通过固定化咯利普兰亲和层析法从大鼠心脏中纯化环磷酸腺苷特异性磷酸二酯酶。
Purification of cAMP-specific phosphodiesterase from rat heart by affinity chromatography on immobilized rolipram.
作者信息
Fougier S, Némoz G, Prigent A F, Marivet M, Bourguignon J J, Wermuth C, Pacheco H
出版信息
Biochem Biophys Res Commun. 1986 Jul 16;138(1):205-14. doi: 10.1016/0006-291x(86)90267-6.
Affinity chromatography on a cAMP-specific phosphodiesterase inhibitor related to Rolipram, immobilized to AH Sepharose allowed to perform an efficient purification of the cAMP-specific phosphodiesterase isoenzyme from rat heart cytosol (102-fold purification with a 35% yield in a single step). This affinity chromatography involved a biospecific interaction since a 2 mM cAMP elution step at 30 degrees C was necessary for releasing the cAMP specific form tightly bound on the affinity gel. The cAMP eluate fraction exhibited a high specificity towards cAMP (cAMP/cGMP hydrolysis ratio 5-10), a marked sensitivity to Rolipram inhibition and could be resolved in two cAMP-specific, highly Rolipram-sensitive peaks of pI 6.7 and 4.8 by IEF on polyacrylamide gel plates. Protein stain of the IEF gel revealed a single band at pI 6.7.
与咯利普兰相关的一种环磷酸腺苷（cAMP）特异性磷酸二酯酶抑制剂固定在琼脂糖凝胶珠4B上进行亲和层析，从而能够从大鼠心脏细胞溶质中高效纯化cAMP特异性磷酸二酯酶同工酶（一步纯化102倍，产率35%）。这种亲和层析涉及生物特异性相互作用，因为在30℃下用2 mM cAMP洗脱步骤对于释放紧密结合在亲和凝胶上的cAMP特异性形式是必要的。cAMP洗脱级分对cAMP表现出高特异性（cAMP/cGMP水解率为5 - 10），对咯利普兰抑制具有显著敏感性，并且通过在聚丙烯酰胺凝胶板上进行等电聚焦可以在pI 6.7和4.8的两个cAMP特异性、对咯利普兰高度敏感的峰中解析出来。等电聚焦凝胶的蛋白质染色显示在pI 6.7处有一条带。
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