Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Departamento de Física e Química, Universidade de São Paulo, Av. do Café s/n, 14040-903 Ribeirão Preto-SP, Brazil.
Toxicon. 2012 Dec 1;60(7):1263-76. doi: 10.1016/j.toxicon.2012.08.008. Epub 2012 Aug 31.
The aim of this study was the isolation of the LAAO from Lachesis muta venom (LmLAAO) and its biochemical, functional and structural characterization. Two different purification protocols were developed and both provided highly homogeneous and active LmLAAO. It is a homodimeric enzyme with molar mass around 120 kDa under non-reducing conditions, 60 kDa under reducing conditions in SDS-PAGE and 60852 Da by mass spectrometry. Forty amino acid residues were directly sequenced from LmLAAO and its complete cDNA was identified and characterized from an Expressed Sequence Tags data bank obtained from a venom gland. A model based on sequence homology was manually built in order to predict its three-dimensional structure. LmLAAO showed a catalytic preference for hydrophobic amino acids (K(m) of 0.97 mmol/L with Leu). A mild myonecrosis was observed histologically in mice after injection of 100 μg of LmLAAO and confirmed by a 15-fold increase in CK activity. LmLAAO induced cytotoxicity on AGS cell line (gastric adenocarcinoma, IC₅₀: 22.7 μg/mL) and on MCF-7 cell line (breast adenocarcinoma, IC₅₀:1.41 μg/mL). It presents antiparasitic activity on Leishmania brasiliensis (IC₅₀: 2.22 μg/mL), but Trypanosoma cruzi was resistant to LmLAAO. In conclusion, LmLAAO showed low systemic toxicity but important in vitro pharmacological actions.
本研究旨在从矛头蝮蛇毒液（Lachesis muta venom，LmLAAO）中分离 LAAO 并对其进行生化、功能和结构特征分析。开发了两种不同的纯化方案，均提供了高度均一和活性的 LmLAAO。它是一种同二聚体酶，在非还原条件下的摩尔质量约为 120 kDa，在 SDS-PAGE 还原条件下为 60 kDa，通过质谱法测定为 60852 Da。从 LmLAAO 直接测序了 40 个氨基酸残基，并从从毒液腺获得的表达序列标签数据库中鉴定和表征了其完整的 cDNA。为了预测其三维结构，手动构建了基于序列同源性的模型。LmLAAO 对疏水性氨基酸表现出催化偏好（用 Leu 作为底物时的 K(m)为 0.97 mmol/L）。在给小鼠注射 100 μg LmLAAO 后，在组织学上观察到轻微的肌坏死，并通过 CK 活性增加 15 倍得到证实。LmLAAO 对 AGS 细胞系（胃腺癌，IC₅₀：22.7 μg/mL）和 MCF-7 细胞系（乳腺腺癌，IC₅₀：1.41 μg/mL）具有细胞毒性。它对 Leishmania brasiliensis 具有抗寄生虫活性（IC₅₀：2.22 μg/mL），但 Trypanosoma cruzi 对 LmLAAO 具有抗性。总之，LmLAAO 表现出低系统毒性，但具有重要的体外药理作用。
