Comparison of putative cGMP-binding regions in bovine brain and cardiac cGMP-stimulated phosphodiesterases.
After photolabelling of purified bovine brain particulate cGMP stimulated phosphodiesterase (PDE) with [32P]cGMP, incubation with V8 Endoproteinase Glu-C produced several fragments; most of the [32P] was associated with smaller fragments (approximately 12-14 kDa), and some occasionally with larger fragments (approximately 55-57 kDa). Partial amino acid sequences were determined for the smaller photolabelled fragments and other peptides. On Western immunoblots, affinity-purified antibodies against a synthetic peptide with a sequence matching part of that of the approximately 12-14 kDa photolabelled material reacted with intact PDE and the approximately 12-14 kDa and approximately 55-57 kDa fragments. Several partial cDNA clones encoding the cGMP-stimulated PDE were isolated from a Lambda Zap II bovine brain cDNA library. Deduced amino acid sequence from one cDNA clone, lambda cGS 3-1, as well as the partial sequence of the approximately 12-14 kDa and other fragments, exhibited considerable identity with amino acid residues 311-921 of the cardiac cGMP-stimulated PDE (Trong et al., 1990), including the putative cGMP-binding domain (Charbonneau et al., 1990). These results further define this cGMP-binding domain and suggest that different cGMP-stimulated PDEs will exhibit considerable homology, at least in their cGMP-binding region(s) and catalytic domains.
Tanaka
T
T
Laboratory of Cellular Metabolism, NHLBI, NIH.
Hockman
S
S
Moos
M
M
Taira
M
M
Meacci
E
E
Murashima
S
S
Manganiello
V C
VC
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D002417:Cattle; D006152:Cyclic GMP / Q000302:isolation & purification*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D015345:Oligonucleotide Probes; D009842:Oligopeptides / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology; D010446:Peptide Fragments; D011817:Rabbits
D000345:Affinity Labels; D015345:Oligonucleotide Probes; D009842:Oligopeptides; D010446:Peptide Fragments; D004247:DNA; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D006152:Cyclic GMP