Isolation, biochemical characterization and N‐terminal sequence of rolipram‐sensitive cAMP phosphodiesterase from human mononuclear leukocytes

FEBS Letters

Truong, V.H.¹  Müller, T.¹ 

[1] Sandoz Research Institute Berne Ltd., Monbijoustrasse 115, CH-3007 Berne, Switzerland

关键词: 3′: 5′ Cyclic AMP phosphodiesterase (mononuclear leukocyte;    human blood);    Rolipram;    Sequence homology;    Amino acid;    PDE;    3′:5′-cyclic nucleotide phosphodiesterase;    PBS;    phospate-buffered saline;    EDTA;    ethylenediaminetetraacetic acid disodium salt dihydrate;    PMSF;    phenylmethylsuphonylflouride;    CHAPS;    3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate;   

DOI  :  10.1016/0014-5793(94)01025-0

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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.

【 授权许可】

Unknown