Menard A, Mamoun R Z, Geoffre S, Castroviejo M, Raymond S, Precigoux G, Hospital M, Guillemain B
Unité INSERM 328, Université de Bordeaux II, Structures et Fonctions des Rétrovirus Humains, fondation Bergonié, France.
Virology. 1993 Apr;193(2):680-9. doi: 10.1006/viro.1993.1176.
To develop efficient bovine leukemia virus (BLV) protease (PR) inhibitors, pure enzyme is required. For this, we have developed a two-step chromatographic nondenaturing purification protocol of PR from virions. As expected, the purified protein presents a molecular weight (14 kDa) and a NH2 terminal end fitting with previously reported data. The enzymatic activity of BLV PR was characterized using a synthetic peptide containing a potential cleavage site of the BLV gag-pro polypeptide precursor as substrate. The protease was most active at pH 6, 40 degrees, and high salt concentration (1-2 M NaCl or ammonium sulfate). In contrast, using a natural substrate such as a human T-cell leukemia virus recombinant gag precursor, BLV PR activity was higher at a low salt concentration (0.5 M NaCl). Besides, the use of different potentially cleavable molecules revealed that PR activity may be influenced by the substrate conformational structure around the cleavage site. Replacement of the two amino acids of a synthetic substrate cleavable site by a statin residue completely inhibited the enzymatic activity of the BLV PR.
为开发高效的牛白血病病毒（BLV）蛋白酶（PR）抑制剂，需要纯酶。为此，我们开发了一种从病毒粒子中两步色谱非变性纯化PR的方案。正如预期的那样，纯化后的蛋白质呈现出的分子量（14 kDa）和氨基末端与先前报道的数据相符。使用含有BLV gag-pro多肽前体潜在切割位点的合成肽作为底物来表征BLV PR的酶活性。该蛋白酶在pH 6、40℃和高盐浓度（1-2 M NaCl或硫酸铵）下活性最高。相比之下，使用天然底物如人T细胞白血病病毒重组gag前体时，BLV PR在低盐浓度（0.5 M NaCl）下活性更高。此外，使用不同的潜在可切割分子表明，PR活性可能受切割位点周围底物构象结构的影响。用他汀类残基取代合成底物切割位点的两个氨基酸完全抑制了BLV PR的酶活性。
