DiSanto M E, Heaslip R J
Division of Inflammation, Wyeth-Ayerst Research, Princeton, NJ 08543-8000.
Biochem Biophys Res Commun. 1993 Dec 30;197(3):1126-31. doi: 10.1006/bbrc.1993.2594.
Cytosolic cyclic nucleotide phosphodiesterases (PDEs) from human (promonocytic) U937 cells were rapidly resolved by DEAE-Sepharose CL-6B anion exchange chromatography into two major peaks of cAMP-specific activity possessing average Kms of 1.70 microM (Peak 1) and 1.65 microM (Peak 2). Both peaks were predominantly PDE-IV, but possessed molecular weights higher than those generally reported for partially purified PDE-IVs. Storage of Peak 2 for 24 h at 4 degrees C resulted in a doubling of its Vmax and an apparent decrease in its molecular weight. Activation of Peak 2 PDE-IV was prevented when the sodium acetate concentration in its buffer was reduced by dilution immediately following isolation. Although the relevance of this activation to cellular regulation of PDE-IV is undefined, the isolation and stabilization of PDE-IV in its large molecular weight form will be critical to future investigations of PDE-IV regulation.
通过DEAE-琼脂糖CL-6B阴离子交换色谱法，可将人（前单核细胞）U937细胞中的胞质环核苷酸磷酸二酯酶（PDEs）快速分离为两个具有cAMP特异性活性的主要峰，其平均Km值分别为1.70微摩尔（峰1）和1.65微摩尔（峰2）。这两个峰主要是PDE-IV，但分子量高于部分纯化的PDE-IV通常报道的分子量。将峰2在4℃下储存24小时，其Vmax增加一倍，分子量明显降低。分离后立即通过稀释降低其缓冲液中的乙酸钠浓度时，可防止峰2的PDE-IV激活。尽管这种激活与PDE-IV的细胞调节的相关性尚不清楚，但以其大分子形式分离和稳定PDE-IV对于未来PDE-IV调节的研究至关重要。
