Oliveira M G A, De Simone S G, Xavier L P, Guedes R N C
Departamento de Bioquímica e Biologia Molecular, Instituto de Biotecnologia Aplicada a Agropecuária (BIOAGRO), Universidade Federal de Viçosa, Viçosa, MG 36571-000, Brazil.
Comp Biochem Physiol B Biochem Mol Biol. 2005 Mar;140(3):369-80. doi: 10.1016/j.cbpc.2004.10.018.
Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hubner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris-HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-alpha-benzoyl-L-Arg-p-nitroanilide (L-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates L-BApNA and N-alpha-p-tosyl-L-Arg methyl ester (L-TAME). Higher activity was observed at pH 8.5 and 35 degrees C when using L-BApNA as substrate and at pH 8.0 and 30 degrees C when using L-TAME. Maximum enzyme activity against L-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for L-BApNA and 52.5 microM for L-TAME.
从棉铃虫（鳞翅目：夜蛾科）中肠提取的类胰蛋白酶在抑肽酶 - 琼脂糖柱上进行纯化，该柱用含5 mM氯化钙的0.01 M Tris - HCl（pH 7.5）平衡。产率为66.7%，纯化倍数为107，以N - α - 苯甲酰 - L - 精氨酸 - 对硝基苯胺（L - BApNA）为底物时，最终比活性为6.88 mM/分钟/毫克蛋白。纯化后的组分在十二烷基硫酸钠（SDS） - 聚丙烯酰胺凝胶电泳（PAGE）中显示出三条具有蛋白水解活性且分子量分别为66,000、71,000和91,000的条带。使用七种含13个氨基酸残基但仅在第5个残基（K、R、Y、L、W或P）不同的合成肽进行酶特异性测定。肽裂解仅在第5位为氨基酸K或R时发生，这是胰蛋白酶的典型特征。部分纯化的酶可水解酪蛋白以及合成胰蛋白酶底物L - BApNA和N - α - 对甲苯磺酰 - L - 精氨酸甲酯（L - TAME）。以L - BApNA为底物时，在pH 8.5和35℃观察到较高活性；以L - TAME为底物时，在pH 8.0和30℃观察到较高活性。反应混合物中加入20 mM氯化钙时，对L - BApNA的酶活性最高。显示胰蛋白酶活性的部分纯化酶对乙二胺四乙酸（EDTA）、苯甲基磺酰氟（PMSF）、N - α - 甲苯磺酰 - L - 赖氨酸氯甲基酮（TLCK）、苯甲脒和抑肽酶的抑制敏感。TLCK和苯甲脒的抑制作用最强。对于L - BApNA，获得的米氏常数（KM）值为0.32 mM，对于L - TAME为52.5 μM。
