Hedeager-Sørensen S, Kenny A J
Biochem J. 1985 Jul 1;229(1):251-7. doi: 10.1042/bj2290251.
Carboxypeptidase P has been purified by immunoaffinity chromatography from pig kidneys. A single-step assay with Z-Pro-Met (where Z represents benzyloxycarbonyl) as substrate was used, methionine being determined by using L-amino acid oxidase and horseradish peroxidase. The enzyme constitutes about 1.5% of the kidney microvillar proteins. Triton X-100-solubilized and papain-released forms of the enzyme were isolated. The former had an apparent subunit Mr of 135 000, and the latter form contained two polypeptide chains of Mr 128 000 and 95 000. The undenatured forms were dimeric proteins. In common with other microvillar hydrolases, carboxypeptidase P was a glycoprotein and each subunit contained one Zn atom. MnCl2 (1 mM) in the assay was necessary for maximum activity; in its absence, 0.5 mM-ZnSO4 produced a limited activation, but was inhibitory at higher concentrations. The Km for Z-Pro-Met, in the presence of MnCl2, was 4.1 mM, and the kcat. for freshly prepared enzyme was 1230 min-1. The enzyme lost activity during storage at -20 degrees C. In a limited survey of peptides, hydrolysis was observed only with substrates containing a proline, alanine or glycine residue in the P1 position, and these included angiotensins II and III. The best substrate in this series was Val-Ala-Ala-Phe.
羧肽酶P已通过免疫亲和层析从猪肾中纯化出来。采用以Z-脯氨酰-蛋氨酸（其中Z代表苄氧羰基）为底物的一步分析法，利用L-氨基酸氧化酶和辣根过氧化物酶来测定蛋氨酸。该酶约占肾微绒毛蛋白的1.5%。分离出了经Triton X-100增溶和木瓜蛋白酶释放的酶形式。前者的表观亚基分子量为135000，后者形式包含两条分子量分别为128000和95000的多肽链。未变性的形式为二聚体蛋白。与其他微绒毛水解酶一样，羧肽酶P是一种糖蛋白，每个亚基含有一个锌原子。测定中1 mM的MnCl₂对最大活性是必需的；在没有它的情况下，0.5 mM的ZnSO₄产生有限的激活作用，但在较高浓度时具有抑制作用。在存在MnCl₂的情况下，Z-脯氨酰-蛋氨酸的Km为4.1 mM，新制备的酶的kcat为1230 min⁻¹。该酶在-20℃储存期间会失去活性。在对肽的有限研究中，仅观察到对P1位含有脯氨酸、丙氨酸或甘氨酸残基的底物有水解作用，这些底物包括血管紧张素II和III。该系列中最佳底物是缬氨酰-丙氨酰-丙氨酰-苯丙氨酸。
