Jankiewicz Urszula, Bielawski Wieslaw
Department of Biochemistry, Warsaw Agricultural University, 02-528 Warsaw, Poland.
J Basic Microbiol. 2002;42(4):260-7. doi: 10.1002/1521-4028(200208)42:4<260::AID-JOBM260>3.0.CO;2-9.
Intracellular aminopeptidase synthesized by a soil strain of Pseudomonas sp. was purified 323-fold using the following procedure: saturation with ammonium sulfate, separation by preparative electrophoresis, anion-exchange chromatography and gel filtration chromatography. Molecular weight of the enzyme determined according to the latter method was 57 kDa. Aminopeptidase showed a high substrate specificity and affinity to Phe-beta-naphtylamide (Phe-beta-NA) as a substrate. A considerable inhibition of the enzymatic activity by iodoacetamide and p-chloromercuribenzoate (p-CMB) led to the conclusion that it was a cysteine aminopeptidase. Hydrosulphide compounds markedly stabilised the enzyme. Ethylenediaminetetra-acetic acid (EDTA), a metalloenzyme inhibitor, caused a double increase in the phenylalanyl aminopeptidase activity.( )Mg(2+) ions activated the enzyme to a negligible extent, whereas Co(2+), Cu(2+), Cd(2+) and Pb(2+) ions contributed to its inhibition. The highest enzymatic activity was observed at 37 degrees C and pH 7.0.
由假单胞菌属的一株土壤菌株合成的细胞内氨肽酶，通过以下步骤纯化了323倍：硫酸铵饱和、制备电泳分离、阴离子交换色谱和凝胶过滤色谱。根据后一种方法测定的酶分子量为57 kDa。氨肽酶对作为底物的苯丙氨酸-β-萘酰胺（Phe-β-NA）表现出高底物特异性和亲和力。碘乙酰胺和对氯汞苯甲酸（p-CMB）对酶活性有显著抑制作用，由此得出结论，它是一种半胱氨酸氨肽酶。硫化氢化合物能显著稳定该酶。金属酶抑制剂乙二胺四乙酸（EDTA）使苯丙氨酰氨肽酶活性增加了两倍。（ ）镁离子对该酶的激活作用可忽略不计，而钴离子、铜离子、镉离子和铅离子则对其有抑制作用。在37℃和pH 7.0时观察到最高酶活性。
