Identification and characterization of a Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in a human lymphoblastoid cell line.
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.
Epstein
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Moraski
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Hachisu
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D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases / Q000276:immunology / Q000378:metabolism*; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D002460:Cell Line; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D006801:Humans; D007945:Leukemia, Lymphoid / Q000201:enzymology*; D008214:Lymphocytes / Q000201:enzymology; D013347:Subcellular Fractions / Q000201:enzymology
D002147:Calmodulin; D015087:2',3'-Cyclic-Nucleotide Phosphodiesterases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D002118:Calcium