肌醇五磷酸。结构、生物学存在形式以及磷酸化生成肌醇六磷酸。
myo-inositol pentakisphosphates. Structure, biological occurrence and phosphorylation to myo-inositol hexakisphosphate.
作者信息
Stephens L R, Hawkins P T, Stanley A F, Moore T, Poyner D R, Morris P J, Hanley M R, Kay R R, Irvine R F
机构信息
Biochemistry Department, AFRC, Babraham, Cambridge, U.K.
出版信息
Biochem J. 1991 Apr 15;275 ( Pt 2)(Pt 2):485-99. doi: 10.1042/bj2750485.
Abstract
- Standard and high-performance anion-exchange-chromatographic techniques have been used to purify myo-[3H]inositol pentakisphosphates from various myo-[3H]inositol-prelabelled cells. Slime mould (Dictyostelium discoideum) contained 8 microM-myo-[3H]inositol 1,3,4,5,6-pentakisphosphate, 16 microM-myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and 36 microM-D-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate [calculated intracellular concentrations; Stephens & Irvine (1990) Nature (London) 346, 580-583]; germinating mung-bean (Phaseolus aureus) seedlings contained both D- and L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate (which was characterized by 31P and two-dimensional proton n.m.r.) and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate; HL60 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 500-fold excess over the other species), myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate; and NG-115-401L-C3 cells contained myo-[3H]inositol 1,3,4,5,6-pentakisphosphate (in a 100-fold excess over the other species), D- and/or L-myo-[3H]inositol 1,2,4,5,6-pentakisphosphate, myo-[3H]inositol 1,2,3,4,6-pentakisphosphate and D- and/or L-myo-[3H]inositol 1,2,3,4,5-pentakisphosphate. 2. Multiple soluble ATP-dependent myo-inositol pentakisphosphate kinase activities have been detected in slime mould, rat brain and germinating mung-bean seedling homogenates. In slime-mould cytosolic fractions, the three myo-inositol pentakisphosphates that were present in intact slime moulds could be phosphorylated to myo-[3H]inositol hexakisphosphate: the relative first-order rate constants for these reactions were, in the order listed above, 1:8:31 respectively (with first-order rate constants in the intact cell of 0.1, 0.8 and 3.1 s-1, assuming a cytosolic protein concentration of 50 mg/ml), and the Km values of the activities for their respective inositol phosphate substrates (in the presence of 5 mM-ATP) were 1.6 microM, 3.8 microM and 1.4 microM. At least two forms of myo-inositol pentakisphosphate kinase activity could be resolved from a slime-mould cytosolic fraction by both pharmacological and chromatographic criteria. Rat brain cytosol and a soluble fraction derived from germinating mung-bean seedlings could phosphorylate myo-inositol D/L-1,2,4,5,6-, D/L-1,2,3,4,5-, 1,2,3,4,6- and 1,3,4,5,6-pentakisphosphates to myo-inositol hexakisphosphate: the relative first-order rate constants were 57:27:77:1 respectively for brain cytosol (with first-order rate constants in the intact cell of 0.0041, 0.0019, 0.0056 and 0.000073 s-1 respectively, assuming a cytosolic protein concentration of 50 mg/ml) and 1:11:12:33 respectively for mung-bean cytosol (with first-order rate constants in a supernatant fraction with a protein concentration of 10 mg/ml of 0.0002, 0.0022, 0.0024 and 0.0066 s-1 respectively).
摘要
- 采用标准和高效阴离子交换色谱技术，从各种预先用³H标记肌醇的细胞中纯化了肌醇-[³H]五磷酸。黏菌（盘基网柄菌）含有8微摩尔/升的肌醇-1,3,4,5,6-五磷酸、16微摩尔/升的肌醇-1,2,3,4,6-五磷酸和36微摩尔/升的D-肌醇-1,2,4,5,6-五磷酸[计算出的细胞内浓度；斯蒂芬斯和欧文（1990年），《自然》（伦敦）346, 580 - 583]；发芽的绿豆（菜豆）幼苗含有D-和L-肌醇-1,2,4,5,6-五磷酸（通过³¹P和二维质子核磁共振进行了表征）以及D-和/或L-肌醇-1,2,3,4,5-五磷酸；HL60细胞含有肌醇-[³H]1,3,4,5,6-五磷酸（比其他种类过量500倍）、肌醇-[³H]1,2,3,4,6-五磷酸以及D-和/或L-肌醇-[³H]1,2,4,5,6-五磷酸；NG-115-401L-C3细胞含有肌醇-[³H]1,3,4,5,6-五磷酸（比其他种类过量100倍）、D-和/或L-肌醇-[³H]1,2,4,5,6-五磷酸、肌醇-[³H]1,2,3,4,6-五磷酸以及D-和/或L-肌醇-[³H]1,2,3,4,5-五磷酸。2. 在黏菌、大鼠脑和发芽绿豆幼苗匀浆中检测到多种可溶性ATP依赖的肌醇五磷酸激酶活性。在黏菌胞质部分，完整黏菌中存在的三种肌醇五磷酸均可被磷酸化为肌醇-[³H]六磷酸：这些反应的相对一级速率常数，按上述顺序分别为1:8:31（假设胞质蛋白浓度为50毫克/毫升，完整细胞中的一级速率常数分别为0.1、0.8和3.1秒⁻¹），并且这些活性对其各自肌醇磷酸底物（在5毫摩尔/升ATP存在下）的Km值分别为1.6微摩尔/升、3.8微摩尔/升和1.4微摩尔/升。通过药理学和色谱标准，至少可从黏菌胞质部分分辨出两种形式的肌醇五磷酸激酶活性。大鼠脑胞质溶胶和发芽绿豆幼苗的可溶性部分可将肌醇D/L-1,2,4,5,6-、D/L-1,2,3,4,5-、1,2,3,4,6-和1,3,4,5,6-五磷酸磷酸化为肌醇六磷酸：脑胞质溶胶的相对一级速率常数分别为57:27:77:1（假设胞质蛋白浓度为50毫克/毫升，完整细胞中的一级速率常数分别为0.0041、0.0019、0.0056和0.000073秒⁻¹），绿豆胞质溶胶的相对一级速率常数分别为1:11:12:33（假设蛋白质浓度为10毫克/毫升的上清液部分中的一级速率常数分别为0.0002、0.0022、0.0024和0.0066秒⁻¹）。
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