Souness J E, Thompson W J, Strada S J
J Cyclic Nucleotide Protein Phosphor Res. 1985;10(4):383-96.
The addition of vanadate (Na3VO4) to intact isolated rat adipocytes stimulated cAMP phosphodiesterase activity (Type IV) in the particulate (P2) fraction. Vanadate increased the Vmax of the Type IV phosphodiesterase activity without affecting its apparent substrate affinity. Na3VO4 also stimulated cAMP hydrolysis of cell-free particulate and cytosolic fractions, but this activation required the presence of reduced glutathione (GSH). The mixture of vanadate and glutathione appeared as an emerald green solution (V-GSH complex), which was shown by EPR to contain vanadyl ion. No effect of either GSH or Na3VO4 alone on cell-free particulate cAMP phosphodiesterase activity was observed; however, Na3VO4, alone or in combination with GSH, stimulated cGMP hydrolysis in this subcellular fraction. The V-GSH complex increased the Vmax of the particulate cAMP phosphodiesterase activity without affecting its apparent Km. The activating effect of the complex was rapid in onset, persistent over 30 minutes, and reversible. The EC50 for activation of the particulate cAMP phosphodiesterase was approximately 5 microM Na3VO4 (maintaining the GSH:Na3VO4 molar ratio at 2:1); maximal stimulation was achieved at 0.1 mM Na3VO4. Purified microsomal membranes showed activation similar to that of the P2 fraction, while only a 60% stimulation was observed in purified plasma membranes. The V-GSH complex increased basal insulin-activated Type IV phosphodiesterase activity to a common maximal level. Detergent-solubilized cAMP-phosphodiesterase from the P2 fraction was stimulated 2.5-fold by the V-GSH complex. Limited trypsin treatment of P2 membranes activated cAMP phosphodiesterase and abolished the stimulatory effect of the V-GSH complex. These results are generally consistent with the hypothesis that V-GSH complex activates Type IV phosphodiesterase by an indirect mechanism, which appears to involve predominantly membrane bound components that may be biologically important enzyme regulatory elements.
向完整分离的大鼠脂肪细胞中添加钒酸盐（Na3VO4）可刺激微粒体（P2）组分中的环磷酸腺苷磷酸二酯酶活性（IV型）。钒酸盐增加了IV型磷酸二酯酶活性的Vmax，而不影响其表观底物亲和力。Na3VO4还刺激了无细胞微粒体和胞质组分中环磷酸腺苷的水解，但这种激活需要还原型谷胱甘肽（GSH）的存在。钒酸盐和谷胱甘肽的混合物呈现出翠绿色溶液（V-GSH复合物），电子顺磁共振显示其含有钒酰离子。单独的GSH或Na3VO4对无细胞微粒体环磷酸腺苷磷酸二酯酶活性均无影响；然而，单独或与GSH联合使用的Na3VO4可刺激该亚细胞组分中环磷酸鸟苷的水解。V-GSH复合物增加了微粒体环磷酸腺苷磷酸二酯酶活性的Vmax，而不影响其表观Km。该复合物的激活作用起效迅速，持续超过30分钟且可逆。激活微粒体环磷酸腺苷磷酸二酯酶的EC50约为5 microM Na3VO4（保持GSH:Na3VO4摩尔比为2:1）；在0.1 mM Na3VO4时达到最大刺激。纯化的微粒体膜显示出与P2组分相似的激活，而在纯化的质膜中仅观察到60%的刺激。V-GSH复合物将基础胰岛素激活的IV型磷酸二酯酶活性提高到共同的最大水平。来自P2组分的经去污剂增溶的环磷酸腺苷磷酸二酯酶受到V-GSH复合物2.5倍的刺激。对P2膜进行有限的胰蛋白酶处理可激活环磷酸腺苷磷酸二酯酶并消除V-GSH复合物的刺激作用
