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| Expression of an active, monomeric catalytic domain of the cGMP-binding cGMP-specific phosphodiesterase (PDE5) |
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| Author(s): Fink TL, Francis SH, Beasley A, Grimes KA, Corbin JD |
| Source: JOURNAL OF BIOLOGICAL CHEMISTRY Volume: 274 Issue: 49 Pages: 34613-34620 Published: DEC 3 1999 |
| Times Cited: 36 References: 57 |
| Abstract: Phosphodiesterases (PDEs) comprise a superfamily of phosphohydrolases that degrade 3',5'-cyclic nucleotides. All known mammalian PDEs are dimeric, but the functional significance of dimerization is unknown, A deletion mutant of cGMP-binding cGMP-specific PDE (PDE5), encoding the 357 carboxyl-terminal amino acids including the catalytic domain, has been generated, expressed, and purified. The K-m of the catalytic fragment for cGMP (5.5 +/- 0.51 mu M) compares well with those of the native bovine lung PDE5 (5.6 mu M) and full-length wild type recombinant PDE5 (2 +/- 0.4 mu M). The catalytic fragment and full-length PDE5 have similar IC50 values for the inhibitors 3-isobutyl-1-methylxanthine (20 mu M) and sildenafil (Viagra (TM)) (4 nm). Based on measured values for Stokes radius (29 Angstrom) and sedimentation coefficient (2.9 S), the PDE5 catalytic fragment has a calculated molecular mass of 35 kDa, which agrees well with that predicted by amino acid content (43.3 kDa) and with that estimated using SDS-polyacrylamide gel electrophoresis (39 kDa), The combined data indicate that the recombinant PDE5 catalytic fragment is monomeric, and retains the essential catalytic features of the dimeric, full-length enzyme. Therefore, the catalytic activity of PDE5 holoenzyme requires neither interaction between the catalytic and regulatory domains nor interactions between subunits of the dimer. |
| Document Type: Article |
| Language: English |
| Reprint Address: Corbin, JD (reprint author), Vanderbilt Univ, Sch Med, Dept Mol Physiol & Biophys, 702 Light Hall,21st & Garland Ave, Nashville, TN 37232 USA |
Addresses:
1. Vanderbilt Univ, Sch Med, Dept Mol Physiol & Biophys, Nashville, TN 37232 USA |
| Publisher: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 9650 ROCKVILLE PIKE, BETHESDA, MD 20814 USA |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: 260XW |
| ISSN: 0021-9258 |
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