Ogasawara W, Abe N, Hagio T, Okada H, Morikawa Y
Department of Bioengineering, Nagaoka University of Technology, Niigata, Japan.
Biosci Biotechnol Biochem. 1997 May;61(5):858-63. doi: 10.1271/bbb.61.858.
A dipeptidyl carboxypeptidase (DCP) activity was detected in cell-free extracts of Pseudomonas sp. WO24. After purification and characterization the enzyme was found to be homogeneous by SDS-PAGE, and had a molecular mass of 74,000 Da by SDS-PAGE and 72,000 Da by gel filtration, indicating that it is monomeric. The isoelectric point was 5.2 and optimum pH was 6.5-7.0. It showed a specific activity of 780 mumol/min/mg, which is the highest of the values shown by known enzymes. The enzyme hydrolyzed angiotensin I to angiotensin II and sequentially released Phe-Arg and Ser-Pro from the C-terminus bradykinin. The DCP could not cleave imido-bonds, Gly-Gly bonds, or tripeptides. The enzymatic activity was completely inhibited by 0.001 mM EDTA and 0.1 mM O-phenanthroline, but it was not affected by general serine and cysteine protease inhibitors. Addition of Zn2+ completely restored the original activity of the inactivated DCP treated with EDTA. These results suggest that this enzyme is a zinc metalloprotease. The characteristics of the purified enzyme are slightly different from those of the DCPs from Escherichia coli, Pseudomonas maltophilia, and Corynebacterium equi, and considerably from those of the DCP from Bacillus pumilus.
在假单胞菌属WO24的无细胞提取物中检测到二肽基羧肽酶（DCP）活性。经过纯化和特性鉴定，该酶经SDS-PAGE分析显示为均一性，SDS-PAGE测得其分子量为74,000 Da，凝胶过滤测得为72,000 Da，表明它是单体。其等电点为5.2，最适pH为6.5 - 7.0。它的比活性为780 μmol/min/mg，这是已知酶所显示的值中最高的。该酶将血管紧张素I水解为血管紧张素II，并从缓激肽的C末端依次释放苯丙氨酸 - 精氨酸和丝氨酸 - 脯氨酸。该DCP不能切割亚氨基键、甘氨酰 - 甘氨酸键或三肽。酶活性被0.001 mM的EDTA和0.1 mM的邻菲罗啉完全抑制，但不受一般丝氨酸和半胱氨酸蛋白酶抑制剂的影响。添加Zn2+可完全恢复经EDTA处理的失活DCP的原始活性。这些结果表明该酶是一种锌金属蛋白酶。纯化酶的特性与大肠杆菌、嗜麦芽窄食单胞菌和马棒状杆菌的DCP略有不同，与短小芽孢杆菌的DCP有很大差异。
