National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, Tallinn 12618, Estonia.
Biochimie. 2011 Feb;93(2):321-30. doi: 10.1016/j.biochi.2010.10.004. Epub 2010 Oct 13.
Vipera lebetina venom contains different metallo- and serine proteinases that affect coagulation and fibrin(ogen)olysis. A novel serine proteinase from V. Lebetina venom having ChymoTrypsin Like Proteolytic activity (VLCTLP) was purified to homogeneity from the venom using Sephadex G-100sf, DEAE-cellulose, heparin-agarose and FPLC on Superdex 75 chromatographies. VLCTLP is a glycosylated serine proteinase with a molecular mass of 41926 Da. It reacts with N-acetyl-L-tyrosine ethyl ester (ATEE) but not with Suc-Ala-Ala-Pro-Phe-pNA or Suc-Ala-Ala-Pro-Leu-pNA. The complete amino acid sequence of the VLCTLP is deduced from the nucleotide sequence of the cDNA encoding this protein. The full-length cDNA sequence of the VLCTLP encodes open reading frame of 257 amino acid residues that includes a putative signal peptide of 18 amino acids, a proposed activation peptide of six amino acid residues and serine proteinase of 233 amino acid residues. VLCTLP belongs to the S1 (chymotrypsin) subfamily of proteases. The multiple alignment of its deduced amino acid sequence showed structural similarity with other serine proteases from snake venoms. The protease weakly hydrolyses azocasein, Aα-chain and more slowly Bβ-chain of fibrinogen. VLCTLP does not cleave fibrin and has no gelatinolytic activity. Specificity studies against peptide substrates (angiotensin I and II, oxidized insulin B-chain, glucagon, fibrinogen fragments etc.) showed that VLCTLP catalysed the cleavage of peptide bonds after tyrosine residues. VLCTLP is the only purified and characterized serine proteinase from snake venoms that catalyses ATEE hydrolysis. We detected ATEE-hydrolysing activities also in 9 different Viperidae and Crotalidae venoms.
蝰蛇毒含有不同的金属蛋白酶和丝氨酸蛋白酶，这些酶影响凝血和纤维蛋白（原）的水解。一种新型的丝氨酸蛋白酶，即来自蝰蛇毒液的类糜蛋白酶样蛋白酶（VLCTLP），使用葡聚糖 G-100sf、DEAE-纤维素、肝素琼脂糖和 FPLC 在 Superdex 75 层析上从毒液中被纯化至均一性。VLCTLP 是一种糖基化的丝氨酸蛋白酶，分子量为 41926 Da。它与 N-乙酰-L-酪氨酸乙酯（ATEE）反应，但不与 Suc-Ala-Ala-Pro-Phe-pNA 或 Suc-Ala-Ala-Pro-Leu-pNA 反应。VLCTLP 的完整氨基酸序列是从编码该蛋白的 cDNA 的核苷酸序列推导出来的。VLCTLP 的全长 cDNA 序列编码 257 个氨基酸残基的开放阅读框，其中包括 18 个氨基酸的假定信号肽、6 个氨基酸的假定激活肽和 233 个氨基酸残基的丝氨酸蛋白酶。VLCTLP 属于蛋白酶 S1（糜蛋白酶）亚家族。其推导的氨基酸序列的多重比对显示与来自蛇毒的其他丝氨酸蛋白酶具有结构相似性。该蛋白酶弱水解偶氮酪蛋白、Aα-链和更缓慢的纤维蛋白原 Bβ-链。VLCTLP 不切割纤维蛋白，没有明胶酶活性。对肽底物（血管紧张素 I 和 II、氧化胰岛素 B 链、胰高血糖素、纤维蛋白原片段等）的特异性研究表明，VLCTLP 催化酪氨酸残基后的肽键断裂。VLCTLP 是唯一从蛇毒中分离和表征的丝氨酸蛋白酶，能够催化 ATEE 水解。我们还在 9 种不同的蝰蛇科和响尾蛇科毒液中检测到 ATEE 水解活性。
