The Effects Of Substrates And Inhibitor On The Submolecular Structure Of Acetylcholinesterase Tetramer Incorporated In A Mica-supported Artificial Phospholipid Membrane

Objective: To explore the possible mechanism of high efficiency of acetylcholinesterase (AChE) hydrolyzing substrates, and study the effects of substrates and inhibitor on the submolecular structures of AChE tetramer (AChE G4) incorporated in a mica-supported artificial phospholipid membrane. Contents:The preparation of artificial phospholipid membrane reconstituted on freshly cleaved mica.The incorporation of AChE in a mica-supported phospholipids membrane.The morphologic study of the molecular isoforms of AChE. The separation and purification of AChE G4 and AChE G1.The effects of ACh and inhibitor on the submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane. The submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane and the substrate inhibition of AChE. The effects of BCh on the submolecular structures of AChE G4 incorporated in a mica-supported artificial phospholipid membrane. Methods: Ice-bath ultrasound was used to prepare phospholipid membrane. Ves-fusion technique was applied to the reconstitution of AChE G4 in the phospholipid membrane on mica.The phase imaging of AFM-Tapping mode was applied to verify whether AChEs were reconstituted in the phospholipid membrane or not. The molecular isoforms of AChE were observed with atomic force microscope (AFM) and transmission electron microscopy (TEM). FPLC was used to separate and purify AChE G4 and AChE G1. The changes of submolecular structure of AChE G4 incorporated in a mica-supported artificial lipid layer were imaged with AFM before and after reacted with substrate acetylcholine (ACh) with or without the existence of propidium (PAS inhibitor).The changes of submolecular structure of AChE G4 were also imaged with AFM before and after reacted with 7μM butyrocholine (BCh). Results: The mica-supported artificial phospholipid membrane was even, flat and there was not obvious defects, and the height of phospholipid membrane was (2.3±0.3) nm, which was the thickness of phospholipid monolayer. The ability of forming membrane of soybean lecithin is inferior to ovolecithin at the same concentration, and soybean lecithin had more big sizes of defects and higher roughness than ovolecithin. The borders of phospholipid membrane displayed by AFM phase imaging were highlighted, and enzyme particles were seen clearly to be inlayed uniformly in the phospholipid membrane compared with those of AFM height imaging, and enzyme particles were darker than phospholipid membrane, demonstrating the elasticity and viscosity of enzymes were bigger than those of phospholipid membrane. Before reacted with substrates, single AChE G4 particle was ellipsoid in shape, and had smooth surface with a central projection and clear border and the four subunits of single enzyme particle were arranged tightly, no separated subunits being seen, with a mean size of (89±7) nm long, (68±9) nm wide and (6±3) nm high. The size of single AChE G1 particle was(18±5)nm long×( 15±4)nm wide×(4.02±0.67)nm high. After reacted with 7μM S-ACh, loose arrangement of subunits of G4 AChE was seen, with the mean size of (104±7) nm long, (91±5) nm wide and (8±2) nm high, and there was an apparent free space in the middle of the four subunits of the AChE G4, which was consistent with the results of theΧ–ray diffraction crystallography and molecular dynamics study. The apparent free space was the central path of AChE G4, changing from small to big to small to lateral door appearance, with the mean size of (60±5)nm long and (51±9)nm wide. The mean size of lateral door was (52±5) nm wide and (32±3) nm deep. In the presence of PAS inhibitor, ACh couldn't cause topological structure changes of AChE G4.The changes of submolecular structure AChE G4 reacted with 50μM S-ACh were obviously different from those reacted with 7μM S-ACh, forming some ring-like structures among some proteins.The diameters of these structures were from 350nm to 400nm, and there were linkages between them. At the same time, there were some separated proteins with the diameter of 150nm to 170nm, not forming analogous structures like rings among proteins. The sizes of AChE G4 reacted with 125μM S-ACh were apparently smaller than those reacted with 7μM and 50μM S-ACh, with the mean size of (16±2) nm long, (15±1) nm wide and (1±0.2) nm high. The enzymes after reacted with 7μM S-BCh remained to be distributed on the artificial phospholipid membranes, with mean sizes of (100±5) nm long, (87±6)nm wide and (7±3)nm high, and there was a (38±4)nm long and (35±2)nm wide apparent free space among the subunits of the enzyme. Conclusion: The microscopic forming procedures of phospholipid membrane could be observed with AFM, which is from the adsorption of liposomes to the adherence to form the point of touching between the phospholipid layers to the ruptures of liposomes to form big phospholipid layer and to the fusion among big phospholipid layers. AChE could be verified to be reconstituted successfully in the phospholipid membrane by the difference of elasticity and viscosity between proteins and membranes and the highlight borders of membranes. According to the changes of submolecular structure AChE G4 after reacted with 7μM S-ACh, we raised the possible mechanism of high efficiency AChE of hydrolyzing substrates and the possible key procures as follows:①there are only two PASs of tightly wild AChE G4 exposed, and cationic substrate AChs are attracted by the strong electrostatic field of AChE to combine with exposed PASs at the presence of ACh,②ACG of monomer of AChE G4 is open, and AChs enter the bottom of ACG,③substrates are hydrolyzed,④the repulsion among subunits of AChE G4 are increased flashily, forming a central path among them, and the four PASs are even more exposed by the allosterism of subunits,⑤the products choline and acetic acid combined with the active site of monomer of AChE G4 arrive at the central path via the"back door"of monomer,⑥"lateral door"of the enzyme is open, and enzyme tightens further,⑦products leave enzyme,⑧enzyme recovers to the normal state. The changes of the submolecular structure of AChE G4 are adapted to the high efficiency of AChE hydrolyzing substrates. AFM verified the central path might govern the turnover of the enzyme morphologically and the interactions between PAS and ACh might gate the creation of central path and the open of ACG in monomer; and the combination of ACh with PAS is conducive to the open of ACG while PAS inhibitor can inhibit this action. Some AChE G4s after reacted with 50μM S-ACh form big ring-like structures might be an open path among 2～3 AChE G4 to achieve the rapid hydrolysis of ACh, which is in accordance with the high efficiency of AChE. The phenomenon of inhibition of substrates of AChE G4 after reacted with 125μM S- ACh is brought about from the depolymerization of AChE G4, and the result of the inhibition of substrates is originated from monomer of AChE. The arrangement of four subnnits of AChE G4 after reacted with 7μM S-BCh is the same loose as those after reacted with 7μM S-ACh, and there is a smaller apparent free space than those after reacted with the same concentrational ACh, which is the possible reason of slower speed of AChE hydrolysing BCh than ACh. Resolution at inframolecular level is favourable to provide substantial information on the relative orientations of the subunits within the polymer of enzyme under the effect of substrates with or without the existence of inhibitor.