De Meester I, Vanhoof G, Hendriks D, Demuth H U, Yaron A, Scharpé S
Department of Clinical Biochemistry, Faculty of Medicine, University of Antwerp, Wilrijk, Belgium.
Clin Chim Acta. 1992 Sep 15;210(1-2):23-34. doi: 10.1016/0009-8981(92)90042-o.
The membrane-bound dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) has been purified 5,400-fold from human peripheral blood mononuclear cells. The purification procedure included detergent solubilization and successive chromatography on DEAE Sepharose Fast Flow, Con A Sepharose, Cu2+ loaded metal-chelating Sepharose, Sephacryl S-300 High Resolution and Q Sepharose Hiload. The molecular mass of the native, detergent solubilized enzyme estimated by gel filtration was 264.kDa. Chromatofocusing indicated a pI of approximately 5.0. The pI optimum was 8.7. The enzymatic activity of the purified preparation was irreversibly inhibited by N-(H-Phe-Pro)-O-(4-nitrobenzoyl)hydroxylamine hydrochloride in the micromolar range. The binding of purified DPP IV to CD26 monoclonal antibodies confirmed the identity between CD26 and dipeptidyl peptidase IV. The purification and characterization of lymphocytic dipeptidyl peptidase IV is of great value for the identification of its natural substrates and for the study of its physiological significance in the T-lymphocyte function.
膜结合二肽基肽酶IV（DPP IV，EC 3.4.14.5）已从人外周血单核细胞中纯化了5400倍。纯化过程包括用去污剂溶解，然后依次在DEAE琼脂糖快速流动柱、伴刀豆球蛋白A琼脂糖柱、负载Cu2+的金属螯合琼脂糖柱、Sephacryl S-300高分辨率柱和Q琼脂糖高负载柱上进行层析。通过凝胶过滤估计，天然的、经去污剂溶解的酶的分子量为264 kDa。色谱聚焦显示其pI约为5.0。最适pI为8.7。纯化制剂的酶活性在微摩尔范围内被N-（H-苯丙氨酸-脯氨酸）-O-（4-硝基苯甲酰基）羟胺盐酸盐不可逆地抑制。纯化的DPP IV与CD26单克隆抗体的结合证实了CD26与二肽基肽酶IV的同一性。淋巴细胞二肽基肽酶IV的纯化和特性鉴定对于确定其天然底物以及研究其在T淋巴细胞功能中的生理意义具有重要价值。
