Christine Hubert

An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels.

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, 299.3 +/- 49, 392.6 +/- 66.8, and 494.1 +/- 88.3 micrograms/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.

The testicular transcript of the angiotensin I-converting enzyme encodes for the ancestral, non-duplicated form of the enzyme

The endothelial angiotensin I‐converting enzyme (ACE) is organized in two large homologous domains, each bearing a putative active site. However, only one of these sites is probably involved in catalysing the conversion of angiotensin I into angiotensin II. The testicular form of ACE is equally active, encoded by the same gene, but translated from a shorter mRNA. Molecular cloning of the human testicular ACE cDNA indicates that the mRNA codes for 732 residues (vs 1306 in endothelium). The testicular transcript corresponds to the 3′ half of the endothelial transcript and encodes one of the two homologous domains of endothelial ACE, preceded by a short specific sequence. This 5′ specific sequence contains 228 nucleotides and encodes 67 amino acids, including the putative signal peptide followed by a serine/threonine‐enriched region, presumably glycosylated. The testicular transcript corresponds to the ancestral, non‐duplicated form of the ACE gene. Since the carboxyl‐terminal domain of the endothelial ACE is expressed in the testicular enzyme, it is likely that it bears the active site in both forms.

Molecular biology of the angiotensin I converting enzyme. I: Biochemistry and structure of the gene

ACE (kininase II, dipeptidyl carboxypeptidase I; EC 3.4.15.1) acts primarily as a dipeptidyl carboxypeptidase and is involved in the metabolism of two major vasoactive peptides, angiotensin II and bradykinin. ACE generates the potent vasopressor hormone angiotensin II by cleaving the carboxyl-terminal dipeptide from angiotensin I and inactivates the vasodepressor hormone bradykinin, by the sequential removal of two carboxyl-terminal dipeptides [1]. The favoured ACE substrate is bradykinin, for which the Michaelis constant (Km) is approximately 80 times lower than for angiotensin I (0.2 versus 16 (imol/1, respectively).

Angiotensin-Converting Enzyme C-Terminal Catalytic Domain Is the Main Site of Angiotensin I Cleavage In Vivo

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.

Male fertility is dependent on dipeptidase activity of testis ACE.

c Figure 2 ACE overexpression in CHO and HEK cells does not affect the shedding of multiple GPI-anchored proteins. (a) CHO cells were stably transfected with vector alone (mock), full-length wild-type (FL-ACE) or GPI-ACE10. Endogenous alkaline phosphatase activity shed into the media was determined using p-nitrophenylphosphate as substrate. Results are means ± s.d. (n = 3) and are expressed as a percentage of activity shed into media of mock-transfected cells. (b) HEK cells stably transfected with either doppel or prion protein were transiently transfected with vector alone (mock), FL-ACE or GPI-ACE. Endogenous alkaline phosphatase activity shed into the media was determined using p-nitrophenylphosphate as substrate. Doppel and prion protein shed into media were determined by immunoblotting followed by densitometric analysis. Results are means ± s.d. (n = 3 or 6 (alkaline phosphatase)) and are expressed as a percentage of protein shed into the media of mock-transfected cells. (c) ACE activity determined using BzGly-His-Leu as substrate in lysates from the HEK cells transiently transfected with vector alone (mock), FL-ACE or GPI-ACE as in b. Results are means ± s.d. (n = 3).

The angiotensin converting enzyme in the kidney.

Immunohistochemical studies and experiments with microdissected nephron segments indicate that the angiotensin I converting enzyme (ACE) in the kidney is expressed in the vascular endothelial cells of the renal vessels and in the epithelial cells of the proximal convoluted tubule and the pars recta. Angiotensin converting enzyme is a membrane-bound zinc metallopeptidase and the primary structure has recently been determined by protein sequencing and molecular cloning. It is probably anchored to the cell membrane by a single, short, transmembrane domain located near the carboxy-terminal extremity. The larger, externally situated, amino-terminal part of the molecule is organized in two large, highly homologous domains, each with a putative active site. The function of the endothelial enzyme in the renal vessels is primarily related to angiotensin II (Ang II) formation. However, its level of expression in renal vessels, especially at the glomerular level, appears to be very low in the adult human kidney, and there is evidence that the conversion of angiotensin I (Ang I) may be a rate-limiting step in Ang II formation in the kidney. The vascular enzyme may also contribute to the inactivation of kinins in the peritubular circulation. In the epithelial cells of the proximal tubule, ACE is present in both the brush border and the basolateral membrane. Although the basolateral enzyme may be involved in Ang II formation in the peritubular interstitium, the function of the enzyme on the brush border is unknown. The effects of ACE inhibitors on renal function are primarily, if not exclusively, related to Ang II suppression and perhaps kinin potentiation in the renal circulation.

Molecular biology of the angiotensin I converting enzyme

The discovery of orally active angiotensin converting enzyme (ACE) inhibitors is one of the major therapeutic advances of the last decade. However, the structure of this enzyme was not determined until recently. Molecular cloning of the enzyme has now opened up new and fascinating areas of research with important clinical implications.