大鼠心脏中环核苷酸磷酸二酯酶的等电聚焦图谱。
Isoelectric-focusing patterns of cyclic nucleotide phosphodiesterase from rat heart.
作者信息
Némoz G, Prigent A F, Pageaux J F, Pacheco H
出版信息
Biochem J. 1981 Oct 1;199(1):113-9. doi: 10.1042/bj1990113.
Abstract
- Isoelectric focusing on a flat gel bed of the rat heart cytosolic fraction resolved cyclic nucleotide phosphodiesterase activity into several forms, characterized by their substrate specificity, kinetic constants and dependence towards Ca2+ and calmodulin. A peak of pI 4.9 displayed 20 times more affinity for cyclic GMP than for cyclic AMP and was markedly inhibited by EGTA. A less substrate-specific form, only slightly sensitive to EGTA inhibition, focused at pH 5.45. Several overlapping peaks detected between pH 5.55 and pH6 specifically hydrolysed cyclic AMP, with non-Michaelian kinetics; these peaks were insensitive to Ca2+ chelation. 2. Isoelectric focusing did not dissociate enzyme-calmodulin complexes, as none of the resulting peaks was activatable by calmodulin plus Ca2+. 3. Some new information on rat cardiac phosphodiesterase is obtained with this technique, which is convenient for routine analytical studies of phosphodiesterase, as well as for preparative purposes.
摘要
- 在大鼠心脏胞质部分的平板凝胶床上进行等电聚焦，可将环核苷酸磷酸二酯酶活性解析为几种形式，其特征在于它们的底物特异性、动力学常数以及对Ca2+和钙调蛋白的依赖性。等电点为4.9的峰对环鸟苷酸的亲和力比对环腺苷酸高20倍，并且被乙二醇双四乙酸（EGTA）显著抑制。一种底物特异性较低、对EGTA抑制仅稍有敏感的形式聚焦在pH 5.45处。在pH 5.55和pH 6之间检测到的几个重叠峰特异性水解环腺苷酸，具有非米氏动力学；这些峰对Ca2+螯合不敏感。2. 等电聚焦不会使酶 - 钙调蛋白复合物解离，因为产生的峰均不能被钙调蛋白加Ca2+激活。3. 用该技术获得了一些关于大鼠心脏磷酸二酯酶的新信息，该技术便于对磷酸二酯酶进行常规分析研究以及制备目的。
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