Williams T A, Barnes K, Kenny A J, Turner A J, Hooper N M
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Biochem J. 1992 Dec 15;288 ( Pt 3)(Pt 3):875-81. doi: 10.1042/bj2880875.
Angiotensin converting enzyme (ACE; EC 3.4.15.1) was purified from porcine kidney and lung (endothelial isoenzyme) and testis (testicular isoenzyme) by affinity chromatography on lisinopril-2.8 nm-Sepharose. Atomic-absorption spectroscopy revealed that ACE purified from kidney and lung contained 2.58 and 2.35 atoms of zinc per molecule of enzyme (M(r) 147,000) respectively. In contrast, ACE purified from testis contained only 1.58 atoms of zinc per molecule of enzyme (M(r) 80,000). Thus it would appear that both putative zinc-binding sites in endothelial ACE contain zinc and may therefore be catalytically active. No differences were observed in the pattern of products generated on hydrolysis of benzoyl (Bz)-Gly-His-Leu, substance P, luteinizing-hormone-releasing hormone (LH-RH) and its analogue, des-Gly10-LH-RH-ethylamide, by kidney and testicular ACE. There was also no difference in the initial rates of hydrolysis of Bz-Gly-His-Leu or substance P by the two isoenzymes, although LH-RH and its analogue were hydrolysed twice as rapidly by kidney ACE. It is therefore unlikely that the N-terminal catalytic site in porcine endothelial ACE is predominantly responsible for the atypical cleavage of LH-RH generating the N-terminal tripeptide. Two polyclonal antisera were raised to the affinity-purified forms of pig kidney and testicular ACE. Isoenzyme-specific antisera were then isolated from these by absorbing out those antibodies recognizing determinants on the other isoenzyme. Immunoelectrophoretic blot analyses and immunofluorescent staining of sections of pig kidney were used to demonstrate the specificity of the antisera. Immunofluorescent staining of sections of pig testis with the antiserum specific to testicular ACE localized testicular ACE solely to the lumen of the seminiferous tubules, whereas the antiserum specific to endothelial ACE revealed the presence of this isoenzyme only in blood vessels. The antiserum to endothelial ACE, which recognizes determinants in the unique N-terminal domain, was investigated as a possible specific inhibitor of the N-terminal catalytic site. Although this antiserum failed to inhibit testicular ACE, the effect on the activity of endothelial ACE appeared to be due to inhibition of both the N- and C-terminal catalytic sites.
通过在赖诺普利 - 2.8nm - 琼脂糖凝胶上进行亲和层析，从猪肾、肺（内皮同工酶）和睾丸（睾丸同工酶）中纯化血管紧张素转换酶（ACE；EC 3.4.15.1）。原子吸收光谱显示，从肾和肺中纯化的ACE每分子酶（分子量147,000）分别含有2.58和2.35个锌原子。相比之下，从睾丸中纯化的ACE每分子酶（分子量80,000）仅含有1.58个锌原子。因此，内皮ACE中两个假定的锌结合位点似乎都含有锌，因此可能具有催化活性。在用肾和睾丸ACE水解苯甲酰（Bz）-甘氨酰 - 组氨酰 - 亮氨酸、P物质、促黄体生成素释放激素（LH - RH）及其类似物去甘氨酰10 - LH - RH - 乙酰胺时，未观察到产物生成模式的差异。两种同工酶对Bz - 甘氨酰 - 组氨酰 - 亮氨酸或P物质的初始水解速率也没有差异，尽管肾ACE对LH - RH及其类似物的水解速度是睾丸ACE的两倍。因此，猪内皮ACE的N端催化位点不太可能是导致LH - RH产生N端三肽的非典型切割的主要原因。针对猪肾和睾丸ACE的亲和纯化形式制备了两种多克隆抗血清。然后通过去除那些识别另一种同工酶上决定簇的抗体，从这些抗血清中分离出同工酶特异性抗血清。利用免疫电泳印迹分析和猪肾切片的免疫荧光染色来证明抗血清的特异性。用睾丸ACE特异性抗血清对猪睾丸切片进行免疫荧光染色，结果显示睾丸ACE仅定位于生精小管腔，而内皮ACE特异性抗血清显示该同工酶仅存在于血管中。研究了识别独特N端结构域中决定簇的内皮ACE抗血清作为N端催化位点可能的特异性抑制剂的作用。虽然这种抗血清未能抑制睾丸ACE，但对内皮ACE活性的影响似乎是由于对N端和C端催化位点的抑制。
