Mineyama R, Saito K
Department of Oral Microbiology, School of Dentistry at Niigata, Nippon Dental University, Japan.
Microbios. 1991;67(274):37-52.
Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.
用N-乙酰胞壁酸酶裂解唾液链球菌HHT细胞壁后，通过阴离子交换色谱、凝胶过滤和亲和色谱从该菌中纯化出二肽基肽酶IV（DAP IV）。DAP IV从粗提物的总活性中纯化了114倍，产率为16.6%。通过圆盘凝胶电泳显示纯化后的酶是均一的。通过凝胶过滤法估计该酶的分子量约为109,000，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法估计为47,000，这表明天然酶是二聚体形式。在甘氨酸-氢氧化钠缓冲液中，该反应的最适pH为8.7，酶的等电点为pH 4.2。该酶能特异性地从X-脯氨酸-对硝基苯胺中水解N端的X-脯氨酸。该酶的活性几乎不受各种阳离子、巯基封闭试剂和金属螯合剂的影响。1 mM二异丙基氟磷酸能显著抑制该酶的活性，去唾液酸酶会被蛋白酶攻击。
