Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Hydrolase
Patent
1998-11-16
2000-11-21
Carlson, Karen Cochrane
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Hydrolase
435196, C12N 916, C12N 922
Patent
active
061501506
ABSTRACT:
Isolated cDNA clones from human brain (frontal cortex) cDNA libraries that encode a unique subtype of the low K.sub.m, cAMP-specific phosphodiesterases (PDE IVs) are disclosed. Analysis of the distribution of hPDE IV.sub.B mRNA expression in various human tissues using a nonconserved fragment of the cDNA as a probe revealed a restricted pattern of expression, with an .about.4-kb mRNA detected in brain, heart, lung and skeletal muscle and not in placenta, liver, kidney or pancreas. Furthermore, an additional .about.5-kb hPDE IV.sub.B.sup.- related mRNA species was detected in brain tissue. Expression of hPDE IV.sub.B in a genetically-engineered PDE-deficient strain of the yeast Saccharomyces cerevisiae resulted in the overproduction of cAMP PDE activity which displayed the expected kinetic characteristics for a PDE IV: 1) low K.sub.m (4.3 .mu.M) for cAMP, 2) high K.sub.m (>3 mM) for cGMP, and 3) sensitivity to rolipram (K.sub.i =0.085 .mu.M), a selective inhibitor of PDE IV. Recombinant hPDE IV.sub.B also bound [.sup.3 H] R-rolipram saturably and with a high affinity. Analysis of [.sup.3 H] R-rolipram binding data revealed curvilinear Scatchard plots, suggesting the presence of two non-interacting high affinity rolipram binding sites (K.sub.d =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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Livi George P.
McLaughlin Megan M.
Torphy Theodore J.
Bugaisky Gabriele E.
Carlson Karen Cochrane
Hecht Elizabeth J.
King William T.
Kinzig Charles M.
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