Salzet M, Stefano G
Centre de Biologie Cellulaire, Laboratoire de Phylogénie moléculaire des Annélides, Université des Sciences et Technologies de Lille, Villeneuve d' Ascq, France.
Mol Cell Endocrinol. 1997 Jul 4;131(1):1-8. doi: 10.1016/s0303-7207(97)04060-4.
We report on the biochemical isolation and characterization of a 32 kDa aspartyl protease from the leech Theromyzon tessulatum. Following a three step purification (gel permeation chromatography, pepstatin A-sepharose affinity column separation followed by reversed-phase HPLC) a renin-like enzyme was purified to homogeneity. The first 124 amino acid residues of the N-terminal part of the purified S-pyridylethylated leech renin exhibits a 26.5-35.5% sequence identity with that of mammals. The 20-81 region of leech renin exhibits a 80% sequence homology with the 175-232 region in mammals. This highly conserved region, which is also found in all aspartic proteases, possesses the aspartyl catalytic residue (D11TGSS). Leech renin hydrolyses at neutral pH and at 37 degrees C the Leu10-Leu11 bond of synthetic porcine angiotensinogen tetradecapeptide yielding the angiotensin I and the Leu11-Val12-Tyr13-Ser14 peptides, with a specific activity of 115 microg AI/min/mg (K[M] 22 microM; K[cat], 2.7). This hydrolysis is inhibited by pepstatin A (IC50: 4.6 microM). Moreover, this enzyme is found on a multiple hormone precursor of 19 kDa which exhibits a specific activity of 850 pmol AI/min/mg of renin. This is the first biochemical characterization of a renin-like enzyme in invertebrates and non-mammalian vertebrates.
我们报道了从医蛭Theromyzon tessulatum中生化分离和鉴定一种32 kDa天冬氨酸蛋白酶的过程。经过三步纯化（凝胶渗透色谱、胃蛋白酶抑制剂A-琼脂糖亲和柱分离，随后进行反相高效液相色谱），一种肾素样酶被纯化至同质。纯化后的S-吡啶基乙基化医蛭肾素N端部分的前124个氨基酸残基与哺乳动物的相应序列具有26.5 - 35.5%的序列同一性。医蛭肾素的20 - 81区域与哺乳动物的175 - 232区域具有80%的序列同源性。这个在所有天冬氨酸蛋白酶中都存在的高度保守区域含有天冬氨酸催化残基（D11TGSS）。医蛭肾素在中性pH和37℃下能水解合成猪血管紧张素原十四肽的Leu10 - Leu11键，产生血管紧张素I和Leu11 - Val12 - Tyr13 - Ser14肽，比活性为115 μg AI/min/mg（米氏常数K[M]为22 μM；催化常数K[cat]为2.7）。这种水解作用受到胃蛋白酶抑制剂A的抑制（IC50：4.6 μM）。此外，这种酶存在于一种19 kDa的多种激素前体上，其比活性为850 pmol AI/min/mg肾素。这是对无脊椎动物和非哺乳动物脊椎动物中肾素样酶的首次生化鉴定。
