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Biomedical subjects

A Michaud

Publications and source records attributed to A Michaud.

At least 19 recordsLinked to original sources
PubMed2005

Hypoxaemia enhances peripheral muscle oxidative stress in chronic obstructive pulmonary disease.

BACKGROUND: Because oxidative stress affects muscle function, the underlying mechanism to explain exercise induced peripheral muscle oxidative stress in patients with chronic obstructive pulmonary disease (COPD) is clinically relevant. This study investigated whether chronic hypoxaemia in COPD worsens peripheral muscle oxidative stress and whether an abnormal muscle inflammatory process is associated with it. METHODS: Nine chronically hypoxaemic and nine non-hypoxaemic patients performed repeated knee extensions until exhaustion. Biopsy specimens were taken from the vastus lateralis muscle before and 48 hours after exercise. Muscle oxidative stress was evaluated by lipid peroxidation (lipofuscin and thiobarbituric acid reactive substances (TBARs)) and oxidised proteins. Inflammation was evaluated by quantifying muscle neutrophil and tumour necrosis factor (TNF)-alpha levels. RESULTS: When both groups were taken together, arterial oxygen pressure was positively correlated with quadriceps endurance time (n = 18, r = 0.57; p < 0.05). At rest, quadriceps lipofuscin inclusions were significantly greater in hypoxaemic patients than in non-hypoxaemic patients (2.9 (0.2) v 2.0 (0.3) inclusions/fibre; p < 0.05). Exercise induced a greater increase in muscle TBARs and oxidised proteins in hypoxaemic patients than in non-hypoxaemic patients (40.6 (9.1)% v 10.1 (5.8)% and 51.2 (11.9)% v 3.7 (12.2)%, respectively, both p = 0.01). Neutrophil levels were significantly higher in hypoxaemic patients than in non-hypoxaemic patients (53.1 (11.6) v 21.5 (11.2) counts per fibre x 10(-3); p < 0.05). Exercise did not alter muscle neutrophil levels in either group. Muscle TNF-alpha was not detected at baseline or after exercise. CONCLUSION: Chronic hypoxaemia was associated with lower quadriceps endurance time and worsened muscle oxidative stress at rest and after exercise. Increased muscle neutrophil levels could be a source of the increased baseline oxidative damage. The involvement of a muscle inflammatory process in the exercise induced oxidative stress of patients with COPD remains to be shown.

Aged↗
PubMed2005

Skeletal muscle microbiopsy: a validation study of a minimally invasive technique.

The study of the peripheral skeletal muscle function in patients with chronic obstructive pulmonary disease (COPD) is of growing interest, but often requires biopsies, usually with the Bergström technique. The current study was designed to test the validity of a minimally invasive technique: the microbiopsy. In 17 patients with COPD and four normal subjects, two specimens of the vastus lateralis were taken percutaneously under local anaesthesia, one with a 16-gauge needle (microbiopsy) and the other with the Bergström needle. The enzymatic activity of citrate synthase (CS) and phosphofructokinase (PFK), and the myosin heavy chain (MyoHC) composition were measured for both techniques. The subjects reported no pain or much less with the microbiopsy compared with the Bergström biopsy. The microbiopsy sample weight reached 55+/-17 mg. The two techniques showed excellent agreement for CS activity and MyoHC composition. The PFK activity did not differ statistically between the techniques, but the agreement was moderate. The agreement between both biopsy techniques was stable over time. The median (range) fibre number within the microbiopsy specimens was 144 (38-286). In conclusion, the current study shows the feasibility and validity of a minimally invasive muscle biopsy technique that appears more comfortable for subjects, compared with the Bergström technique.

Adult↗
PubMed2003

Iron supplementation for unexplained fatigue in non-anaemic women: double blind randomised placebo controlled trial.

