Saikia Debashree, Bordoloi Naba K, Chattopadhyay Pronobesh, Choklingam S, Ghosh Siddhartha S, Mukherjee Ashis K
Microbial Biotechnology and Protein Research Laboratory, Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur-784 028, Assam, India.
Biochim Biophys Acta. 2012 Dec;1818(12):3149-57. doi: 10.1016/j.bbamem.2012.08.005. Epub 2012 Aug 16.
An acidic phospholipase A₂ (RVVA-PLA₂-I) purified from Daboia russelli venom demonstrated dose-dependent catalytic, mitochondrial and erythrocyte membrane damaging activities. RVVA-PLA₂-I was non-lethal to mice at the tested dose, however, it affected the different organs of mice particularly the liver and cardiac tissues as deduced from the enzymatic activities measured in mice serum after injection of this PLA₂ enzyme. RVVA-PLA₂-I preferentially hydrolyzed phospholipids (phosphatidylcholine) of erythrocyte membrane compared to the liver mitochondrial membrane. Interestingly, RVVA-PLA₂-I failed to hydrolyze membrane phospholipids of HT-29 (colon adenocarcinoma) cells, which contain an abundance of phosphatidylcholine in its outer membrane, within 24h of incubation. The gas-chromatographic (GC) analysis of saturated/unsaturated fatty acids' release patterns from intact mitochondrial and erythrocyte membranes after the addition of RVVA-PLA₂-I showed a distinctly different result. The results are certainly a reflection of differences in the outer membrane phospholipid composition of tested membranes owing to which they are hydrolyzed by the venom PLA₂s to a different extent. The chemical modification of essential amino acids present in the active site, neutralization study with polyvalent antivenom and heat-inactivation of RVVA-PLA₂-I suggested the correlation between catalytic and membrane damaging activities of this PLA₂ enzyme. Our study advocates that the presence of a large number of PLA₂-sensitive phospholipid domains/composition, rather than only the phosphatidylcholine (PC) content of that particular membrane may determine the extent of membrane damage by a particular venom PLA₂ enzyme.
从锯鳞蝰蛇毒液中纯化得到的一种酸性磷脂酶A₂（RVVA-PLA₂-I）表现出剂量依赖性的催化活性、线粒体损伤活性和红细胞膜损伤活性。在所测试的剂量下，RVVA-PLA₂-I对小鼠无致死性，然而，从注射该磷脂酶A₂后小鼠血清中测得的酶活性推断，它会影响小鼠的不同器官，尤其是肝脏和心脏组织。与肝线粒体膜相比，RVVA-PLA₂-I优先水解红细胞膜的磷脂（磷脂酰胆碱）。有趣的是，在孵育24小时内，RVVA-PLA₂-I未能水解HT-29（结肠腺癌）细胞的膜磷脂，该细胞外膜含有大量磷脂酰胆碱。添加RVVA-PLA₂-I后，对完整线粒体膜和红细胞膜中饱和/不饱和脂肪酸释放模式的气相色谱（GC）分析显示出明显不同的结果。这些结果肯定反映了所测试膜的外膜磷脂组成的差异，由于这种差异，它们被毒液磷脂酶A₂水解的程度不同。对活性位点中存在的必需氨基酸进行化学修饰、用多价抗蛇毒血清进行中和研究以及对RVVA-PLA₂-I进行热失活处理，表明了这种磷脂酶A₂的催化活性和膜损伤活性之间的相关性。我们的研究表明，特定膜中大量对磷脂酶A₂敏感的磷脂结构域/组成的存在，而不仅仅是该特定膜的磷脂酰胆碱（PC）含量，可能决定了特定毒液磷脂酶A₂对膜的损伤程度。
