A polymorphism consisting of the presence or absence of a 250-bp DNA fragment was detected within the angiotensin I-converting enzyme gene (ACE) using the endothelial ACE cDNA probe. This polymorphism was used as a marker genotype in a study involving 80 healthy subjects, whose serum ACE levels were concomitantly measured. Allele frequencies were 0.6 for the shorter allele and 0.4 for the longer allele. A marked difference in serum ACE levels was observed between subjects in each of the three ACE genotype classes. Serum immunoreactive ACE concentrations were, respectively, 2993±49, 392.6±66.8, and 494.1±883 ,g/liter, for homozygotes with the longer allele (n = 14), and heterozygotes (n = 37) and homozygotes (n = 29) with the shorter allele. The insertion/deletion polymorphism accounted for 47% of the total phenotypic variance of serum ACE, showing that the ACE gene locus is the major locus that determines serum ACE concentration. Concomitant determination of the ACE genotype will improve discrimination between normal and abnormal serum ACE values by allowing comparison with a more appropriate reference interval. (J. Clin. Invest. 1990Invest. . 86:1343Invest. -1346

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The amino-terminal amino acid sequence and several internal peptide sequences of angiotensin I-converting enzyme (ACE; peptidyl-dipeptidase A, kininase II; EC 3.4.15. 1) purified from human kidney were used to design oligonucleotide probes. The nucleotide sequence of ACE mRNA was determined by molecular cloning of the DNA complementary to the human vascular endothelial cell ACE mRNA. The complete amino acid sequence deduced from the cDNA contains 1306 residues, beginning with a signal peptide of 29 amino acids. A highly hydrophobic sequence located near the carboxylterminal extremity of the molecule most likely constitutes the anchor to the plasma membrane. The sequence of ACE reveals a high degree of internal homology between two large domains, suggesting that the molecule resulted from a gene duplication. Each of these two domains contains short amino acid sequences identical to those located around critical residues of the active site of other metallopeptidases (thermolysin, neutral endopeptidase, and collagenase) and therefore bears a putative active site. Since earlier experiments suggested that a single Zn atom was bound per molecule of ACE, only one of the two domains should be catalytically active. The results of genomic DNA analysis with the cDNA probe are consistent with the presence of a single gene for ACE in the haploid human genome. Whereas the ACE gene is transcribed as a 4.3-kilobase mRNA in vascular endothelial cells, a 3.0-kilobase transcript was detected in the testis, where a shorter form of ACE is synthesized.Peptidyl-dipeptidase A (EC 3.4.15.1) plays an important role in blood pressure homeostasis by hydrolyzing angiotensin I, the inactive peptide released after cleavage of angiotensin by renin, into angiotensin II (1). Accordingly, this Zn metallopeptidase is designated angiotensin I-converting enzyme (ACE), although being the same enzyme as kininase II, it is also able to hydrolyze bradykinin and various other peptides (2, 3). This enzyme is a widely distributed peptidase, occurring, for example, as a membrane-bound ectoenzyme on the surface of vascular endothelial cells and renal epithelial cells and as a circulating enzyme in plasma (3-5). We report here the amino acid sequence of ACE as deduced from the nucleotide sequence of DNA complementary to the ACE mRNA.t MATERIALS AND METHODSPurification and Sequencing of ACE and Preparation of Oligodeoxyribonucleotide Probe. The cortex offresh postmortem human kidneys (600 g) was homogenized (54:100, wt/vol) in 20 mM potassium phosphate buffer (pH 8) containing 250 mM sucrose and a mixture of protease inhibitors, cells debris was discarded, and the particulate fraction was sedimented by centrifugation at 105,000 x g for 1 hr. The pellet was resuspended in 200 ml of 150 mM potassium phosphate buffer (pH 8; buffer I) and treated for 18 hr with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, 8 mM; Serva). The supernatant obtained after centrifugation at 105,000 x g for 1 hr was dialyzed extensively against b...

Abstract-Angiotensin-converting enzyme (ACE) plays a central role in the production of the vasoconstrictor angiotensin II. ACE is a single polypeptide, but it contains 2 homologous and independent catalytic domains, each of which binds zinc. To understand the in vivo role of these 2 domains, we used gene targeting to create mice with point mutations in the ACE C-domain zinc-binding motif. Such mice, termed ACE13/13, produce a full-length ACE protein with tissue expression identical to wild-type mice. Analysis of ACE13/13 mice showed that they produce ACE having only N-domain catalytic activity, as determined by the hydrolysis of domain specific substrates and by chloride sensitivity. ACE13/13 mice have blood pressure and blood angiotensin II levels similar to wild-type mice. However, plasma renin concentration is increased 2.6-fold and blood angiotensin I levels are increased 7.5-fold. Bradykinin peptide levels are not different from wild-type levels. ACE13/13 mice have a reduced increase of blood pressure after intravenous infusion of angiotensin I. ACE13/13 mice have a normal renal structure, but they are not able to concentrate urine after dehydration as effectively as wild-type mice. This study shows that the C-domain of ACE is the predominant site of angiotensin I cleavage in vivo. Although mice lacking C-domain activity have normal physiology under laboratory conditions, they respond less well to the stress of dehydration. (Hypertension. 2008;51:267-274.)