Shina R, Crain R C, Rosenberg P, Condrea E
Basil and Gerald Felsenstein Medical Research Center, Petah Tikva, Israel.
Toxicon. 1994 Jun;32(6):675-85. doi: 10.1016/0041-0101(94)90337-9.
Phospholipase A2 (PLA2) toxins act presynaptically to block acetylcholine release and are much more potent and specific in their actions than PLA2 enzymes even though they have lower enzymatic activity. Since their mechanism of action is not completely understood, it was of interest to examine the toxins' effects on phospholipid asymmetry as changes in asymmetry are associated with changes in membrane functioning. Rat brain synaptosomes were treated with the PLA2 toxins beta-bungarotoxin (beta-BuTx) and notexin and with the PLA2 enzymes Naja nigricollis and Naja naja atra under relatively non-disruptive conditions as judged by leakage of lactate dehydrogenase (LDH) and levels of phospholipid hydrolysis. The exposure of phosphatidylcholine (PC) and phosphatidylinositol (PI) on the synaptosomal surface was investigated by means of a specific PC-exchange protein (PCEP) and a PI-specific phospholipase C (PI-PLC), respectively. Treatment of the synaptosomes with N. nigricollis PLA2, beta-BuTx and notexin did not affect the availability of PC to exchange by PCEP, but significantly increased the exposure of PI to hydrolysis by PI-PLC. In contrast, N. n. atra PLA2 slightly decreased the exposure of PC and did not affect that of PI. The differences between N. n. atra PLA2, on the one hand, and N. nigricollis PLA2, beta-BuTx and notexin, on the other hand, parallel differences in their pharmacological activities. Our earlier studies showed that PLA2 enzymes, and possibly PLA2 toxins, have a pharmacological site separate from the enzymatic site. Since in the present study the effect on PI was abolished by EDTA, the presence of an enzymatic site in addition to the pharmacological site may be required or alternatively divalent cations may be required for the effects on PI asymmetry independent of the inhibition of PLA2 by EDTA.
磷脂酶A2（PLA2）毒素在突触前发挥作用，阻断乙酰胆碱释放，尽管其酶活性较低，但其作用比PLA2酶更强且更具特异性。由于其作用机制尚未完全明确，因此研究毒素对磷脂不对称性的影响很有意义，因为不对称性的变化与膜功能的改变相关。在相对不破坏细胞的条件下，用乳酸脱氢酶（LDH）泄漏和磷脂水解水平来判断，将大鼠脑突触体分别用PLA2毒素β-银环蛇毒素（β-BuTx）和诺维毒素以及PLA2酶黑颈眼镜蛇毒和中华眼镜蛇毒进行处理。分别通过一种特异性的磷脂酰胆碱交换蛋白（PCEP）和一种磷脂酰肌醇特异性磷脂酶C（PI-PLC）来研究突触体表面磷脂酰胆碱（PC）和磷脂酰肌醇（PI）的暴露情况。用黑颈眼镜蛇毒PLA2、β-银环蛇毒素和诺维毒素处理突触体并不影响PC通过PCEP进行交换的可及性，但显著增加了PI对PI-PLC水解的暴露。相比之下，中华眼镜蛇毒PLA2略微降低了PC的暴露，且不影响PI的暴露。一方面中华眼镜蛇毒PLA2与另一方面黑颈眼镜蛇毒PLA2、β-银环蛇毒素和诺维毒素之间的差异与它们的药理活性差异相似。我们早期的研究表明，PLA2酶以及可能的PLA2毒素具有一个与酶活性位点分开的药理位点。由于在本研究中EDTA消除了对PI的影响，除了药理位点之外可能还需要一个酶活性位点，或者对PI不对称性的影响可能需要二价阳离子，这与EDTA对PLA2的抑制无关。
