Purification and characterization of guanosine 3':5'-monophosphate-specific phosphodiesterase from guinea pig lung.
Purification and characterization of guanosine 3':5'-monophosphate-specific phosphodiesterase from guinea pig lung.
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豚鼠肺鸟苷 3:5-单磷酸特异性磷酸二酯酶的纯化和表征。
DOI：
10.1016/s0021-9258(17)40235-3
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发表时间：
1977
期刊：
影响因子：
--
通讯作者：
J. Kuo
中科院分区：
文献类型：
--
作者：
C. Davis;J. Kuo
鸟苷3*：采用DEAE-纤维素层析、羟基磷灰石凝胶处理和丙烯酰胺凝胶电泳等步骤，从豚鼠肺中纯化了5*-单磷酸特异性磷酸二酯酶（环GMP-PDE），纯化倍数比粗提物高250倍。分析型凝胶电泳显示存在双重蛋白条带，表明酶制剂至少50%是同质的。使用1 μ M底物浓度时，环GMP和环AMP水解的相对速率为1，000比1。环GMP的表观Km（0.8 μ M）比环AMP的表观Kja（150 μ M）低200倍。未观察到环IMP以及环GMP、环AMP或环IMP的8-溴和8-苄氨基衍生物的显著水解。酶的特异性不受pH值、金属离子、蛋白质激活剂或温度的影响。
A guanosine 3*: 5*-monophosphate-specific phosphodiesterase (cyclic GMP-PDE) from guinea pig lung was purified 250-fold over the activity present in crude extracts using steps of DEAE-cellulose chromatography, hydroxylapatite gel treatment and preparatory acrylamide gel electrophoresis. Analytical gel electrophoresis revealed the existence of a doublet protein band indicating that the enzyme preparation was at least 50% homogenous. The relative rate of hydrolysis of cyclic GMP and cyclic AMP, using 1 yM substrate concentrations, was 1,000 to 1. The apparent Km for cyclic GMP (0.8 yM) was 200 times lower than the apparent Kja for cyclic AMP (150 yM). No significant hydrolysis of cyclic IMP and the 8-bromo and 8-benzylamino derivatives of cyclic GMP, cyclic AMP or cyclic IMP was noted. The specificity of the enzyme was unaltered by pH, by metal ions, by the protein activator, or by temperature.