| The concept of receptor was proposed 100 years ago by John Newort Langley and Paul Ehrlich. All the receptor was activated by the binding of Ligand, which generate the correspondent biology effect. The allosteric effect was first observed by Bohr in the process of the combination of Oxygen and Hemoglobin, in which the binding capacity of Hemoglobin to Oxygen increased when the first Oxygen molecular binds to the protein. The consequence study revealed that the conformation of Hemoglobin changes when the oxygen molecular binds to hemoglobin which preferred the consequence binding of other oxygen molecular. The concept of allosterism was first coined by Monod and colleagues in his paper about the mechanism of action between enzyme and substrate. Monod suggested that the proteins involved enzymes and receptors are consists of many subunits. Two conformation R(active state) and R*(inactive state) existing in protein matrix keep the balance, both of the states have the specific allosteric ligand binding site, when the allosteric ligand binds to the site, the balance is broken and the protein change to the ligand preferred conformation by the cooperation of subunits. It is presumed that there are second site-allosteric sites which are different from the agonist recognized site-orthosteric sites. When allosteric regulator binds to the site, usually do not generate effect. The regulation is classified into two effects, positive and negative regulation. Which domain or amino acid sites are involved in the allosteric interactions? Receptors engage in many physiological functions by forming allosteric sites which decide the low conservation of these domains. Many drugs do its effect by the recognization of receptors and ligands to simulate the effect endogenous ligands or inhibit its effect. Orthosteric sites keep high conservation that cause the drug targeting at this type of site doing many side effects.The phenomenon of allosterism has been known for a long time. Recently, more and more studies revealed that allosteric regulator can increase the binding capacity of receptor to specific ligand and effect. Low conservation of allosteric sites offer us a novel way to explore drugs which have allosteric properties. 8021 is a anti-acetylcholine drug which was synthesized in our institute. DM8021 (De-methyl-phencynonate hydrochloride) is one of its methyl metabolin in vitro,which are chiral drug with one chiral centre,thus have two optical isomoters. Our study focused on its L-isomer.Methods:The allosteric interaction of DM8021 was proved through saturation essay, competitive essay and dissociation essay by radioligand binding essay, and we established a new way to compare a competitive interaction with a negative allosteric interaction by studing the relationship between the concentration of the very regulator and its correspondent Kapp. On this foundation, we constructed M2/M5 chimera to study the allosteric site of Gallamine/DM8021 on muscarinic receptors which direct the development of new drugs targeting on GPCR.Results:1. Effect of gallamine on the binding properties of [H3]-NMS to M receptorsa) Saturation essay, Bmax of control group=12.61±1.58 pmol/mg.protein, kd = 0.27±0.14 nM; added a series of concentration of Gallamine: 10μM, Bmax = 5.57±1.33 pmol/mg.protein, kd = 0.85±0.16 nM; 1 mM, Bmax = 8.88±0.15 pmol/mg.protein, kd = 0.83±0.04 nM; 10 mM, Bmax = 9.06±0.90 pmol/mg.protein, kd = 0.37±0.13 nM.b) Competitive essay, we can not fit a classical curve because of Gallamine's biphasic regulation.c) Dissociation essay, 0.01μM dissociation rate=0.078/s, 100μM dissociation rate=0.005/s.2. Effect of R-DM8021 on the binding properties of [H3]-NMS to M receptorsa) Saturation essay, the control groups'Bmax = 15.69±1.20 pmol/mg.protein, kd= 0.98±0.2 nM; added a series of concentration of R-DM8021: 0.1μM, Bmax = 13.54±4.18 pmol/mg.protein, kd = 3.59±1.90 nM; 10 mM, Bmax = 37.73±3.14 pmol/mg.protein, kd = 1.21±0.25 nM.b) Competitive essay, EC50 = 10-6.65M.c) Dissociation essay, 0.01μM dissociation rate = 0.115/s, 100μM dissociation rate = 0.0045/s.3. M2 and M5 recombinate proteins were expressed successfully in Sf9-Baculovirus systems.Conclusions:1. Gallamine shows negative allosteric regulation in whole essay, also shows biphasic regulation. The boundary is 10-8M. When the concentration is less than the boundary, the Bmax shows positive correlation to the concentration, and vice verse.2. DM8021 also shows biphasic allosteric regulation, the boundary is 10-6M. When the concentration is higher than the boundary, the binding of NMS is greatly increased.3. By the result of function essay, we proposed a thorey: The allosterism of DM8021 to NMS and Ach is different. When the concentration is higher than the boundary, the binding of NMS is increased compared with the relaxation of intestinal canal which means the bound of Ach decreased.4. A new way was established to compare a classical competitive reaction with a negative allosteric reaction in which we fitted a new curve about Kapp and its correspondent regulator concentration.5. We expressed M2 and M5 recombinated receptor in Sf9-Baculovirus system which established the foundation preferred the next study.
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