Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6.
Purified rat brain calcium- and phospholipid-dependent protein kinase phosphorylates ribosomal protein S6.
复制标题
纯化的大鼠脑钙和磷脂依赖性蛋白激酶磷酸化核糖体蛋白 S6。
DOI：
10.1073/pnas.80.22.6858
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发表时间：
1983
影响因子：
11.1
通讯作者：
Rosen,OM
中科院分区：
文献类型：
--
作者：
LePeuch,CJ;Ballester,R;Rosen,OM
Ca ~（2+）-磷脂调节的蛋白激酶已从100，000 × g大鼠脑匀浆的上清液中纯化至均一，所用的方法包括DEAE-纤维素层析和在EGTA和磷脂酰丝氨酸和Ca ~（2+）中的Ultrogel AcA 34上连续过滤。一个更快速的纯化组成的DEAE-纤维素层析，Ultrogel AcA 34凝胶过滤层析，和DEAE-trisacryl层析，所有在EGTA的存在下，也被开发。虽然通过后一种方法获得的酶是不均匀的，但它表现出与纯酶相似的性质，并且更稳定。此外，DEAE-trisacryl步骤允许解析污染的Ca 2 +-可降解蛋白激酶，其可以干扰Ca 2 +-磷脂刺激的酶的研究。纯化约300倍的均质酶估计具有84，000的Mr。其活性在磷脂和Ca 2+存在下比在磷脂和EGTA、EGTA或单独Ca 2+存在下高20- 30倍。活化激酶的比活性为在20 ℃下每分钟/mg掺入组蛋白的P为852 nmol。纯酶进行自磷酸化的Ca 2+和磷脂依赖性的方式。在组蛋白的存在下，该反应被抑制，而不影响酶的动力学性质。在最佳测定条件下，均相酶被10 μ M二油酸甘油酯或100 nM佛波醇12-肉豆蔻酸酯13-乙酸酯激活10-20%。二油酸甘油酯或佛波醇酯的纯化的酶的激活是更大的（3至4倍）时，聚集而不是新鲜的超声波处理的磷脂被使用，这表明这些化合物影响的酶与磷脂和Ca 2+的相互作用。纯化的酶催化40 S核糖体亚基蛋白S6的磷酸化。S6的Km约等于1 μ M，估计每摩尔S6掺入2摩尔磷酸盐。蛋白S6可以被纯化的钙磷脂依赖性蛋白激酶磷酸化的观察结果可能与最近的报道有关，即佛波酯肿瘤促进剂在体外激活钙磷脂依赖性蛋白激酶，并在体内刺激核糖体蛋白S6的磷酸化。
The Ca2+-phospholipid-regulated protein kinase has been purified to homogeneity from a 100,000 X g supernatant fluid of rat brain homogenate by a procedure that includes DEAE-cellulose chromatography and successive filtrations on Ultrogel AcA 34 in EGTA and in phosphatidylserine and Ca2+. A more rapid purification consisting of DEAE-cellulose chromatography, Ultrogel AcA 34 gel filtration chromatography, and DEAE-trisacryl chromatography, all in the presence of EGTA, was also developed. Although the enzyme obtained by the latter procedure is not homogeneous, it exhibits properties similar to those of the pure enzyme and is more stable. In addition, the DEAE-trisacryl step permitted resolution of a contaminating Ca2+-inhibitable protein kinase that can interfere with studies of the Ca2+-phospholipid-stimulated enzyme. The homogeneous enzyme, purified about 300-fold, was estimated to have a Mr of 84,000. Its activity was 20- to 30-fold higher in the presence of phospholipid and Ca2+ than in the presence of phospholipid and EGTA, EGTA, or Ca2+ alone. The specific activity of the activated kinase was 852 nmol of P incorporated into histone per min/mg at 20 degrees C. The pure enzyme underwent autophosphorylation in a Ca2+- and phospholipid-dependent manner. This reaction was inhibited in the presence of histones without affecting the kinetic properties of the enzyme. Under optimal assay conditions, the homogeneous enzyme was activated 10-20% by either 10 microM diolein or 100 nM phorbol 12-myristate 13-acetate. Activation of the purified enzyme by diolein or the phorbol ester was far greater (3- to 4-fold) when aggregated instead of freshly sonicated phospholipids were used, suggesting that these compounds affect the interaction of the enzyme with phospholipids and Ca2+. The purified enzyme catalyzed the phosphorylation of the 40S ribosomal subunit protein S6. The Km for S6 was approximately equal to 1 microM and it was estimated that 2 mol of phosphate were incorporated per mol of S6. The observation that protein S6 can be phosphorylated by the purified Ca2+-phospholipid-dependent protein kinase may link recent reports that phorbol ester tumor promoters activate the Ca2+-phospholipid-dependent protein kinase in vitro and stimulate phosphorylation of the ribosomal protein S6 in vivo.