OBJECTIVE: To determine the subjective response to iron therapy in non-anaemic women with unexplained fatigue. DESIGN: Double blind randomised placebo controlled trial. SETTING: Academic primary care centre and eight general practices in western Switzerland. PARTICIPANTS: 144 women aged 18 to 55, assigned to either oral ferrous sulphate (80 mg/day of elemental iron daily; n=75) or placebo (n=69) for four weeks. MAIN OUTCOME MEASURES: Level of fatigue, measured by a 10 point visual analogue scale. RESULTS: 136 (94%) women completed the study. Most had a low serum ferritin concentration; <or= 20 microg/l in 69 (51%) women. Mean age, haemoglobin concentration, serum ferritin concentration, level of fatigue, depression, and anxiety were similar in both groups at baseline. Both groups were also similar for compliance and dropout rates. The level of fatigue after one month decreased by -1.82/6.37 points (29%) in the iron group compared with -0.85/6.46 points (13%) in the placebo group (difference 0.95 points, 95% confidence interval 0.32 to 1.62; P=0.004). Subgroups analysis showed that only women with ferritin concentrations <or= 50 microg/l improved with oral supplementation. CONCLUSION: Non-anaemic women with unexplained fatigue may benefit from iron supplementation. The effect may be restricted to women with low or borderline serum ferritin concentrations.

Adolescent↗
PubMed2002

The security of urban women: practice, research, and partnerships.

All public bodies whose mandate includes responsibility for the quality of urban life cannot ignore the question of security. On a global level, it is agreed that action needs to be taken locally, with the input of everyone involved. Since women have more at stake where safety issues are concerned, it is logical that security plans be drawn up that prioritize them. The interaction of public bodies, community organizations, and individual women is in itself innovative, enriching everyone involved, despite the inherent drawbacks and demands. International information networks on women's security are expanding and can help the new partnerships refine their working methods, to the benefit of all, regardless of gender. Still, the transfer of knowledge between partners and among the population is fundamental and demanding. The same applies to the need to develop methods and criteria of evaluation that are adapted to the specific realities of the partnership for prevention of crime and violence towards women.

Community-Institutional Relations↗
PubMed2001

Study of asparagine 353 in aminopeptidase A: characterization of a novel motif (GXMEN) implicated in exopeptidase specificity of monozinc aminopeptidases.

Aminopeptidase A (EC 3.4.11.7, APA) is a 160 kDa membrane-bound zinc enzyme that contains the HEXXH consensus sequence found in members of the zinc metalloprotease family, the zincins. In addition, the monozinc aminopeptidases are characterized by another conserved motif, GXMEN, the glutamate residue of which has been shown to be implicated in the exopeptidase specificity of aminopeptidase A [Vazeux G. (1998) Biochem. J. 334, 407-413]. In carboxypeptidase A (EC 3.4.17.1, CPA), the exopeptidase specificity is conferred by an arginine residue (Arg-145) and an asparagine residue (Asn-144). Thus, we hypothesized that Asn-353 of the GXMEN motif in APA plays a similar role to Asn-144 in CPA and contributes to the exopeptidase specificity of APA. We investigated the functional role of Asn-353 in APA by substituting this residue with a glutamine (Gln-353), an alanine (Ala-353) or an aspartate (Asp-353) residue by site-directed mutagenesis. Expression of wild-type and mutated APAs revealed that Gln-353 and Ala-353 are similarly routed and glycosylated to the wild-type APA, whereas Asp-353 is trapped intracellularly and partially glycosylated. Kinetic studies, using alpha-L-glutamyl-beta-naphthylamide (GluNA) as a substrate showed that the K(m) values of the mutants Gln-353 and Ala-353 were increased 11- and 8-fold, respectively, whereas the k(cat) values were decreased (2-fold) resulting in a 24- and 14-fold reduction in cleavage efficiency. When alpha-L-aspartyl-beta-naphthylamide or angiotensin II were used as substrates, the mutations had a greater effect on k(cat), leading to a similar decrease in cleavage efficiencies as that observed with GluNA. We then measured the inhibitory potencies of several classes of inhibitors, glutamate thiol, glutamine thiol and two isomers (L- or D-) of glutamate phosphonate to explore the functional role of Asn-353. The data indicate that Asn-353 is critical for the integrity and catalytic activity of APA. This residue is involved in substrate binding via interactions with the free N-terminal part and with the P1 carboxylate side chain of the substrate. In conclusion, Asn-353 of the GXMEN motif, together with Glu-352, contributes to the exopeptidase specificity of APA and plays an equivalent role to Asn-144 in CPA.

