Isolation, biochemical characterization and N-terminal sequence of rolipram-sensitive cAMP phosphodiesterase from human mononuclear leukocytes.
A cyclic AMP specific phosphodiesterase (type IV) was purified 450,000-fold from human peripheral blood mononuclear cells through a sequence of chromatographic steps involving anion exchange, affinity chromatography on a matrix coupled to a derivative of the type IV inhibitor rolipram, and gel filtration. The enzyme showed apparent molecular masses of 70 kDa on gel filtration and 35 kDa on denaturing or native PAGE, indicating a possible dimerization or cleavage under certain conditions. The isoelectric point was 4.6. Kinetic parameters were Km = 2.2 microM, Ki = 1.2 microM (rolipram) and vmax = 80 mumol/min per mg protein. The most probable N-terminal sequence was determined as SLTNTNIPRF, 80% identical to part of the deduced amino acid sequence from cDNA sequences of PDE IVA and PDE IVD.
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Sandoz Research Institute Berne Ltd., Switzerland.
Müller
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D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000201:enzymology*; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D011760:Pyrrolidinones / Q000494:pharmacology*; D020889:Rolipram; D017386:Sequence Homology, Amino Acid
D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram