Villarreal Fernando D, Kültz Dietmar
EcoPhysiological Proteomics Laboratory, Department of Animal Science, University of California Davis, One Shields Avenue, Davis, California 95616, United States of America.
PLoS One. 2015 Jun 11;10(6):e0123212. doi: 10.1371/journal.pone.0123212. eCollection 2015.
Myo-inositol (Ins) is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus). Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS) and inositol monophosphatase (IMPase), by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1) were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P), mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P) is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.
肌醇（Ins）是许多细胞中的一种主要相容性渗透溶质，包括莫桑比克罗非鱼（Oreochromis mossambicus）的细胞。在暴露于高渗应激的罗非鱼和其他广盐性鱼类中，肌醇生物合成被高度上调。在本研究中，分析了直接离子效应对肌醇生物合成的两种酶，即肌醇磷酸合酶（MIPS）和肌醇单磷酸酶（IMPase）的酶促调节作用。基于支持它们在高渗应激期间对肌醇生物合成作用的实验、系统发育和结构证据，选择了莫桑比克罗非鱼的特定MIPS和IMPase同工型（MIPS-160和IMPase 1）。在模拟高渗应激期间细胞内环境变化的离子条件下，体外测定重组罗非鱼IMPase 1和MIPS-160的活性。MIPS-160和IMPase 1的体外活性在pH 8.8的碱性条件下最高。在高渗状态下，IMPase 1的催化效率显著提高（特别是对于底物D-肌醇-3-磷酸，Ins-3P），这主要是由于[Na+]升高所致。此外，IMPase 1对D-肌醇-1-磷酸（Ins-1P）的底物特异性低于对Ins-3P的底物特异性。由于MIPS催化产生Ins-3P，这一结果为IMPase 1是介导罗非鱼肌醇生物合成的同工型提供了额外证据。我们的数据共同表明，肌醇生物合成酶在细胞在高渗状态下所暴露的离子条件下被激活，导致肌醇积累，进而导致细胞内离子稳态的恢复。我们提出，肌醇生物合成酶活性的独特直接离子调节代表了一种有效的生化反馈回路，用于在高渗应激期间调节细胞内生理离子稳态。
