Ross T S, Majerus P W
Division of Hematology-Oncology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1991 Jan 15;266(2):851-6.
Glycerophosphoinositol (GroPIns) is a major inositol phosphate in many cell types. In this study we have determined the optimal conditions (pH 8.0 and 0.5 mM MnCl2) for the metabolism of this molecule in an extract from human placenta, and we show that the major product is inositol (1)-phosphate (Ins(1)P). The enzyme activity that catalyzes this reaction is contained in the same protein designated previously as inositol-(1,2)-cyclic-phosphate 2-inositolphosphohydrolase (cyclic hydrolase), a phosphodiesterase that catalyzes the conversion of inositol-(1,2)-cyclic phosphate (cIns(1,2)P) to Ins(1)P. In addition, the enzyme also catalyzes the production of Ins(1)P from inositol (1)-methylphosphate. All of these substrates, (cIns(1,2)P, GroPIns, and inositol (1)-methylphosphate), contain a phosphodiester bond at the 1-position of the inositol ring. Additional phosphate groups on the 4- or 5-positions of the inositol ring prevent hydrolysis by cyclic hydrolase. The Km of the enzyme for GroPIns is 0.67 mM, and the Vm is 5 mumol/min/mg of protein. GroPIns competitively inhibits cIns(1,2)P hydrolysis with a Ki equal to its Km as a substrate. Hydrolysis of GroPIns and cIns(1,2)P is stimulated by MnCl2, phosphatidylserine, and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA). However, whereas cIns(1,2)P hydrolysis is increased 5-8-fold by phosphatidylserine and EGTA only a 2-fold increase of GroPIns hydrolysis occurs under the same conditions. Hydrolysis of both GroPIns and cIns(1,2)P is inhibited by Ins(2)P; the ID50 values are 12 and 1 microM, respectively. There are significant quantities of GroPIns and Ins(2)P in 3T3 cells, indicating that these compounds that alter cIns(1,2)P hydrolase activity may modulate intracellular levels of cIns(1,2)P. Finally, we present evidence suggesting that the substrate specificity of this enzyme is altered during cell transformation.
甘油磷酸肌醇（GroPIns）是许多细胞类型中的主要肌醇磷酸酯。在本研究中，我们确定了人胎盘提取物中该分子代谢的最佳条件（pH 8.0和0.5 mM氯化锰），并且我们表明主要产物是肌醇（1）-磷酸酯（Ins(1)P）。催化此反应的酶活性包含在先前称为肌醇-（1,2）-环磷酸酯2-肌醇磷酸水解酶（环水解酶）的同一蛋白质中，该磷酸二酯酶催化肌醇-（1,2）-环磷酸酯（cIns(1,2)P）转化为Ins(1)P。此外，该酶还催化从肌醇（1）-甲基磷酸酯产生Ins(1)P。所有这些底物（cIns(1,2)P、GroPIns和肌醇（1）-甲基磷酸酯）在肌醇环的1位都含有一个磷酸二酯键。肌醇环4位或5位上的额外磷酸基团可防止环水解酶的水解作用。该酶对GroPIns的Km为0.67 mM，Vm为5 μmol/min/mg蛋白质。GroPIns竞争性抑制cIns(1,2)P水解，其Ki等于其作为底物的Km。GroPIns和cIns(1,2)P的水解受到氯化锰、磷脂酰丝氨酸和[乙二胺双（氧乙烯腈）]四乙酸（EGTA）的刺激。然而，虽然磷脂酰丝氨酸和EGTA使cIns(1,2)P水解增加5至8倍，但在相同条件下GroPIns水解仅增加2倍。Ins(2)P抑制GroPIns和cIns(1,2)P的水解；ID50值分别为12和1 μM。3T3细胞中存在大量的GroPIns和Ins(2)P，表明这些改变cIns(1,2)P水解酶活性的化合物可能调节细胞内cIns(1,2)P的水平。最后，我们提供的证据表明该酶的底物特异性在细胞转化过程中发生改变。
