Identification and stabilization of large molecular weight PDE-IVs from U937 cells.
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.
DiSanto
M E
ME
Division of Inflammation, Wyeth-Ayerst Research, Princeton, NJ 08543-8000.
Heaslip
R J
RJ
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D003600:Cytosol / Q000201:enzymology; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D013379:Substrate Specificity; D013997:Time Factors; D014407:Tumor Cells, Cultured
D015105:3',5'-Cyclic-AMP Phosphodiesterases