Höer A, Höer D, Oberdisse E
Institut für Pharmakologie, Freien Universität Berlin, Federal Republic of Germany.
Biochem J. 1990 Sep 15;270(3):715-9. doi: 10.1042/bj2700715.
We have previously shown that Ins(1,3,4,5)P4 is degraded to Ins(1,4,5)P3 by a soluble Ins(1,3,4,5)P4 3-phosphatase from pig brain [Höer, Kwiatkowski, Seib, Rosenthal, Schultz & Oberdisse (1988) Biochem. Biophys. Res. Commun. 154, 668-675]. Here we present some properties of this enzyme using [5-32P]Ins(1,3,4,5)P4 as substrate. The molecular mass, estimated by gel filtration chromatography on a Superose 6 column, was determined to be 36 kDa. The 3-phosphatase showed a high affinity towards the substrate Ins(1,3,4,5)P4 (Km approximately 400 nM); the Vmax. of the freshly prepared enzyme was 2 nmol/min per mg of protein. The influence of Ins(1,4,5)P3 and Ins(1,3,4)P3, the reaction products of Ins(1,3,4,5)P4 hydrolysis by either 3- or 5-phosphatase respectively, on the 3-phosphatase was tested. Both isomers inhibited the enzyme, with Ki values of about 2 microM and 1.75 microM for Ins(1,3,4)P3 and Ins(1,4,5)P3 respectively. Enzyme activity was not influenced by Mg2+ up to 30 mM or Ca2+ up to 1 mM. Commercially available Ins(3,4,5,6)P4 from turkey erythrocytes produced a marked inhibition of the 3-phosphatase (Ki approximately 500 nM). Significant inhibitory effects on enzyme activity were also found with GTP and the pyrimidine nucleotides UTP and CTP. The kinetic data presented here suggest that the Ins(1,3,4,5)P4 3-phosphatase may be regulated by the intracellular concentrations of inositol tris- and tetrakis-phosphates.
我们之前的研究表明，猪脑可溶性肌醇-1,3,4,5-四磷酸3-磷酸酶可将肌醇-1,3,4,5-四磷酸（Ins(1,3,4,5)P4）降解为肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）[赫厄、克维亚特科夫斯基、塞布、罗森塔尔、舒尔茨和奥伯迪斯（1988年），《生物化学与生物物理研究通讯》154卷，668 - 675页]。在此，我们以[5-32P]肌醇-1,3,4,5-四磷酸为底物，介绍这种酶的一些特性。通过在Superose 6柱上进行凝胶过滤色谱法估算，该酶的分子量为36 kDa。这种3-磷酸酶对底物肌醇-1,3,4,5-四磷酸表现出高亲和力（Km约为400 nM）；新制备的酶的Vmax为每毫克蛋白质2 nmol/分钟。分别测试了肌醇-1,3,4,5-四磷酸经3-或5-磷酸酶水解的反应产物肌醇-1,4,5-三磷酸（Ins(1,4,5)P3）和肌醇-1,3,4-三磷酸（Ins(1,3,4)P3）对3-磷酸酶的影响。两种异构体均抑制该酶，肌醇-1,3,4-三磷酸和肌醇-1,4,5-三磷酸的Ki值分别约为2 microM和1.75 microM。高达30 mM的Mg2+或高达1 mM的Ca2+对酶活性均无影响。来自火鸡红细胞的市售肌醇-3,4,5,6-四磷酸（Ins(3,4,5,6)P4）对3-磷酸酶有显著抑制作用（Ki约为500 nM）。还发现鸟苷三磷酸（GTP）以及嘧啶核苷酸尿苷三磷酸（UTP）和胞苷三磷酸（CTP）对酶活性有显著抑制作用。此处给出的动力学数据表明，肌醇-1,3,4,5-四磷酸3-磷酸酶可能受细胞内肌醇三磷酸和四磷酸浓度的调节。
