IDENTIFICATION AND CHARACTERIZATION OF A CA-2+-CALMODULIN-SENSITIVE CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE IN A HUMAN-LYMPHOBLASTOID CELL-LINE
IDENTIFICATION AND CHARACTERIZATION OF A CA-2+-CALMODULIN-SENSITIVE CYCLIC-NUCLEOTIDE PHOSPHODIESTERASE IN A HUMAN-LYMPHOBLASTOID CELL-LINE
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DOI：
10.1042/bj2430533
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发表时间：
1987-04-15
影响因子：
4.1
通讯作者：
HACHISU, R
中科院分区：
文献类型：
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作者：
EPSTEIN, PM;MORASKI, S;HACHISU, R
本研究探讨的模式和监管性质的环核苷酸磷酸二酯酶在人类淋巴母细胞样B细胞系（RPMI 8392）建立从急性淋巴细胞白血病患者。在该细胞系中，在0.25 μ M-环AMP下测得的磷酸二酯酶活性约为0.15 μ M。7-倍以上，在分离的人外周血淋巴细胞，和16%的磷酸二酯酶活性在RPMI 8392细胞与颗粒馏分。该细胞系的粗级分中的磷酸二酯酶活性可重复地被Ca 2 +-钙调蛋白刺激约60-80%。在存在20 nM-钙调蛋白的情况下，半最大刺激发生在0.7 μ M-Ca 2+处。用DEAE-Sephacel柱层析法将RPMI 8392细胞胞浆磷酸二酯酶活性分离为两种形式。第一种形式在约100 ° C下洗脱。0.2 M-乙酸钠，催化环AMP和环GMP的水解，并被Ca 2 +-钙调蛋白刺激3倍。该形式在不存在钙调蛋白的情况下表现出环AMP的非线性动力学，外推的Km值为0.8和4 μ M，而在存在钙调蛋白的情况下表现出非线性动力学，外推的Km值为0.5和1 μ M。Vmax值增加约。3-被钙调素折叠。第二种形式在约100 ° C下洗脱。0.6 M-乙酸钠，对环AMP具有特异性，对Ca 2 +-钙调蛋白的刺激不敏感。从DEAE-Sephacel柱上分离的钙调素敏感的磷酸二酯酶可以吸附到钙调素-Sepharose亲和柱上，用EGTA洗脱。这种酶活性也可以通过针对钙调蛋白-牛心脏磷酸二酯酶复合物的单克隆抗体进行免疫沉淀。本研究记录了来自白血病患者的培养的淋巴母细胞系中存在Ca 2+钙调素敏感的磷酸二酯酶。
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 .mu.M-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 .mu.M-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 .mu.M, and non-linear kinetics in the presence of calmodulin, with extrapolated Km values of 0.5 and 1 .mu.M. 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.