Molecular cloning and expression of human myocardial cGMP-inhibited cAMP phosphodiesterase.
We have cloned a cDNA for a myocardial cGMP-inhibited cAMP phosphodiesterase (cGI PDE) from a human heart cDNA library in lambda Zap II. The open reading frame [3.5 kilobases (kb)] of cDNA clone n.13.2 (7.7 kb) encodes a protein of 125 kDa. In Northern blots of total human ventricle RNA, a single mRNA species (8.3 kb) hybridized with a 4-kb EcoRI restriction fragment of clone n.13.2 cDNA (containing the entire open reading frame). The carboxyl-terminal region of the deduced amino acid sequence of the cGI PDE contains the putative catalytic domain conserved among mammalian PDE families. A partial cDNA clone, n.2, encoding a truncated, 54-kDa cGI PDE containing the conserved domain was expressed as a catalytically active fusion protein in Escherichia coli. cAMP hydrolytic activity was inhibited by cGMP and OPC 3911 but not by rolipram. Thus, this report provides direct proof that the conserved domain contains the catalytic core of cGI PDEs.
Meacci
E
E
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, Bethesda, MD 20892.
Taira
M
M
Moos
M
M
Smith
C J
CJ
Movsesian
M A
MA
Degerman
E
E
Belfrage
P
P
Manganiello
V
V
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000494:pharmacology; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D015345:Oligonucleotide Probes / Q000737:chemistry; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D004247:DNA; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases