Harden T K, Stephens L, Hawkins P T, Downes C P
J Biol Chem. 1987 Jul 5;262(19):9057-61.
Phosphoinositides of human, rabbit, rat, and turkey erythrocytes were radiolabeled by incubation of intact cells with [32P]Pi. Guanosine 5'-O-(thiotriphosphate) (GTP gamma S) and NaF, which are known activators of guanine nucleotide regulatory proteins, caused a large increase in [32P]inositol phosphate release from plasma membranes derived from turkey erythrocytes, but had no effect on inositol phosphate formation by plasma membranes prepared from the mammalian erythrocytes. High performance liquid chromatography analysis indicated that inositol bisphosphate, inositol 1,3,4-trisphosphate, inositol 1,4,5-trisphosphate, and inositol 1,3,4,5-tetrakisphosphate all increased by 20-30-fold during a 10-min incubation of turkey erythrocyte membranes with GTP gamma S. The increase in inositol phosphate formation was accompanied by a similar decrease in radioactivity in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2). GTP gamma S increased inositol phosphate formation with a K0.5 of 600 nM; guanosine 5'-(beta, gamma-imido)trisphosphate was 50-75% as efficacious as GTP gamma S and expressed a K0.5 of 36 microM. Although GTP alone had little effect on inositol phosphate formation, it blocked GTP gamma S-stimulated inositol phosphate formation, as did guanosine 5'-O-(2-thiodiphosphate). Turkey erythrocytes were also shown to express phosphatidylinositol synthetase activity in that incubation of cells with [3H] inositol resulted in incorporation of radiolabel into phosphatidylinositol, PIP, and PIP2. Incubation of membranes derived from [3H]inositol-labeled erythrocytes with GTP gamma S resulted in large increases in [3H] inositol phosphate formation and corresponding decreases in radiolabel in PIP and PIP2. The data suggest that, in contrast to mammalian erythrocytes, the turkey erythrocyte expresses a guanine nucleotide-binding protein that regulates phospholipase C, and as such, should provide a useful model system for furthering our understanding of hormonal regulation of this enzyme.
通过完整细胞与[32P]Pi孵育，对人、兔、大鼠和火鸡红细胞的磷酸肌醇进行放射性标记。已知鸟嘌呤核苷酸调节蛋白的激活剂鸟苷5'-O-(硫代三磷酸)(GTPγS)和NaF，可使源自火鸡红细胞的质膜中[32P]肌醇磷酸释放大幅增加，但对由哺乳动物红细胞制备的质膜中肌醇磷酸的形成没有影响。高效液相色谱分析表明，在火鸡红细胞膜与GTPγS孵育10分钟期间，肌醇二磷酸、肌醇1,3,4-三磷酸、肌醇1,4,5-三磷酸和肌醇1,3,4,5-四磷酸均增加了20-30倍。肌醇磷酸形成的增加伴随着磷脂酰肌醇4-磷酸(PIP)和磷脂酰肌醇4,5-二磷酸(PIP2)中放射性的类似降低。GTPγS以600 nM的K0.5增加肌醇磷酸的形成；鸟苷5'-(β,γ-亚氨基)三磷酸的效力为GTPγS的50-75%，K0.5为36 μM。虽然单独的GTP对肌醇磷酸的形成几乎没有影响，但它像鸟苷5'-O-(2-硫代二磷酸)一样，阻断了GTPγS刺激的肌醇磷酸形成。火鸡红细胞还显示出表达磷脂酰肌醇合成酶活性，因为用[3H]肌醇孵育细胞会导致放射性标记掺入磷脂酰肌醇、PIP和PIP2中。用GTPγS孵育源自[3H]肌醇标记红细胞的膜会导致[3H]肌醇磷酸形成大幅增加，以及PIP和PIP2中放射性标记相应减少。数据表明，与哺乳动物红细胞不同，火鸡红细胞表达一种调节磷脂酶C的鸟嘌呤核苷酸结合蛋白，因此，应该为增进我们对该酶的激素调节的理解提供一个有用的模型系统。
