Purification to homogeneity of protein kinase C from bovine brain–identity with the phorbol ester receptor.
Purification to homogeneity of protein kinase C from bovine brain–identity with the phorbol ester receptor.
复制标题
从牛脑中纯化同质蛋白激酶 C——与佛波酯受体一致。
DOI：
10.1002/j.1460-2075.1984.tb01913.x
复制
发表时间：
1984
期刊：
影响因子：
--
通讯作者：
M. Waterfield
中科院分区：
文献类型：
--
作者：
P. Parker;S. Stabel;M. Waterfield
通过结合 DEAE 纤维素层析、凝胶过滤和辛基琼脂糖凝胶和苯基琼脂糖凝胶疏水层析，从牛脑中分离出钙和磷脂依赖性激酶活性（蛋白激酶 C）。佛波酯受体在整个过程中与蛋白激酶 C 共同纯化，在 SDS 聚丙烯酰胺凝胶上产生 79 500 道尔顿的均质蛋白。纯化的激酶在 Ca2+ 和磷脂酰丝氨酸的饱和浓度下将大约 5000 nmol 磷酸盐掺入底物/分钟/mg 蛋白质中。不同磷脂酰丝氨酸浓度下蛋白激酶活性的倒数图是双相的，并产生磷脂酰丝氨酸的两个表观 Ka 值（0.6-2 和 35-80 微克/毫升）。二油精（20 微克/毫升）或佛波醇-12,13-二丁酸酯（10 微克/毫升）可将这些表观 Ka 值降低 2 至 3 倍。该蛋白以磷脂酰丝氨酸依赖性方式以高亲和力 (Ka = 15 nM) 结合 [3H]佛波醇-12,13-二丁酸酯 ([3H]PDB)。在饱和磷脂酰丝氨酸浓度下，每摩尔蛋白质结合 0.89 摩尔 [3H]PDB。蛋白激酶 C 作为佛波酯受体的鉴定讨论了该蛋白的功能和调节。
The calcium‐ and phospholipid‐dependent kinase activity (protein kinase C) was isolated from bovine brains by a combination of DEAE‐cellulose chromatography, gel filtration and hydrophobic chromatography on octyl‐Sepharose and phenyl‐Sepharose. The phorbol ester receptor co‐purifies with the protein kinase C throughout the procedure yielding a homogeneous protein of 79 500 daltons on SDS‐polyacrylamide gels. The purified kinase incorporated approximately 5000 nmol phosphate into substrate/min/mg protein at saturating concentrations of Ca2+ and phosphatidyl serine. Reciprocal plots of protein kinase activity at varying phosphatidyl serine concentrations were biphasic and yielded two apparent Ka values for phosphatidyl serine of 0.6‐2 and 35‐80 micrograms/ml). These apparent Ka values were reduced 2‐ to 3‐fold by either diolein (20 micrograms/ml) or phorbol‐12,13‐dibutyrate (10 micrograms/ml). The protein binds [3H]phorbol‐12,13‐dibutyrate ([3H]PDB) with high affinity (Ka = 15 nM) in a phosphatidyl serine‐dependent manner. At saturating phosphatidyl serine concentrations 0.89 mol [3H]PDB are bound per mol protein. The identification of protein kinase C as the phorbol ester receptor is discussed with respect to the function and regulation of this protein.