I2-imidazoline binding sites: relationship with different monoamine oxidase domains and identification of histidine residues mediating ligand binding regulation by H+1.
We have shown that I2-imidazoline binding sites (I2BSs) are located on both monoamine oxidases A (MAO-A) and B (MAO-B) and are selectively regulated by H+ and K+ in vitro. In the present study we used chemical modifying agents to investigate the localization of I2BSs with respect to different MAO domains and the mechanisms of ligand binding regulation by K+ and H+. In mitochondrial or solubilized preparations from rabbit kidney and liver, modification of cysteine residues, which are critical for MAO activity, did not affect [3H]idazoxan binding, indicating that I2BS is not associated to the cysteine-containing flavin adenine dinucleotide (FAD) prosthetic group or to the catalytic site of MAOs. Among various chemical modifying agents, only diethylpyrocarbonate and 4-bromophenacyl bromide, two histidine modifying agents, inhibited [3H]idazoxan binding to I2BS. The pH profile of diethylpyrocarbonate effect was consistent with the specific modification of histidine residues. In protection experiments, the effect of diethylpyrocarbonate was not prevented in the presence of saturating concentrations of amiloride, guanabenz or KCl, suggesting that these residues are not located within the ligand or K+ binding sites. In contrast, histidine residues appear to be within a MAO domain involved in regulation of [3H]idazoxan binding by H+. Indeed, the pH-dependent increase in [3H]idazoxan binding was fully abolished after treatment of solubilized material with diethylpyrocarbonate. In conclusion, our results show that MAO I2BSs are not located within the flavin adenine dinucleotide prosthetic group or the catalytic site. Histidine(s) residue(s) involved in the regulation of ligand binding to I2BS by H+ also has been identified.
Limon-Boulez
I
I
INSERM U388, Institut Louis Bugnard, CHU Rangueil, Toulouse, France.
Tesson
F
F
Gargalidis-Moudanos
C
C
Parini
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D004146:Dioxanes / Q000378:metabolism; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D019329:Idazoxan; D007093:Imidazoles / Q000378:metabolism; D054755:Imidazoline Receptors; D008024:Ligands; D008297:Male; D008969:Molecular Sequence Data; D008995:Monoamine Oxidase / Q000378:metabolism*; D011817:Rabbits; D011955:Receptors, Drug / Q000378:metabolism*; D013329:Structure-Activity Relationship
D004146:Dioxanes; D007093:Imidazoles; D054755:Imidazoline Receptors; D008024:Ligands; D011955:Receptors, Drug; D006639:Histidine; D008995:Monoamine Oxidase; D019329:Idazoxan