Lee S Y, Sim S S, Kim J W, Moon K H, Kim J H, Rhee S G
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
J Biol Chem. 1990 Jun 5;265(16):9434-40.
A new, rapid method for purification of inositol(1,4,5)P3 3-kinase in high yield from rat brain is described. Purified enzyme exhibited a polypeptide of Mr = 53,000 on sodium dodecyl sulfate-polyacrylamide gel and a specific activity of 29 mumol/min/mg at 37 degrees C in the absence of calmodulin. Inclusion of calpain inhibitors was critical for obtaining the 53-kDa protein as the major product and 0.1% of the zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylamino]-2-propanesulfonate, was necessary to stabilize enzyme activity. In the absence of calpain inhibitors, the 53-kDa protein degraded progressively during purification and yielded a mixture containing polypeptides of various sizes. Relative intensity of these degradation products on sodium dodecyl sulfate-polyacrylamide gel varied from one preparation to another. However, broad band(s) at the 42-45 kDa region and a band at 35 kDa were always weak, while bands of 53, 51, 40 (sometimes doublets), 33, and 32 KDa were usually strong. The fact that all of these polypeptides including the weak bands of 42-45 and 35 kDa were derived from the 53 kDa form was confirmed by their immunocross-reactivity with monoclonal antibodies to the 53 kDa form. When the 51, 40, and a mixture of the 33 and 32 kDa forms were obtained separately and nearly free from other forms, each of them exhibited catalytic activity. Nevertheless, calmodulin binds to polypeptides larger than 35,000 but not to the 33 and 32 kDa forms. Incubation of the purified 53 kDa form with calpain generated a fragmentation pattern nearly identical to that generated during purification in the absence of calpain inhibitors. Incubation with five other endoproteases produced proteolytic fragments slightly different from those by calpain. However, the general fragmentation patterns generated by the proteases were similar, suggesting that inositol(1,4,5)P3 3-kinase contains several motifs susceptible to a variety of proteases.
本文描述了一种从大鼠脑中高产纯化肌醇（1,4,5）三磷酸3 - 激酶的新的快速方法。纯化后的酶在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上显示出一条分子量为53,000的多肽，在37℃、无钙调蛋白的条件下比活性为29μmol/分钟/毫克。加入钙蛋白酶抑制剂对于获得以53 - kDa蛋白为主要产物至关重要，并且需要0.1%的两性离子去污剂3 - [（3 - 胆酰胺丙基）二甲基氨基] - 2 - 丙烷磺酸盐来稳定酶活性。在没有钙蛋白酶抑制剂的情况下，53 - kDa蛋白在纯化过程中逐渐降解，产生了含有各种大小多肽的混合物。这些降解产物在十二烷基硫酸钠 - 聚丙烯酰胺凝胶上的相对强度因制备方法而异。然而，42 - 45 kDa区域的宽带和35 kDa处的条带总是较弱，而53、51、40（有时为双峰）、33和32 kDa的条带通常较强。通过它们与针对53 kDa形式的单克隆抗体的免疫交叉反应性，证实了所有这些多肽，包括42 - 45和35 kDa的弱条带，均源自53 kDa形式。当分别获得几乎不含其他形式的51、40以及33和32 kDa形式的混合物时，它们各自都表现出催化活性。然而，钙调蛋白与大于35,000的多肽结合，但不与33和32 kDa形式结合。将纯化的53 kDa形式与钙蛋白酶一起孵育产生的片段化模式与在没有钙蛋白酶抑制剂的纯化过程中产生的模式几乎相同。与其他五种内切蛋白酶一起孵育产生的蛋白水解片段与钙蛋白酶产生的略有不同。然而，蛋白酶产生的一般片段化模式相似，表明肌醇（1,4,5）三磷酸3 - 激酶包含几个易受多种蛋白酶作用的基序。
