Purification Of Phospholipase A₂ From The Venom Of Naja Atra And Its Action On Muscarinic Acetylcholine Receptor

Snake venom contains many components which interact with the cholinergic system. Although snake toxins which associate with the nAChR have been extensively studied and documented, very few studies on the interaction of snake toxins with the mAChR have been carried out. A group of components isolated from the venom of Dendroaspis angusticeps (green mamba), named muscarinic toxins (MTs), which bind selectively to mAChR, caught our attentions. With their selectivity for some subtype of mAChR, a few of them are used as some pharmacological tools. Furthermore, some of them were shown to act as agonists at mAChR, and improve memory consolidation, which may be useful in the therapy of cognition disorder like Alzheimer's disease. The mamba genus belongs to the Elapidae family, which also includes the cobras. The venom of mambas and cobras has much in common. Therefore, one can assume some components like MTs could be present in the venom of cobras.Snake venom phospholipase A₂ (vPLA₂), as a sort of component with special activities, have been extensively studied, which shares with mammalian secreted phospholipase A₂ (msPLA₂) similar structure and common ability to hydrolyze at the sn-2 position of phospholipids. In addition to their activity of hydrolization, vPLA₂ exhibit a wide variety of pharmacological effects by interfering in normal physiological processes. It was proposed that their unique ability to'target'themselves to a specific organ or tissue is due to their high affinity binding to specific proteins which act as receptors. By using vPLA₂s as tools, a number of mammalian protein targets including receptors which bind vPLA₂s with high affinities have been identified. The study of the interaction between the target protein of vPLA₂ and msPLA₂ led to the discovery of the PLA₂ receptor (PLA₂R), which has expanded our concept of the functions of the msPLA₂ family. Recently, a few of vPLA₂s, like MTs, were found to have the ability to bind with mAChR. However, their pharmacological function with mAChR has never been known. Using the method of chromatography, we have isolated a vPLA₂ with the similar activity to MTs, designated as muscarinic protein (MP) from the venom of Naja atra. Radioligand-binding studies have showed MP possesses high affinity for mAChR and selectivity for their subtype. In isolated muscle preparations with mAChR, it shows that MP active mAChR (especially for M₁ receptor). The study proceeded with three parts.1 Purification and characterization of phospholipase A₂ from the venom of Naja atraThe active principle was isolated by column chromatography on Sephadex G-50, Sephadex G-150, CM-Sepharose Fast Flow, and POROS? CM 4.6/100 Perfusion Chromatography column from the venom of Naja atra. The final preparation was homogeneous as determined by SDS-PAGE and HPLC, with a yield of 10%. The isolated active component, which was designated"muscarinic protein"(MP), was found to induce the contraction of isolated guinea-pig ileal longitudinal muscle-myenteric plexus. MP was determined to be a 13.3 kDa, and have an N-terminal amino acid sequence (NLYQFKNMIQCTVPSR), which is highly homologous with PLA₂ from the venom of genus Naja. MP could hydrolyze phosphatidylcholine in a dose- and temperature-dependent manner. MP was shown to have acute toxicity with LD50 of 14.3 mg/kg.2 The binding to mAChR of the isolated phospholipase A₂ from the venom of Naja atra and its selectivities for subtypeUsing radioligand-binding studies, a model of mAChR-ligand bound was established by the saturation binding experiment with [3H]Quinuclidinylbenzi- late ([3H]QNB) binding to rat telencephalon. The dissociation rate constant (KD) was fixed at 0.17 nmol/L, and the maximumal saturation binding (Bmax) was 1313 fmol/mg. Hill coefficient was obatined as 1.0078. It showed that [3H]-QNB binding to muscarinic cholinergic receptors of rat telencephalon tissue fit into the Clark model. Competition experiments showed that MP suppressed QNB-receptor interaction in a dose-dependent manner. The value of IC₅₀ was determined as 28.0 nmol/L, and then KI (apparent) value was 10.1 nmol/L from the transformation of the value of IC50. It suggests that MP interact with high affinity for mAChR like MI from the venom of Naja naja sputatrix, and are much efficient than atropine and MT2 from the venom of Dendroaspis angusticeps.The selectivity of MP for M₁ or M₂ receptor subtype was determined by assaying the inhibition of [3H]QNB binding to rat cerebrum cortex and rat heart by MP. MP suppressed the binding of QNB to rat cerebrum cortex and rat heart in a dose-dependent manner, completely with enough concentration. The value of IC50 in assays with rat cerebrum cortex is 248.8 nmol/L, while the IC50 value in assays with rat heart is 22.1 nmol/L. It suggests that MP have higher affinity for M₂ receptor than for M₁ receptor.3 Pharmacological action of the isolated phospholipase A₂ from the venom of Naja atra on mAChR3.1 Distinct effect of MP on M₁, M₂ and M3 receptorSeveral pharmacological properties of MP in the electrically stimulated