Nemecek G M, Butcher R W
J Cyclic Nucleotide Res. 1979 Dec;5(6):449-61.
Broken cell preparations of WI-38 and SV40-transformed WI-38 (VA13) fibroblasts were used to compare the cyclic nucleotide phosphodiesterase activities of the two cell strains. The bulk of the cAMP or cGMP phosphodiesterase activity of WI-38 and VA13 homogenates was found in the 100,000 x g fibroblast supernatant fractions. WI-38 and VA13 soluble phosphodiesterase activities showed anomalous kinetic behavior with either cAMP or cGMP as the substrate. At low substrate concentrations, e.g., 0.1 muM, WI-38 supernatant fractions hydrolyzed cGMP much more rapidly than cAMP. At high substrate concentrations, e.g., 100muM, the same enzyme preparations degraded cAMP more than twice as fast as cGMP. In contrast, VA13 soluble phosphodiesterase activity catalyzed the hydrolysis of a wide range of cAMP and cGMP concentrations at similar rates. Phosphodiesterase activity in WI-38 supernatant fractions was generally more sensitive than that of the comparable VA13 enzyme activity to inhibition by MIX and papaverine. The cAMP phosphodiesterase activity of both WI-38 and VA13 supernatant preparations was decreased by cGMP in a concentration-dependent manner. cAMP was an effective inhibitor of cGMP hydrolysis by VA13 soluble phosphodiesterase activity. Yet, the cGMP phosphodiesterase activity of WI-38 supernatant fractions was only slightly reduced in the presence of cAMP. DEAE-cellulose chromatography of WI-38 and VA13 supernatant preparations revealed two major peaks of phosphodiesterase activity for each cell type. WI-38 peak I showed much greater activity with 1muM cGMP than with 1muM cAMP and appeared to be composed of two different phosphodiesterase activities. WI-38 peak Ia included phosphodiesterase activity which could be stimulated by boiled, dialyzed fibroblast homogenates while WI-38 peak Ib coincided with column fractions which contained most of the cyclic GMP hydrolytic activity. VA13 peak I phosphodiesterase activity was eluted from DEAE cellulose columns at the same ionic strength as WI-38 peak Ia and hydrolyzed these two substrates at nearly identical rates. This enzyme activity was also increased in the presence of boiled, dialyzed fibroblast preparations. Peak II phosphodiesterase activities from both WI-38 and VA13 fibroblasts were relatively specific for cAMP as the substrate. Phosphodiesterase activity with the properties of WI-38 peak Ib was not isolated from VA13 supernatant fractions. These results suggested that the dissimilar patterns of cAMP accumulation in WI-38 and VA13 cultures may be at least partially related to different phosphodiesterase activities in the normal and the transformed fibroblasts.
使用WI - 38和SV40转化的WI - 38（VA13）成纤维细胞的破碎细胞制剂来比较这两种细胞株的环核苷酸磷酸二酯酶活性。发现WI - 38和VA13匀浆中大部分的cAMP或cGMP磷酸二酯酶活性存在于100,000 x g的成纤维细胞上清液组分中。WI - 38和VA13的可溶性磷酸二酯酶活性以cAMP或cGMP为底物时表现出异常的动力学行为。在低底物浓度下，例如0.1μM，WI - 38上清液组分水解cGMP的速度比cAMP快得多。在高底物浓度下，例如100μM，相同的酶制剂降解cAMP的速度比cGMP快两倍多。相比之下，VA13可溶性磷酸二酯酶活性以相似的速率催化各种cAMP和cGMP浓度的水解。WI - 38上清液组分中的磷酸二酯酶活性通常比VA13的可比酶活性对MIX和罂粟碱的抑制更敏感。WI - 38和VA13上清液制剂的cAMP磷酸二酯酶活性以浓度依赖的方式被cGMP降低。cAMP是VA13可溶性磷酸二酯酶活性水解cGMP的有效抑制剂。然而，在存在cAMP的情况下，WI - 38上清液组分的cGMP磷酸二酯酶活性仅略有降低。对WI - 38和VA13上清液制剂进行DEAE - 纤维素色谱分析，发现每种细胞类型都有两个主要的磷酸二酯酶活性峰。WI - 38峰I对1μM cGMP的活性比对1μM cAMP的活性高得多，并且似乎由两种不同的磷酸二酯酶活性组成。WI - 38峰Ia包括可被煮沸、透析的成纤维细胞匀浆刺激的磷酸二酯酶活性，而WI - 38峰Ib与含有大部分环GMP水解活性的柱馏分一致。VA13峰I磷酸二酯酶活性在与WI - 38峰Ia相同的离子强度下从DEAE纤维素柱上洗脱，并以几乎相同的速率水解这两种底物。这种酶活性在存在煮沸、透析的成纤维细胞制剂时也会增加。WI - 38和VA13成纤维细胞的峰II磷酸二酯酶活性对cAMP作为底物相对特异。未从VA13上清液组分中分离出具有WI - 38峰Ib特性的磷酸二酯酶活性。这些结果表明，WI - 38和VA13培养物中cAMP积累的不同模式可能至少部分与正常和转化成纤维细胞中不同的磷酸二酯酶活性有关。
