Background: Inosine monophosphate dehydrogenase (IMPDH) controls the biosynthetic pathway of guanine nucleotides. The IMPDH-catalyzed conversion of IMP to XMP is the rate-limiting step in guanine nucleotide biosynthesis. It is nowadays accepted that binding of purine nucleotides to the allosteric regulatory domains leads to overall conformational changes of the enzyme. In this report, we are aimed to evaluate the mode of allosteric regulation of IMPDH1 (514) and IMPDH1 (546), via their structural variation, using mainly chromatography techniques. Methods: Following cloning, expression and affinity purification of the mouse retinal enzymes, we added identical concentrations of each enzyme to the assembly buffer (50mM Tris, 100mM KCl, 1mM DTT, pH 7.4 containing 3mM IMP, 5mM NAD and 0.1mM of either ATP, GTP and/or MPA), incubated for 15 min at room temperature, followed by chromatography/spectroscopy overall structural evaluations. Results: Our results clearly indicated that ATP augmented the association of the monomeric subunits of both IMPDH1 isoforms to octameric format with activity enhancement. However, MPA treatments led to macromolecular aggregates devoid of catalytic activity. Despite ATP, GTP treatments led to dispersion of the macromolecular associates and activity quenching. Conclusion: Based on our data, it can be concluded that modulation of IMPDH1 (514,546 variants) activities by the allosteric modulators (ATP or GTP) and the MPA inhibitor mainly occur via subunit association/disassociation of the enzyme subunits. Different responses of the 514 and 546 isoforms to these treatments were seen.
