PURIFICATION OF CYCLIC 3',5'-NUCLEOTIDE PHOSPHODIESTERASE INHIBITORY PROTEIN BY AFFINITY CHROMATOGRAPHY ON ACTIVATOR PROTEIN COUPLED TO SEPHAROSE
PURIFICATION OF CYCLIC 3',5'-NUCLEOTIDE PHOSPHODIESTERASE INHIBITORY PROTEIN BY AFFINITY CHROMATOGRAPHY ON ACTIVATOR PROTEIN COUPLED TO SEPHAROSE
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DOI：
10.1021/bi00594a017
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发表时间：
1978-01-01
期刊：
影响因子：
2.9
通讯作者：
KRINKS, MH
中科院分区：
文献类型：
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作者：
KLEE, CB;KRINKS, MH
钙依赖性，可逆的，相互作用的环[c]AMP磷酸二酯酶与其激活剂被用来纯化[牛脑]酶的亲和层析。激活剂依赖性cAMP磷酸二酯酶仅是在Ca 2+存在下由偶联至琼脂糖凝胶的Ca 2+依赖性激活剂蛋白特异性吸附并随后由[亚乙基双（氧乙烯基氮）]四乙酸释放的蛋白质的次要组分。通过Sephadex G-200凝胶过滤，可以从酶中部分分离出主要的蛋白组分。该蛋白质被纯化至表观均一性，并显示由分子量分别为61，000和15，000的2条多肽链组成。这种蛋白本身没有磷酸二酯酶活性，并通过其激活剂抑制cAMP磷酸二酯酶的激活，而不影响基础活性。因此，激活cAMP磷酸二酯酶的Ca 2+依赖性激活蛋白可以通过与酶复合物的第三组分的相互作用来控制。
The Ca2+-dependent, reversible, interaction of cyclic[c]AMP phosphodiesterase with its activator was used to purify the [bovine brain] enzyme by affinity chromatography. Activator-dependent cAMP phosphodiesterase is only a minor component of the proteins specifically adsorbed in the presence of Ca2+ by the Ca2+-dependent activator protein coupled to Sepharose and subsequently released by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid. The major protein component can be partially resolved from the enzyme by gel filtration on Sephadex G-200. This protein was purified to apparent homogeneity and shown to be composed of 2 polypeptide chains with molecular weights of 61,000 and 15,000, respectively. This protein is, by itself, devoid of phosphodiesterase activity and inhibits the activation of cAMP phosphodiesterase by its activator without affecting the basal activity. Thus, activation of cAMP phosphodiesterase by the Ca2+-dependent activator protein may be controlled by interactions with yet a 3rd component of the enzyme complex.