Duttagupta C, Rifas L, Makman M H
Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Biochim Biophys Acta. 1978 Apr 12;523(2):385-94. doi: 10.1016/0005-2744(78)90041-4.
Cyclic nucleotide phosphodiesterase activity (3', 5'-cyclic-nucleotide 5'-nucleotidohydrolase, 3.1.2.17) was studied in homogenates of WI-38 human lung fibroblasts using 0.1--200 microgram cyclic nucleotides. Activities were observed with low Km for cyclic AMP(2--5 micron) and low Km for cyclic GMP (1--2 micron) as well as with high Km values for cyclic AMP (100--125 micron) and cyclic GMP (75--100 micron). An increased low Km cyclic AMP phosphodiesterase activity was found upon exposure of intact fibroblasts to 3-isobutyl-1-methylxanthine, an inhibitor of phosphodiesterase activity in broken cell preparations, as well as to other agents which elevate cyclic AMP levels in these cells. The enhanced activity following exposure to 3-isobutyl-1-methylxanthine was selective for the low Km cyclic AMP phosphodiesterase since there was no change in activity of low Km cyclic GMP phosphodiesterase activity or in high Km phosphodiesterase activity with either nucleotide as substrate. The enhanced activity due to 3-isobutyl-1-methylxanthine appeared to involve de novo synthesis of a protein with short half-life (30 min), based on experiments involving cycloheximide and actinomycin D. This activity was also enhanced with increased cell density and by decreasing serum concentration. Studies of some biochemical properties and subcellular distribution of the enzyme indicated that the induced enzyme was similar to the non-induced (basal) low Km cyclic AMP phosphodiesterase.
使用0.1 - 200微克环核苷酸，对WI - 38人肺成纤维细胞匀浆中的环核苷酸磷酸二酯酶活性（3',5'-环核苷酸5'-核苷酸水解酶，3.1.2.17）进行了研究。观察到对环磷酸腺苷（cAMP）的低Km值（2 - 5微米）和对环磷酸鸟苷（cGMP）的低Km值（1 - 2微米）的活性，以及对cAMP（100 - 125微米）和cGMP（75 - 100微米）的高Km值的活性。完整的成纤维细胞暴露于3 - 异丁基 - 1 - 甲基黄嘌呤（破碎细胞制剂中磷酸二酯酶活性的抑制剂）以及其他能提高这些细胞中cAMP水平的试剂后，发现低Km的cAMP磷酸二酯酶活性增加。暴露于3 - 异丁基 - 1 - 甲基黄嘌呤后增强的活性对低Km的cAMP磷酸二酯酶具有选择性，因为以任何一种核苷酸为底物时，低Km的cGMP磷酸二酯酶活性或高Km磷酸二酯酶活性均无变化。基于涉及环己酰亚胺和放线菌素D的实验，3 - 异丁基 - 1 - 甲基黄嘌呤引起的活性增强似乎涉及半衰期短（30分钟）的蛋白质的从头合成。随着细胞密度增加和血清浓度降低，该活性也增强。对该酶的一些生化特性和亚细胞分布的研究表明，诱导的酶与未诱导的（基础的）低Km的cAMP磷酸二酯酶相似。
