title: Metal ion stimulators of PDE5 cause similar conformational changes in the enzyme as does cGMP or sildenafil.
abstract: Purified PDE5 preparations exhibited variable proportions of two mobility forms (Bands 2 and 3) by native PAGE. Treatment of recombinant or native PDE5 with either cGMP or a substrate analog such as sildenafil, each of which is known to produce stimulatory effects on enzyme functions, caused a similar native PAGE band-shift to the lower mobility form (shift of Band 2 to Band 3). Incubation of PDE5 with Mg(++) or Mn(++), which is known to stimulate activity, caused a similar shift of the enzyme from Band 2 to Band 3 as did cGMP or sildenafil, but incubation with EDTA caused a time- and concentration-dependent shift to higher mobility (shift of Bands 2 and 3 to Band 1). A slow time course of the EDTA-induced band-shift suggested removal of a pre-bound metal ion (Me(++)) with affinity of ~0.1 nM, which was similar to the previously determined affinity of PDE5 for Zn(++). The EDTA-treated enzyme (Band 1) could be shifted to Bands 2 and 3 by addition of cGMP, sildenafil, or Me(++); however, the cGMP- or sildenafil-induced shift was inhibited and the Me(++)-induced shift was facilitated by treatment with EDTA. Results suggested that Me(++) removal from PDE5 produces a unique apoenzyme form (Band 1, more globular, negatively charged, or both) of PDE5 that can be partially converted to forms (Band 2, less globular or negatively charged, or both; and Band 3, more elongated/positively charged, or both) by addition of Me(++), substrate, or substrate analog. It is concluded that Me(++) causes conversion of PDE5 to similar conformational forms as caused by substrate or inhibitor binding to the catalytic site.
authors.affiliation: Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615, USA. Jackie.corbin@vanderbilt.edu
authors.forename: Jackie D
authors.initials: JD
authors.lastname: Corbin
authors.forename: Teri-Lee
authors.initials: TL
authors.lastname: Foster
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Bessay
authors.forename: Jennifer
authors.initials: J
authors.lastname: Busch
authors.forename: Mitsi
authors.initials: M
authors.lastname: Blount
authors.forename: Sharron H
authors.initials: SH
authors.lastname: Francis
mesh_terms: D020134:Catalytic Domain; D006152:Cyclic GMP / Q000494:pharmacology*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 / Q000737:chemistry* / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007477:Ions / Q000737:chemistry; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology*; D058986:Phosphodiesterase 5 Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D010879:Piperazines / Q000737:chemistry / Q000494:pharmacology*; D011687:Purines / Q000737:chemistry / Q000494:pharmacology; D000068677:Sildenafil Citrate; D013450:Sulfones / Q000737:chemistry / Q000494:pharmacology*
chemical_list: D007477:Ions; D008670:Metals; D058986:Phosphodiesterase 5 Inhibitors; D010879:Piperazines; D011687:Purines; D013450:Sulfones; D008345:Manganese; D004492:Edetic Acid; D000068677:Sildenafil Citrate; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; C517381:PDE5A protein, human; D006152:Cyclic GMP; D008274:Magnesium