The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance -: Distinct enzymic characteristics and alternative expression during pupal development
The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance -: Distinct enzymic characteristics and alternative expression during pupal development
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DOI：
10.1046/j.1432-1327.1998.2570599.x
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发表时间：
1998-11-01
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影响因子：
--
通讯作者：
Corvol, P
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文献类型：
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作者：
Houard, X;Williams, TA;Corvol, P
黑胃果蝇表达两种不同的血管紧张素- i转换酶（ace），称为Ance和Acer，它们显示出高度的初级结构相似性。我们在毕赤酵母中表达了Acer，并纯化了重组酶，以期开发生化工具来区分Acer和Ance。用酵母中表达的纯化的Acer和Ance培养抗Acer Ig和抗Ance Ig，它们在免疫印迹上与各自的酶特异性交叉反应，但不作为特异性抑制剂。Acer从苯甲酰甘酰基-组酰亮氨酸和[Leu5]脑啡肽中切割c端二肽，Acer和Ance都能作为内肽酶，从[Leu5]脑啡肽中释放c端二肽酰胺。然而，Acer水解这种底物的速度比[Leu5]脑啡肽略快，而Ance水解带有游离c末端的肽，其k（cat）比[Leu5]脑啡肽高15倍。此外，Acer不裂解血管紧张素i，而Ance在低8倍的酶浓度下水解了25%的血管紧张素i。此外，Acer不能水解合成底物Phc-Ser-Pro-Arg-Leu-Gly-Arg-Arg和phee - ser - pro - arg - leu - gly - lys - arg，这两种合成底物在Ance产生82%底物水解的条件下，被推测为是部分加工的原肌促肽前体。卡托普利、曲多普利和依那普利对Acer有明显的抑制作用，K-i值在纳摩尔范围内，赖诺普利和福辛普利的抑制作用较弱。我们发现这两种果蝇ace在蛹发育的pi（白蛹）-P15（羽化）阶段交替表达；ACE在P9-p12期表达，而Ance主要在p4-P7期表达，表明这两种酶在蛹发育过程中的作用不同。
Drosophila melanogaster express two distinct angiotensin-I-converting enzymes (ACEs) called Ance and Acer, which display a high level of primary structure similarity. We have expressed Acer in the yeast Pichia pastoris and purified the recombinant enzyme with a view to developing biochemical tools to distinguish between Acer and Ance. purified Acer and Ance expressed in yeast were used to raise anti-Acer Ig and anti-Ance Ig that specifically cross-reacted with the respective enzyme on immunoblotting, but did not act as specific inhibitors. Acer cleaves the C-terminal dipeptides from benzoylglycyl-histidyl-leucine and [Leu5]enkephalin, and Acer and Ance are both able to act as endopeptidases, releasing the C-terminal dipeptideamide from [Leu5]enkephalinamide. However, Acer hydrolyses this substrate at a slightly faster rate than [Leu5]enkephalin, whereas Ance hydrolyses the peptide with a free C-terminus with a k(cat) 15-fold higher than [Leu5] enkephalinamide. In addition, Acer did not cleave angiotensin I. In contrast, Ance hydrolysed 25% of this substrate at an 8-fold lower enzyme concentration. Furthermore, Acer did not hydrolyse the synthetic substrates Phc-Ser-Pro-Arg-Leu-Gly-Arg-Arg and Phe-Ser-Pro-Arg-Leu-Gly-Lys-Arg, two partially processed putative locustamyotropin precursors, under conditions where Ance produced 82% substrate hydrolysis. Acer was inhibited by captopril, trandolaprilat and enalaprilat, with apparent K-i values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages pi (white puparium)-P15 (eclosion) of pupal development; Ance is expressed predominantly during stages p4-P7, whereas the ACE activity expressed during stages P9-p12 is mainly due to,Acer suggesting different roles for the two enzymes during pupal development.