The Drosophila melanogaster-related angiotensin-I-converting enzymes Acer and Ance -: Distinct enzymic characteristics and alternative expression during pupal development

被引:72
作者
Houard, X
Williams, TA
Michaud, A
Dani, P
Isaac, RE
Shirras, AD
Coates, D
Corvol, P
机构
[1] Coll France, INSERM U36, F-75005 Paris, France
[2] Univ Leeds, Dept Biol, Leeds, W Yorkshire, England
[3] Inst Canc Res, Turin, Italy
[4] Univ Lancaster, Div Biol Sci, Lancaster LA1 4YQ, England
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1998年 / 257卷 / 03期
关键词
angiotensin I-converting enzyme; Drosophila melanogaster; Pichia pastoris; angiotensin I-converting enzyme substrates; pupal development;
D O I
10.1046/j.1432-1327.1998.2570599.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:599 / 606
页数:8
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