Lovell-Smith C J, Manganiello V C, Vaughan M
Biochim Biophys Acta. 1977 Apr 27;497(2):447-58. doi: 10.1016/0304-4165(77)90202-1.
Fat cells particulate phosphodiesterase activity can be solubilized in high yield (80--100%) in a buffer system (30 mM Tris - HCl, pH 8.0) containing non-ionic detergents (0.1% Brij 30, 1.0% Triton X-100), salt (3.0 mM MgSO4, 5.0 mM NaBr) and dithiothreitol (5.0 mM). Polyacrylamide gel electrophoresis of the solubilized enzyme activity indicated the presence of two bands of activities of different electrophoretic mobilities, both of which hydrolyzed cyclic AMP and cyclic GMP. The solubilized activity eluted from DEAE Bio-Gel columns as a somewhat broad profile with at least two peaks of activity. Activity against both cyclic AMP and cyclic GMP eluted in similar but not identical patterns. The solubilized enzyme and DEAE column eluates wxhibited low (less than 1 micronM) Michaelis constants for cyclic AMP and cyclic GMP. In addition, the increases in phosphodiesterase activity induced by incubation of intact fat cells with insulin or adrenocorticotropic hormone are maintained in the solubilized state.
脂肪细胞中的颗粒性磷酸二酯酶活性能够在含有非离子去污剂（0.1% 布里杰30、1.0% 吐温X - 100）、盐（3.0 mM 硫酸镁、5.0 mM 溴化钠）和二硫苏糖醇（5.0 mM）的缓冲体系（30 mM 三羟甲基氨基甲烷 - 盐酸，pH 8.0）中以高产率（80 - 100%）溶解。对溶解后的酶活性进行聚丙烯酰胺凝胶电泳显示存在两条具有不同电泳迁移率的活性条带，二者均能水解环磷酸腺苷（cAMP）和环磷酸鸟苷（cGMP）。从二乙氨基乙基琼脂糖凝胶（DEAE Bio - Gel）柱上洗脱下来的溶解活性呈现出较为宽泛的图谱，至少有两个活性峰。针对cAMP和cGMP的活性以相似但不完全相同的模式洗脱。溶解后的酶以及DEAE柱洗脱液对cAMP和cGMP的米氏常数较低（小于1微摩尔）。此外，完整脂肪细胞与胰岛素或促肾上腺皮质激素孵育所诱导的磷酸二酯酶活性增加在溶解状态下得以维持。
