Monoclonal antibody 3F3 against conformational epitope of Torpedo acetylcholinesterase.

AIM: To study the type of epitope of native Torpedo acetylcholinesterase (AChE) directed by its monoclonal antibody (McAb) 3F3. METHODS: Enzyme-linked immunosorbent assay (ELISA) was used for the assay of the reaction between antigen and antibody. RESULTS: McAb 3F3 immunoreacted well with the native AChE, but not with the reduced- and alkylated-AChE (RA-AChE) at all. Soman did not interfere the binding of 3F3 with AChE molecule. The synthesized 24-peptide containing the active serine residue of the AChE active center did not react with McAb 3F3. CONCLUSION: 3F3 is a monoclonal antibody against the conformational epitope of Torpedo AChE active center, but dose not occupy the active serine residue of the enzyme.

Anionic subsite of active center of Torpedo acetylcholinesterase constructs a part of its conformational epitope.

AIM: To study the structure-activity relationship of Torpedo acetylcholinesterase (AChE) and explore whether the anionic subsite of the active center is a constituent of the conformational epitope of enzyme. METHODS: Using ELISA and enzyme inhibition test to examine the effect of 1-methyl-2-hydroxyiminomethylpyridium chloride (2-PAM), an anionic subsite probe of AChE, on the immunoreactivity between Torpedo AChE and its monoclonal antibody (McAb) 3F3. RESULTS: McAb 3F3 did not react with 2-PAM-AChE complex. 2-PAM decreased the inhibitory rate of McAb 3F3 on AChE in a concentration-dependent fashion, but did not dissociate the McAb 3F3-AChE complex. CONCLUSION: Anionic subsite of the active center of Torpedo AChE constructs a part of its conformational epitope.

Effects of Ilexonin A on circulatory neuroregulation.

Ilexonin A (IA), a pentacyclic triterpene, has been semisynthesized in china for the first time. It is extracted from the root of Ilicis pubescentis, a commonly used herbal medicine in Guangdong for the treatment of cardiovascular, cerebrovascular and peripheral vascular diseases and heart failure with satisfactory effects. The pharmacokinetic studies indicated that the elimination half-life after oral and i.v. dosing were 17.7 +/- 2.4 h and 22.5 +/- 2.9 h respectively. The total clearance was 4.6 +/- 0.51/h. The bioavailability of IA capsules was 0.39 +/- 0.14 and LD50 was 234 mg/Kg. We have adopted modern techniques, including cellular electrophysiology, isotope tracing methods, molecular biology, electromicroscopy, etc., to probe into the pharmacologic mechanisms of the effects of IA on cardiovascular system. The results indicated that IA can increase the contractility of isolated guinea pig auricular myocardium, attenuate vascular smooth muscle tension induced by noradrenaline in the rabbit aorta. IA can exert a biphasic regulatory effect on arterial blood pressure. IA also can prolong A-V duration of Hiss bundle electrograph (HBE) in rabbits and prolong the action potential duration and the effective refractory period (ERP) of myocardial cells in guinea pigs. The results showed that IA can increase the cAMP content in the smooth muscle of aorta and exert a calcium-blockade effect. Therefore, the peripheral resistance vessels are relaxed and the cardiac afterload is lowered. IA-blocked calcium channels are correlated with both the potential-dependent channel and the receptor operated channel in vascular smooth muscles. IA also increases the cAMP content of myocardium and accelerates the cellular calcium influx and efflux, and this may be responsible for the direct mechanism of the positive inotropic action of IA. Under electron microscopy, it is observed that IA can alleviate the defect of succinate dehydrogenase in the myocardial mitochrondria of rabbit chronic congestive heart failure (CF) model and reduce the microstructural damage of the failed myodardium, therefore the anoxic tolerance of myocardium is increased, the effect of IA on the platelet stretching activity and microstructure in the patients with CF is also studied. It is found that IA can reduce the hypercoagulability of blood, decrease the severity of blood stagnation and improve the status of microcirculation. Effects of IA introventricular and cardiovascular central microinjection (nucleus tractus solitarius, paraventricular nucleus) on arterial blood pressure and heart rate were studied. It demonstrated that IA possess circulatory neuroregular effects by the medium of alpha-receptor and beta-receptor of cardiovascular motoneurons.

Epitopes recognized by anti-reduced and alkylated acetylcholinesterase antibodies.

Peptides of the reduced and alkylated acetylcholinesterase (RA-AChE) from the electric organ of Torpediniformes Torpedo torpedo subjected to bromo-cynogen (CNBr) cleavage or/and peptic digestion conserved well the antigen-antibody reactivity with anti-RA-AChE monoclonal antibodies E9, F6, and F12, whereas peptides produced by CNBr and tryptic treatments lost all the reactivity. Periodate oxidation of the RA-AChE or glycopeptidase digestion of the CNBr cleaved RA-AChE did not change the antigen-antibody reactivity. It implied that the epitopes recognized by the 3 anti-RA-AChE monoclonal antibodies are all peptide determinants rather than carbohydrate determinants.

Purification and properties of acetylcholinesterase from human brain.

Acetylcholinesterase from human caudate nucleus and partial thalamus was purified by using Con A-Sepharose, short-arm and long-arm ligand Sepharose affinity chromatographies. SDS-PAGE of the purified AChE under the reduced condition showed one main band, corresponding to a molecular weight of 66 kD. The purified AChE with a specific activity of 3384 U/mg protein represented 20% activity of the homogenate supernatant. Analysis of purified AChE by gradient slab PAGE and DISC-PAGE with activity staining revealed the existence of monomer, dimer, tetramer, hexamer and octomer of the enzyme. The isoelectric point of AChE ranged between pH 5.6 and 6.0. Con A-Sepharose affinity chromatography retained most of the applied AChE activity implying that the enzyme is a kind of glycoprotein. The isolated human brain AChE had no cross-immunoreactivity with 3F3 and weak cross-immunoreactivity with 2G8 and 1H11 anti-Torpedo AChE antibodies. Balb/c mice were immunized with human cerebellum AChE purified with Con A and short-arm ligand affinity chromatographies. The antiserum produced showed strong cross-immunoreactivity with Torpedo AChE but weak cross-immunoreactivity with human RBC membrane AChE. The purified human brain striatum AChE was reduced and alkylated, and then hydrolyzed by immobilized TPCK-treated trypsin. Trypsin peptides in the hydrolysate was separated by RP-HPLC. Several large peptide peaks and numbers of small peaks were observed. The large peaks showed obvious immunoreactivity with the mouse anti human cerebellum AChE antiserum.