Sadana Rachna, Mittal Ashwani, Khurana Shiwani, Singh Hari, Kamboj Ramesh C
Department of Biochemistry, Kurukshetra University, Kurukshetra 136 119, Haryana, India.
Indian J Biochem Biophys. 2003 Oct;40(5):315-23.
Cathepsin L-like proteinase was purified approximately 1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be approximately 65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbetaNA (benzyloxycarbonyl-L-phenylalanine-L-arginine-4-methoxy-beta-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbetaNA was hydrolyzed maximally by the enzyme (Km value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbetaNA, Z-Phe-Val-Arg-4mbetaNA, Z-Arg-Arg-4mbetaNA and Z-Ala-Arg-Arg-4mbetaNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and beta-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with Ki value 44 x 10(-9) M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg(2+), Ca(2+), Cu(2+), Li(2+), K(+), Cd(2+), Ni(2+), Ba(2+), Mn(2+), Co(2+) and Sn(2+) also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40 degrees C. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbetaNA was approximately 50-55 degrees C with an activation energy Ea of approximately 6.34 KCal mole(-1).
通过匀浆、在pH 4.2下进行酸自溶、30 - 80%硫酸铵分级沉淀、Sephadex G - 100柱层析以及在pH 5.0和5.6条件下于CM - Sephadex C - 50上进行离子交换层析，从山羊脑中纯化出组织蛋白酶L样蛋白酶，纯化倍数约为1708倍，活性回收率为40%，达到表观电泳均一性。通过凝胶过滤层析发现该蛋白酶的分子量约为65,000道尔顿。对于Z - Phe - Arg - 4mbetaNA（苄氧羰基 - L - 苯丙氨酸 - L - 精氨酸 - 4 - 甲氧基 - β - 萘酰胺）和偶氮酪蛋白的水解，最适pH分别为5.9和4.5。在所测试的合成显色底物中，Z - Phe - Arg - 4mbetaNA被该酶水解的程度最大（水解的Km值为0.06 mM），其次是Z - Val - Lys - Lys - Arg - 4mbetaNA、Z - Phe - Val - Arg - 4mbetaNA、Z - Arg - Arg - 4mbetaNA和Z - Ala - Arg - Arg - 4mbetaNA。该蛋白酶在谷胱甘肽与EDTA共同作用下被最大程度激活，其次是半胱氨酸、二硫苏糖醇、巯基乙酸、二硫赤藓糖醇和β - 巯基乙醇。它受到对羟基汞苯磺酸、碘乙酸、碘乙酰胺以及微生物肽抑制剂亮抑蛋白酶肽和抑肽酶的强烈抑制。亮抑蛋白酶肽以Ki值44×10⁻⁹ M竞争性抑制该酶。该酶受到4 M尿素的强烈抑制。金属离子Hg²⁺、Ca²⁺、Cu²⁺、Li²⁺、K⁺、Cd²⁺、Ni²⁺、Ba²⁺、Mn²⁺、Co²⁺和Sn²⁺也抑制该酶的活性。该酶在pH 4.0 - 6.0之间以及高达40℃时稳定。水解Z - Phe - Arg - 4mbetaNA的最适温度约为50 - 55℃，活化能Ea约为6.34千卡·摩尔⁻¹。
