization. The metabolic profile of inositol 1,3,4-trisphosphate is entirely different, and its function and source remain unclear.
通过阴离子交换高效液相色谱法，成功实现了对从人红细胞制备的肌醇1,4,5-[4,5-(32)P]三磷酸和从卡巴胆碱刺激的大鼠腮腺制备的肌醇[2-3H]1,3,4-三磷酸的完全分离[欧文、莱彻、兰德和唐斯（1984年）《生物化学杂志》223卷，237 - 243页]。然后利用这种分离技术研究了卡巴胆碱刺激的大鼠腮腺中这两种肌醇三磷酸异构体的代谢情况。将腺体碎片用肌醇[2-3H]预先标记，洗涤后，再用卡巴胆碱刺激。刺激后5秒，检测到肌醇1,4,5-三磷酸明显增加，而肌醇1,3,4-三磷酸无显著增加。然而在这个初始延迟之后，肌醇1,3,4-磷酸迅速上升；到15秒时它超过了肌醇1,4,5-三磷酸，并持续上升，以至于15分钟后它是1,4,5-异构体放射性标记水平的10 - 20倍。相比之下，在最初快速上升（15秒内达到最大值）之后，肌醇1,4,5-三磷酸水平在1分钟后降至接近对照水平，然后在接下来的15分钟内又非常缓慢地上升。当在卡巴胆碱刺激15分钟后加入毒蕈碱阻断剂（阿托品）时。
