| 摘要 |
Isolated cDNA clones from human brain (frontal cortex) cDNA libraries that encode a unique subtype of the low Km, cAMP-specific phosphodiesterases (PDE IVs) are disclosed. Analysis of the distribution of hPDE IVB mRNA expression in various human tissues using a nonconserved fragment of the cDNA as a probe revealed a restricted pattern of expression, with an ~4-kb mRNA detected in brain, heart, lung and skeletal muscle and not in placenta, liver, kidney or pancreas. Furthermore, an additional ~5-kb hPDE IVB-related mRNA species was detected in brain tissue. Expression of hPDE IVB in a genetically-engineered PDE-deficient strain of the yeast Saccharomym cerevisiae resulted in the overproduction of cAMP PDE activity which displayed the expected kinetic characteristics for a PDE IV: 1) low Km (4.3 muM) for cAMP, 2) high Km (>3 mM) for cGMP, and 3) sensitivity to rolipram (Ki=0.085 muM), a selective inhibitor of PDE IV. Recombinant hPDE IVB also bound [3H] R-rolipram saturably and with a high affinity. Analysis of [3H] R-rolipram binding data revealed curvilinear Scatchard plots, suggesting the presence of two non-interacting high affinity rolipram binding sites (Kd=0.4 and 6 nM) or a negatively cooperative interaction among multiple binding sites.This novel enzyme is particularly useful for screening candidate compounds for their ability to serve as potential anti-depressant, antiasthmatic or anti-inflammatory agents.
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