Calgarotto Andrana K, Damico Daniela C S, Ponce-Soto L A, Baldasso Paulo A, Da Silva Saulo L, Souza Gustavo H M F, Eberlin Marcos N, Marangoni Sergio
Departamento de Bioquímica, Instituto de Biologia, Universidade Estadual de Campinas, Campinas, SP, Brazil.
Toxicon. 2008 Jun 15;51(8):1509-19. doi: 10.1016/j.toxicon.2008.03.030. Epub 2008 Apr 8.
BmTX-I, an Asp49 phospholipase A(2), was purified from Bothrops moojeni venom after only one chromatographic step using reverse-phase HPLC on mu-Bondapak C-18 column. A molecular mass of 14238.71Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The BmTX-I PLA(2) had a sequence of 121 residues of amino acids: DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C and pI value 7.84, and showed a high degree of homology with basic Asp49 PLA(2) myotoxins from other Bothrops venoms. BmTX-I presented PLA(2) activity in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 35-45 degrees C. Maximum PLA(2) activity required Ca(2+) and in the presence of Mg(2+), Cd(2+) and Mn(2+) it was reduced in presence or absence of Ca(2+). Crotapotin from Crotalus durissus colillineatus rattlesnake venom has significantly inhibited (P<0.05) the enzymatic activity of BmTX-I. In vitro, the whole venom and BmTX-I caused a blockade of the neuromuscular transmission in young chick biventer cervicis preparations in a similar way to other bothrops species. In mice, BmTX-I and the whole venom-induced myonecrosis and a systemic interleukin-6 response upon intramuscular injection. Edema-forming activity was also analyzed through injection of the venom and the purified BmTX-I into the subplantar region of the right footpad. Since BmTX-I exert a strong proinflammatory effect; the enzymatic phospholipids hydrolysis might be relevant for these phenomena.
BmTX-I是一种天冬氨酸49型磷脂酶A₂，仅通过在μ-Bondapak C-18柱上进行反相高效液相色谱的一步色谱法，就从矛头蝮蛇毒液中纯化得到。通过基质辅助激光解吸电离飞行时间质谱法测定其分子量为14238.71道尔顿。氨基酸分析表明其含有高含量的疏水和碱性氨基酸以及14个半胱氨酸残基。BmTX-I磷脂酶A₂具有121个氨基酸残基的序列：DLWQFNKMIK KEVGKLPFPF YGAYGCYCGW GGRGEKPKDG TDRCCFVHDC CYKKLTGCPK WDDRYSYSWK DITIVCGEDL PCEEICECDR AAAVCFYENL GTYNKKYMKH LKPCKKADYP C，其等电点值为7.84，与其他矛头蝮蛇毒液中的碱性天冬氨酸49型磷脂酶A₂肌毒素具有高度同源性。BmTX-I在合成底物存在的情况下呈现磷脂酶A₂活性，并表现出最小的S形曲线行为在pH 8.0和35 - 45℃时达到其最大活性。最大磷脂酶A₂活性需要钙离子，并且在镁离子、镉离子和锰离子存在时，无论有无钙离子，其活性都会降低。来自杜氏响尾蛇毒液的响尾蛇毒素显著抑制（P<0.05）BmTX-I的酶活性。在体外，整个毒液和BmTX-I以与其他矛头蝮蛇物种相似的方式导致幼雏双头肌颈肌标本中的神经肌肉传递阻滞。在小鼠中，BmTX-I和整个毒液在肌肉注射后会引起肌坏死和全身性白细胞介素-6反应。还通过将毒液和纯化的BmTX-I注射到右足垫的足底区域来分析其形成水肿的活性。由于BmTX-I具有强烈的促炎作用；酶促磷脂水解可能与这些现象有关。
