Paul S, Volle D J, Beach C M, Johnson D R, Powell M J, Massey R J
Department of Pharmacology, University of Nebraska Medical Center, Omaha 68105.
Science. 1989 Jun 9;244(4909):1158-62. doi: 10.1126/science.2727702.
Vasoactive intestinal peptide (VIP) labeled with 125I, [Tyr10-125I]VIP, can be hydrolyzed by immunoglobulin G (IgG) purified from a human subject, as judged by trichloroacetic acid precipitation and reversed-phase high-performance liquid chromatography (HPLC). The hydrolytic activity was precipitated by antibody to human IgG, it was bound by immobilized protein G and showed a molecular mass close to 150 kilodaltons by gel filtration chromatography, properties similar to those of authentic IgG. The Fab fragment, prepared from IgG by papain treatment, retained the VIP hydrolytic activity of the IgG. Peptide fragments produced by treatment of VIP with the antibody fraction were purified by reversed-phase HPLC and identified by fast atom bombardment-mass spectrometry and peptide sequencing. The scissile bond in VIP deduced from these experiments was Gln16-Met17. The antibody concentration (73.4 fmol per milligram of IgG) and the Kd (0.4 nM) were computed from analysis of VIP binding under conditions that did not result in peptide hydrolysis. Analysis of the antibody-mediated VIP hydrolysis at varying concentrations of substrate suggested conformity with Michaelis-Menton kinetics (Km). The values for Km (37.9 X 10(-9) M) and the turnover number kcat (15.6 min-1) suggested relatively tight VIP binding and a moderate catalytic efficiency of the antibody.
用125I标记的血管活性肠肽（VIP），即[Tyr10 - 125I]VIP，经三氯乙酸沉淀和反相高效液相色谱（HPLC）判断，可被从人类受试者纯化的免疫球蛋白G（IgG）水解。水解活性可被抗人IgG抗体沉淀，能与固定化蛋白G结合，通过凝胶过滤色谱显示分子量接近150千道尔顿，这些性质与天然IgG相似。通过木瓜蛋白酶处理IgG制备的Fab片段保留了IgG的VIP水解活性。用抗体片段处理VIP产生的肽片段通过反相HPLC纯化，并通过快原子轰击质谱和肽测序进行鉴定。从这些实验推断，VIP中的裂解键为Gln16 - Met17。在未导致肽水解的条件下，通过分析VIP结合计算出抗体浓度（每毫克IgG 73.4飞摩尔）和Kd（0.4纳摩尔）。在不同底物浓度下分析抗体介导的VIP水解表明符合米氏动力学（Km）。Km值（37.9×10⁻⁹ M）和周转数kcat（15.6分钟⁻¹）表明VIP结合相对紧密，抗体的催化效率适中。