rabbit vas deferens (mediated via M₁ receptors), guinea-pig ileal longitudinal muscle (mediated via M₂ receptors), and guinea-pig atria (mediated via M3 receptors) were compared.Like McN-A-343, MP inhibited the isometric contraction of rabbit vas deferens induced by electrical stimulation in a dose-dependent manner, almost completely with enough dose. The value of EC50 of MP was determined as 23.7 nmol/L, which is 1/10 of that of McN-A-343. In other words, the efficacy of MP in the electrically stimulated rabbit vas deferens is similar to McN-A-343, while the potency of MP is 10-fold of that of McN-A-343. Pirenzepine (10-7 mol/L) shifted the concentration-response curves of MP in the electrically stimulated rabbit vas deferens to the right in parallel without altering the amplitude of the maximal response. It suggests that the effect of MP in the electrically stimulated rabbit vas deferens is mediated via M₁ receptors, and MP may be an agonist of M₁ receptor.MP with low dose (2×10^-10～2×10^-7.5 mol/L) was shown its weak negative inotropic effects on the contraction of guinea pig isolated atria;while MP with high dose (2×10-7～2×10-5.5 mol/L)increase the force of the contraction of guinea pig isolated atria.The action of MP on guinea pig isolated atria shows that MP is likely a partial agonist of mAChR.MP produced a concentration-dependent contractile response in the guinea pig ileum longitudinal muscle, but the maximum force of contraction was only approximately 43% of the maximum obtained with carbachol. The EC50 value for MP was 29.5 nmol/L, which is similar to that for carbachol (48 nmol/L). The action of MP was inhibited by atropine. But the dose of atropine inhibiting the action of MP was higher than inhibiting the action of carbachol. Atropine shifted the concentration-response curves of MP to the right, but reduced the amplitude of the maximal response. It suggests that the action of MP in the guinea pig ileum longitudinal muscle is multiplicity. In addition of activating M3 receptor, other mechanism is concerned.As the result described above shows that MP may activate all of the three receptor subtype (M₁, M₂ and M3 receptor), but MP show different order of potencies on them: M₁ > M3 > M₂.3.2 Mechanism of the action of MP in the guinea pig ileum longitudinal muscle The amplitude of the maximal responses to MP was not significantly affected by hexamethonium (10^-5 mol/L) indicating that MP-induced contraction is medicated via postsynaptic muscarinic receptor. Furthermore, the amplitude of the maximal responses to carbachol, the EC50 values and the slopes of the concentration-response curves were not significant different in the absence and presence of MP (10^-5.2 mol/L) indicating that MP and carbachol are not compete for the same site. In the isolated frog rectus abdominis, the Kreb's solution in which MP (10^-5.7 mol/L) incubated with the guinea pig ileum longitudinal muscle for 15 min produced significant contraction, while MP almost had no action. It suggests that the release of ACh may be involved in the mechanism of MP's action in the guinea pig ileum longitudinal muscle.3.3 The relationship between the PLA₂ enzymatic activity and the action in the guinea pig ileum longitudinal muscleThe amplitude of the maximal responses to MP, the EC50 values and the slopes of the concentration-response curves were not significant different in the absence and presence of DEDA (10-4 mol/L), a PLA₂ competitive inhibitor. Moreover, the concentration-response curves of MP was not significant different at 27℃and 37℃, while the PLA₂ enzymatic activity varied from 27℃to 37℃. From the result above, it suggests that the effect of MP on the guinea pig ileum longitudinal muscle is independent of its PLA₂ enzymatic activity.The other PLA₂, bee venom PLA₂ and porcine pancreas PLA₂, also produced the contraction in isolated guinea-pig ileal longitudinal muscle indicating that the action may be a common activity of PLA₂.Conclusion: 1. a venom phospholipase A₂, designated as muscarinic protein (MP), has been isolated from the venom of Naja atra, which was homogeneous as determined by SDS-PAGE and HPLC. The molecular weight of the isolated muscarinic protein was determined to be 13.3 kDa and the first 16 residues of its N-terminal amino acid sequence were determined to be NLYQFKNMIQCTVP- SR. It is indicated that MP possesses phospholipase A₂ enzymatic activity. 2. MP has high affinity for mAChR with the value of KI being 10.1 nmol/L, Its affinity for mAChR is higher than those of atropine or MTs from the venom of Dendroaspis angusticeps, and is similar to that of MI from the venom of Naja naja sputatrix. The affinity of MP for M₁ receptor subtype is higher than that for M₂ receptor subtype, indicating that MP possesses the selectivity for subtype. 3. MP may activate mAChR as an agonist. MP may possess a strong agonistic action on M₁ receptor subtype and a weak agonistic action on M₂ receptor subtype. 4. The mechanism underlying contraction action of MP on isolated guinea pig ileum smooth muscle may involved in the activation on mAChR by MP and the release of ACh induced by MP, but not the PLA₂ enzymatic activity of MP.