Ryan J W, Chung A Y, Nearing J A, Valido F A, Shun-Cun C, Berryer P
Department of Medicine, School of Medicine, University of Miami, Florida 33101.
Anal Biochem. 1993 Apr;210(1):27-33. doi: 10.1006/abio.1993.1145.
We developed an assay for aminopeptidase N (AmN) in which substrate, Arg-Phe-[3H]anilide (24.9 Ci/mmol), can be used at concentrations (1-200 nM) well below Km (12 microM) and at or below enzyme concentration ([E]). Such reaction conditions simulate those in vivo where peptide hormones in picomolar concentrations (<< Km) are degraded by nano- or micromolar concentrations of enzyme. The Arg-Phe-[3H]anilide:AmN reaction obeyed first-order enzyme kinetics when human serum, human seminal plasma, guinea pig serum, or homogeneous porcine kidney AmN was used as enzyme source and substrate was within the concentration range of 1-200 nM. For porcine AmN, kcat/Km was 1.47 x 10(9) M-1 min-1, kcat 17,640 min-1. Human serum AmN was in a concentration (about 4.6 nM) in great excess over those reported for substrates such as angiotensin III. Several advantages accrue under conditions of first-order enzyme kinetics: (1) Vmax/Km is measured directly. (2) When kcat/Km is known, [E] can be computed in mol/liter. (3) IC50 values for alternative substrates can be taken as Km values. (4) IC50 values for inhibitors are Ki values when Ki >> [E]. Arg-Phe-[3H]anilide can be used to measure AmN activity in the presence of chromophores and fluorophores that interfere with photometric and fluorometric assays. We have confirmed that alleged substrates such as angiotensin III and Met-Lys- and Lys-bradykinin are bound by AmN with high affinities (Km values, 5.7, 9.1, and 14.3 microM). Bovine pulmonary artery endothelial cell cultures were found to possess AmN-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)
我们开发了一种氨肽酶N（AmN）检测方法，其中底物精氨酸 - 苯丙氨酸 - [³H]苯胺（24.9 Ci/mmol）可在远低于米氏常数（Km，12 μM）的浓度（1 - 200 nM）以及酶浓度（[E]）或低于酶浓度的情况下使用。这样的反应条件模拟了体内的情况，即皮摩尔浓度（远低于Km）的肽激素被纳摩尔或微摩尔浓度的酶降解。当用人血清、人精浆、豚鼠血清或纯化的猪肾AmN作为酶源且底物浓度在1 - 200 nM范围内时，精氨酸 - 苯丙氨酸 - [³H]苯胺与AmN的反应遵循一级酶动力学。对于猪AmN，催化常数与米氏常数的比值（kcat/Km）为1.47×10⁹ M⁻¹ min⁻¹，催化常数（kcat）为17,640 min⁻¹。人血清AmN的浓度（约4.6 nM）大大超过了诸如血管紧张素III等底物所报道的浓度。在一级酶动力学条件下有几个优点：（1）可直接测量最大反应速度与米氏常数的比值（Vmax/Km）。（2）当已知催化常数与米氏常数的比值（kcat/Km）时，可计算出酶浓度（[E]），单位为摩尔/升。（3）替代底物的半数抑制浓度（IC50）值可作为米氏常数（Km）值。（4）当抑制常数（Ki）远大于酶浓度（[E]）时，抑制剂的半数抑制浓度（IC50）值即为抑制常数（Ki）值。精氨酸 - 苯丙氨酸 - [³H]苯胺可用于在存在干扰光度法和荧光法检测的发色团和荧光团的情况下测量AmN活性。我们已证实，诸如血管紧张素III、甲硫氨酸 - 赖氨酸 - 缓激肽和赖氨酸 - 缓激肽等所谓的底物与AmN具有高亲和力（Km值分别为5.7、9.1和14.3 μM）。发现牛肺动脉内皮细胞培养物具有类似AmN的活性。（摘要截短于250字）
