Epstein P M, Moraski S, Hachisu R
Biochem J. 1987 Apr 15;243(2):533-9. doi: 10.1042/bj2430533.
This study examines the pattern and regulatory properties of cyclic nucleotide phosphodiesterases in a human lymphoblastoid B-cell line (RPMI 8392) established from a patient with acute lymphocytic leukaemia. In this cell line, phosphodiesterase activity measured at 0.25 microM-cyclic AMP is approx. 7-fold greater than that in isolated human peripheral-blood lymphocytes, and 16% of the phosphodiesterase activity in RPMI 8392 cells is associated with particulate fractions. Phosphodiesterase activity in crude fractions of this cell line is reproducibly stimulated by about 60-80% by Ca2+-calmodulin. In the presence of 20 nM-calmodulin, half-maximal stimulation occurs at 0.7 microM-Ca2+. The cytosolic phosphodiesterase activity of RPMI 8392 cells is separated into two forms by DEAE-Sephacel chromatography. The first form is eluted at approx. 0.2 M-sodium acetate, catalyses the hydrolysis of both cyclic AMP and cyclic GMP, and is stimulated 3-fold by Ca2+-calmodulin. This form exhibits non-linear kinetics for cyclic AMP in the absence of calmodulin, with extrapolated Km values of 0.8 and 4 microM, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 microM. The Vmax. values are increased approx. 3-fold by calmodulin. The second form is eluted at approx. 0.6 M-sodium acetate, is specific for cyclic AMP, and insensitive to stimulation by Ca2+-calmodulin. The Ca2+-calmodulin-sensitive phosphodiesterase from the DEAE-Sephacel column can be adsorbed to a calmodulin-Sepharose affinity column and eluted with EGTA. This enzymic activity can also be immunoprecipitated by a monoclonal antibody directed against a calmodulin-bovine heart phosphodiesterase complex. This study documents the existence of Ca2+-calmodulin-sensitive phosphodiesterase in a cultured lymphoblastoid cell line derived from a leukaemic patient.
本研究检测了从一名急性淋巴细胞白血病患者建立的人淋巴母细胞样B细胞系（RPMI 8392）中环核苷酸磷酸二酯酶的模式和调节特性。在该细胞系中，以0.25微摩尔/升环磷酸腺苷（cAMP）测定的磷酸二酯酶活性约比分离的人外周血淋巴细胞中的活性高7倍，且RPMI 8392细胞中16%的磷酸二酯酶活性与颗粒部分相关。该细胞系粗提物中的磷酸二酯酶活性可被Ca2+-钙调蛋白可重复地刺激约60 - 80%。在存在20纳摩尔/升钙调蛋白的情况下，半最大刺激发生在0.7微摩尔/升Ca2+时。通过DEAE - 琼脂糖凝胶柱层析将RPMI 8392细胞的胞质磷酸二酯酶活性分离为两种形式。第一种形式在约0.2摩尔/升醋酸钠处洗脱，催化cAMP和环磷酸鸟苷（cGMP）的水解，并被Ca2+-钙调蛋白刺激3倍。在不存在钙调蛋白时，这种形式对cAMP表现出非线性动力学，外推的米氏常数（Km）值为0.8和4微摩尔，在存在钙调蛋白时也表现出非线性动力学，外推的Km值为0.5和1微摩尔。最大反应速度（Vmax）值被钙调蛋白增加约3倍。第二种形式在约0.6摩尔/升醋酸钠处洗脱，对cAMP具有特异性，且对Ca2+-钙调蛋白的刺激不敏感。来自DEAE - 琼脂糖凝胶柱的Ca2+-钙调蛋白敏感的磷酸二酯酶可吸附到钙调蛋白 - 琼脂糖亲和柱上并用乙二醇双四乙酸（EGTA）洗脱。这种酶活性也可被针对钙调蛋白 - 牛心磷酸二酯酶复合物的单克隆抗体免疫沉淀。本研究证明了在源自白血病患者的培养淋巴母细胞样细胞系中存在Ca2+-钙调蛋白敏感的磷酸二酯酶。
