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Isolation, biochemical characterization and N‐terminal sequence of rolipram‐sensitive cAMP phosphodiesterase from human mononuclear leukocytes
Author(s) -
Truong V.H.,
Müller T.
Publication year - 1994
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(94)01025-0
Subject(s) - rolipram , size exclusion chromatography , isoelectric point , phosphodiesterase , complementary dna , affinity chromatography , chemistry , peptide sequence , biochemistry , enzyme , cyclic nucleotide phosphodiesterase , peripheral blood mononuclear cell , microbiology and biotechnology , cleavage (geology) , biology , in vitro , gene , paleontology , fracture (geology)
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 K m = 2.2μM, K i = 1.2 μM (rolipram) and ν max = 80 μmol/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 IV A and PDE IV D .