Yoshimoto T, Matsubara F, Kawano E, Tsuru D
J Biochem. 1983 Dec;94(6):1889-96. doi: 10.1093/oxfordjournals.jbchem.a134542.
Prolidase [iminodipeptidase, EC 3.4.13.9] was highly purified from the cytosol fraction of bovine small intestine by a series of column chromatographies on DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose and hydroxyapatite. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.2 with Gly-Pro as substrate. It was stable between pH 5.5 and 8.5 for 30 min at 30 degrees C and retained half of the activity after 15 min at 40 degrees C. It was completely inactivated by p-chloromercuribenzoate (PCMB) but not inhibited by diisopropylphosphorofluoridate (DFP), phenylmethane sulfonylfluoride (PMSF) and metal chelators. Its amino acid composition was determined. Its molecular weight was estimated to be 116,000 by gel filtration on Sephadex G-150 and 56,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that it is a dimer. It hydrolyzed dipeptides represented as X-Pro (X = amino acid).
通过在DEAE- Toyopearl、Sephadex G - 150、对氯汞苯甲酸 - T - 琼脂糖和羟基磷灰石上进行一系列柱色谱，从牛小肠的胞质溶胶部分高度纯化了脯氨酰二肽酶[亚氨基二肽酶，EC 3.4.13.9]。通过圆盘凝胶电泳判断，纯化后的酶呈现出均一性。该酶以甘氨酰 - 脯氨酸为底物时，在pH 7.2下活性最高。在30℃时，它在pH 5.5至8.5之间稳定30分钟，在40℃下15分钟后仍保留一半活性。它被对氯汞苯甲酸（PCMB）完全灭活，但不受二异丙基氟磷酸酯（DFP）、苯甲基磺酰氟（PMSF）和金属螯合剂的抑制。测定了其氨基酸组成。通过在Sephadex G - 150上进行凝胶过滤，其分子量估计为116,000，通过十二烷基硫酸钠（SDS）凝胶电泳测定为56,000，表明它是一种二聚体。它水解表示为X - Pro（X =氨基酸）的二肽。
