Johanson R A, Hansen C A, Williamson J R
Department of Biochemistry & Biophysics, University of Pennsylvania School of Medicine, Philadelphia 19104.
J Biol Chem. 1988 Jun 5;263(16):7465-71.
The ATP-dependent, calmodulin-sensitive 3-kinase responsible for the conversion of D-myo-inositol 1,4,5-trisphosphate to D-myo-inositol 1,3,4,5-tetrakisphosphate has been purified 2,700-fold from rat brain to a specific activity of 2.3 mumol/min/mg protein. A method of purification is described involving chromatography on phosphocellulose, Orange A dye ligand, calmodulin agarose, and hydroxylapatite columns. Neither the highly purified enzyme nor enzyme eluting from the phosphocellulose column were activated by Ca2+. However, enzyme in the 100,000 x g supernatant from rat brain was activated by Ca2+ over the range from 10(-7) to 10(-6) M and Ca2+ sensitivity of the purified enzyme was restored by the addition of calmodulin. The enzyme has a catalytic subunit Mr of 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Size exclusion chromatography of the purified enzyme on a Superose 12 column gave a Mr value of 70,000, indicating that the purified enzyme was present as a monomer. In contrast, the 100,000 x g supernatant and the purified enzyme after addition of calmodulin and 10(-6) M Ca2+ chromatographed on size exclusion chromatography with a Mr of 150,000-160,000. These results imply that the native enzyme is a dimeric structure of two catalytic subunits plus calmodulin. The purified enzyme showed a Km of 0.21 +/- 0.08 microM for D-myo-inositol 1,4,5-trisphosphate and had a pH optimum of 8.5. Addition of calmodulin increased both the Km and the Vmax of the purified enzyme about 2-fold. The high affinity of the 3-kinase for D-myo-inositol 1,4,5-trisphosphate together with its activation by Ca2+/calmodulin suggests that this enzyme may exert an important regulatory role in inositol phosphate signaling by promoting the formation of additional inositol polyphosphate isomers.
负责将D - 肌醇1,4,5 - 三磷酸转化为D - 肌醇1,3,4,5 - 四磷酸的ATP依赖性、钙调蛋白敏感性3 - 激酶已从大鼠脑中纯化了2700倍，比活性达到2.3 μmol/分钟/毫克蛋白。本文描述了一种纯化方法，该方法包括在磷酸纤维素、橙黄A染料配体、钙调蛋白琼脂糖和羟基磷灰石柱上进行层析。无论是高度纯化的酶还是从磷酸纤维素柱上洗脱下来的酶都不能被Ca²⁺激活。然而，大鼠脑100,000×g上清液中的酶在10⁻⁷至10⁻⁶M的Ca²⁺浓度范围内被激活，并且通过添加钙调蛋白可恢复纯化酶的Ca²⁺敏感性。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳测定，该酶的催化亚基Mr为53,000。在Superose 12柱上对纯化酶进行尺寸排阻色谱分析得到的Mr值为70,000，表明纯化后的酶以单体形式存在。相比之下，100,000×g上清液以及添加钙调蛋白和10⁻⁶M Ca²⁺后的纯化酶在尺寸排阻色谱上的Mr为150,000 - 160,000。这些结果表明天然酶是由两个催化亚基加钙调蛋白组成的二聚体结构。纯化后的酶对D - 肌醇1,4,5 - 三磷酸的Km为0.21±0.08 μM，最适pH为8.5。添加钙调蛋白使纯化酶的Km和Vmax都增加了约2倍。3 - 激酶对D - 肌醇1,4,5 - 三磷酸的高亲和力及其被Ca²⁺/钙调蛋白激活表明，该酶可能通过促进额外的肌醇多磷酸异构体的形成在肌醇磷酸信号传导中发挥重要的调节作用。
