Ongusaha P P, Hughes P J, Davey J, Michell R H
Centre for Clinical Research in Immunology and Signalling, University of Birmingham, Birmingham B15 2TT, UK.
Biochem J. 1998 Nov 1;335 ( Pt 3)(Pt 3):671-9. doi: 10.1042/bj3350671.
Schizosaccharomyces pombe extracts synthesize InsP6 (myo-inositol hexaphosphate) from Ins(1,4,5)P3 plus ATP. An S. pombe soluble fraction converts Ins(1,4,5)P3 into Ins(1,4,5,6)P4 and Ins(1,3,4, 5)P4, in a constant ratio of approximately 5:1, and thence to Ins(1, 3,4,5,6)P5 and InsP6. We have purified a soluble Mg2+-dependent kinase of molecular mass approximately 41 kDa that makes Ins(1,4,5, 6)P4 and Ins(1,3,4,5)P4 in the same ratio and also converts Ins(1,4, 5,6)P4 or Ins(1,3,4,5)P4 into Ins(1,3,4,5,6)P5 and InsP6. Of InsP3 isomers other than Ins(1,4,5)P3, only the non-biological molecule Ins(1,4,6)P3 potently 'competed' with all steps in conversion of Ins(1,4,5)P3 into InsP6. Examination of molecular graphics representations allowed us to draw tentative conclusions about the environment needed for an hydroxyl group to be phosphorylated by this kinase and to predict successfully that the purified kinase would phosphorylate the 5-hydroxyl of Ins(1,4,6)P3. S. pombe that have been cultured with [3H]inositol contains a variety of 3H-labelled inositol polyphosphates, with Ins(1,4,5)P3 and InsP6 the most prominent, and the InsP6 concentration quickly increases in hyper-osmotically stressed S. pombe. This yeast therefore contains InsP6 and Ins(1,4,5)P3 as normal constituents, makes more InsP6 when hyper-osmotically stressed and contains a versatile inositol polyphosphate kinase that synthesizes InsP6 from Ins(1,4,5)P3.
粟酒裂殖酵母提取物可从肌醇-1,4,5-三磷酸(Ins(1,4,5)P3)和ATP合成肌醇六磷酸(InsP6,即myo-肌醇六磷酸)。粟酒裂殖酵母的一种可溶性组分可将Ins(1,4,5)P3转化为肌醇-1,4,5,6-四磷酸(Ins(1,4,5,6)P4)和肌醇-1,3,4,5-四磷酸(Ins(1,3,4,5)P4),二者比例恒定,约为5:1,进而转化为肌醇-1,3,4,5,6-五磷酸(Ins(1,3,4,5,6)P5)和InsP6。我们纯化了一种分子量约为41 kDa的可溶性Mg2+依赖性激酶,它以相同比例生成Ins(1,4,5,6)P4和Ins(1,3,4,5)P4,还能将Ins(1,4,5,6)P4或Ins(1,3,4,5)P4转化为Ins(1,3,4,5,6)P5和InsP6。除Ins(1,4,5)P3外的其他肌醇三磷酸异构体中,只有非生物分子肌醇-1,4,6-三磷酸(Ins(1,4,6)P3)能有效“竞争”Ins(1,4,5)P3转化为InsP6的所有步骤。通过对分子图形表示的研究,我们能够初步推断该激酶磷酸化羟基所需的环境,并成功预测纯化的激酶会磷酸化Ins(1,4,6)P3的5-羟基。用[3H]肌醇培养的粟酒裂殖酵母含有多种3H标记的肌醇多磷酸,其中Ins(1,4,5)P3和InsP6最为突出,在高渗胁迫的粟酒裂殖酵母中InsP6浓度迅速增加。因此,这种酵母正常情况下含有InsP6和Ins(1,4,5)P3,在高渗胁迫时会生成更多InsP6,并且含有一种多功能肌醇多磷酸激酶,可从Ins(1,4,5)P3合成InsP6。