Blanco J, Nguyen C, Callebaut C, Jacotot E, Krust B, Mazaleyrat J P, Wakselman M, Hovanessian A G
ERS 572 CNRS, Department SIDA/Retrovirus, Institut Pasteur, Paris, France.
Eur J Biochem. 1998 Sep 1;256(2):369-78. doi: 10.1046/j.1432-1327.1998.2560369.x.
Dipeptidyl peptidase IV-beta (DPP IV-beta) is a novel protein which shows a peptidase activity similar to the T-cell-activation antigen CD26. To further characterize this DPP IV-beta and confirm its cell surface expression, we have developed a purification strategy using the CD26- cell line C8166. The purification process includes biotinylation of cell surface proteins before preparation of cell extracts and processing by gel-filtration, ion-exchange and lectin chromatographies. Consistent with the molecular mass of DPP IV-beta estimated by gel-filtration chromatography, the final purified fraction, manifesting a typical DPP IV activity, showed a major biotinylated 75-80-kDa band in SDS/PAGE, thus suggesting the monomeric nature of this enzyme. Kinetic parameters of DPP IV-beta and the sensitivity to a new family of irreversible DPP IV inhibitors, were studied in comparison to CD26. Both enzymes followed a Michaelis kinetics with different Km values for Gly-Pro-NH-Np (NH-Np, para-nitroanilide) hydrolysis (0.28+/-0.05 mM and 0.12+/-0.02 mM). More significant differences were observed in the sensitivity to inhibitors, which exerted a much higher activity on CD26 than on DPP IV-beta. These differences permitted us to study DPP IV-beta expression in CD26-expressing cells, showing the expression of this new enzyme in all lymphoid cells tested, and a rapid enhancement in phytohemagglutinin-stimulated or protein-A-stimulated peripheral blood mononuclear cells. Our results indicate that, although DPP IV-beta and CD26 are coexpressed and manifest a typical DPP IV activity, there are distinct features in their catalytic activities that may confer to each enzyme a complementary role in peptide processing.
二肽基肽酶IV-β（DPP IV-β）是一种新型蛋白质，其肽酶活性与T细胞激活抗原CD26相似。为了进一步表征这种DPP IV-β并确认其细胞表面表达，我们开发了一种利用不表达CD26的细胞系C8166的纯化策略。纯化过程包括在制备细胞提取物之前对细胞表面蛋白进行生物素化，并通过凝胶过滤、离子交换和凝集素色谱法进行处理。与通过凝胶过滤色谱法估计的DPP IV-β分子量一致，最终纯化的级分表现出典型的DPP IV活性，在SDS/PAGE中显示出一条主要的生物素化的75 - 80 kDa条带，从而表明该酶的单体性质。与CD26相比，研究了DPP IV-β的动力学参数以及对一类新型不可逆DPP IV抑制剂的敏感性。两种酶都遵循米氏动力学，对甘氨酰 - 脯氨酰 - 对硝基苯胺（NH-Np，对硝基苯胺）水解具有不同的Km值（分别为0.28±0.05 mM和0.12±0.02 mM）。在对抑制剂的敏感性方面观察到更显著的差异，抑制剂对CD26的活性比对DPP IV-β的活性高得多。这些差异使我们能够研究DPP IV-β在表达CD26的细胞中的表达，结果表明这种新酶在所有测试的淋巴细胞中均有表达，并且在植物血凝素刺激或蛋白A刺激的外周血单核细胞中迅速增强。我们的结果表明，尽管DPP IV-β和CD26共表达并表现出典型的DPP IV活性，但它们的催化活性存在明显特征，这可能赋予每种酶在肽加工中互补的作用。
