Guse A H, Emmrich F
Max-Planck-Society, Clinical Research Unit for Rheumatology/Immunology, Institute for Clinical Immunology, Erlangen, Germany.
J Biol Chem. 1991 Dec 25;266(36):24498-502.
Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor/CD3 complex by an anti-CD3 antibody (OKT3) induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate (InsPn) isomers. Quantitative mass analysis by anion-exchange HPLC and a recently developed postcolumn dye system (Mayr, G. W. (1988) Biochem. J. 254, 585-591) revealed basal intracellular concentrations between less than 5 pmol/10(9) cells for Ins(1,3,4,5)P4 and 6380 +/- 355 pmol/10(9) cells for InsP6. Time course analysis of samples from stimulated Jurkat T-cells showed an increase of Ins(1,3,4,5)P4 to 1125 +/- 125 pmol/10(9) cells within 10 min and remained elevated over more than 30 min. Moreover, increases of the intracellular concentrations of Ins(1,3,4,6)P4, Ins(1,4,5,6)P4, and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture), Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5 and InsP6 were observed. In contrast, the concentration of Ins(1,2,4,5,6)P5 and/or Ins(2,3,4,5,6)P5 (determined as the enantiomeric mixture) decreased after stimulation. Using cytosolic extracts from Jurkat T-lymphocytes incubated with purified Ins(1,3,4,5,6)P5, Ins(1,2,3,4,6)P5, or Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 three enzyme activities were observed. Ins(1,3,4,5,6)P5 was dephosphorylated by a phosphatase removing a phosphate group at the 1 and/or 3 position resulting in the formation of Ins(1,4,5,6)P4 and/or Ins(3,4,5,6)P4 (determined as the enantiomeric mixture). Ins(1,2,3,4,6)P5 was metabolized by a specific phosphatase that cleaved the phosphate group at the 2 position, thereby generating the product Ins(1,3,4,6)P4. On the other hand, Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5 was phosphorylated by a 1/3-kinase activity to InsP6. Together novel receptor-mediated metabolic pathways of inositol polyphosphates were demonstrated in human T-lymphocytes, and corresponding enzyme activities for the inositol pentakisphosphate metabolism were found in cell lysates.
通过抗CD3抗体（OKT3）经由T细胞受体/CD3复合物刺激人T淋巴细胞系Jurkat，可诱导多种肌醇多磷酸（InsPn）异构体的细胞内浓度发生时间依赖性变化。通过阴离子交换高效液相色谱和最近开发的柱后染色系统（迈尔，G.W.（1988年）《生物化学杂志》254卷，585 - 591页）进行的定量质量分析显示，Ins(1,3,4,5)P4的基础细胞内浓度低于5 pmol/10⁹个细胞，而InsP6的基础细胞内浓度为6380 ± 355 pmol/10⁹个细胞。对受刺激的Jurkat T细胞样本进行的时间进程分析表明，Ins(1,3,4,5)P4在10分钟内增加至1125 ± 125 pmol/10⁹个细胞，并在30多分钟内保持升高。此外，还观察到Ins(1,3,4,6)P4、Ins(1,4,5,6)P4和/或Ins(3,4,5,6)P4（作为对映体混合物测定）、Ins(1,3,4,5,6)P5、Ins(1,2,3,4,6)P5和InsP6的细胞内浓度增加。相反，Ins(1,2,4,5,6)P5和/或Ins(2,3,4,5,6)P5（作为对映体混合物测定）的浓度在刺激后降低。使用与纯化的Ins(1,3,4,5,6)P5、Ins(1,2,3,4,6)P5或Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5孵育的Jurkat T淋巴细胞的胞质提取物，观察到三种酶活性。Ins(1,3,4,5,6)P5被一种磷酸酶去磷酸化，该磷酸酶在1和/或3位去除磷酸基团，导致形成Ins(1,4,5,6)P4和/或Ins(3,4,5,6)P4（作为对映体混合物测定）。Ins(1,2,3,4,6)P5被一种特异性磷酸酶代谢，该磷酸酶在2位切割磷酸基团，从而产生产物Ins(1,3,4,6)P4。另一方面，Ins(1,2,4,5,6)P5/Ins(2,3,4,5,6)P5被1/3激酶活性磷酸化为InsP6。在人T淋巴细胞中共同证明了肌醇多磷酸的新型受体介导的代谢途径，并且在细胞裂解物中发现了肌醇五磷酸代谢的相应酶活性。