Amino Acid Sequence↗
PubMed2001

Pulmonary function in men after repeated sessions of oxygen breathing at 0.25 MPa for 90 min.

HYPOTHESIS: We wanted to evaluate the pulmonary effects of discontinuous oxygen breathing (15 min O2, 2 min air breaks, 15:2), at 0.25 MPa once a day for 90 min O2 (6 sequences) over 10 d. This sequence, which has never been evaluated, is currently used in our hyperbaric therapy center. METHODS: Clinical and functional pulmonary status (questionnaire, spirometry, flow/volume loop, pulmonary diffusing capacity for carbon monoxide) was assessed in 10 non-smoking healthy volunteers after one exposure at 0.25 MPa consisting of 90 min of discontinuous oxygen breathing (15:2) and in 10 non-smoking patients who received a hyperbaric treatment consisting of 90 min of the same discontinuous O2 breathing (15:2) once a day over 10 d. The patients received daily intravenous methylprednisolone (1 mg x kg(-1)) and nicergoline (60 mg). RESULTS: There were no respiratory symptoms in either group. As expected, for a single exposure of that duration, lung function did not change in volunteers; however, a significant decrease in maximal expiratory flows (MEF) at 50 (-15%) and 25% (-33%) of forced vital capacity (p < 0.05) without change in forced vital capacity (FVC) appeared in patients treated over 10 d. CONCLUSION: Repetition of the 15:2 oxygen breathing sequence for 90 min once a day over 10 d led to greater flow limitation in peripheral airways than reported after continuous oxygen breathing of 210 min at 0.3 MPa which showed a 7% decrement in MEF50 and a 12% decrement in MEF25. No studies reporting these indexes were found in the 0.2-0.25 MPa range. Similar decrements in MEF50 and MEF25 with steady FVC have been reported after 14 d of daily hyperbaric therapy (0.24 MPa) with 30:5 sequence (-9% and -13%, respectively), 80% of the patients were symptom free. Similarily, our patients were all symptom free and remained so 1 yr after the study, hence, this toxicity is of weak clinical significance in subjects free of inflammatory lung diseases. HBO therapy, though safe, is not totally without effect on the lung.

Adult↗
PubMed2001

RXP 407, a selective inhibitor of the N-domain of angiotensin I-converting enzyme, blocks in vivo the degradation of hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro with no effect on angiotensin I hydrolysis.

The phosphinic peptide RXP 407 has recently been identified as the first potent selective inhibitor of the N-active site (domain) of angiotensin-converting enzyme (ACE) in vitro. The aim of this study was to probe the in vivo efficacy of this new ACE inhibitor and to assess its effect on the metabolism of AcSDKP and angiotensin I. In mice infused with increasing doses of RXP 407 (0.1--30 mg/kg/30 min), plasma concentrations of AcSDKP, a physiological substrate of the N-domain, increased significantly and dose dependently toward a plateau 4 to 6 times the basal levels. RXP 407 significantly and dose dependently inhibited ex vivo plasma ACE N-domain activity, whereas it had no inhibitory activity toward the ACE C-domain. RXP 407 (10 mg/kg) did not inhibit the pressor response to an i.v. angiotensin I bolus injection in mice. In contrast, lisinopril infusion (5 and 10 mg/kg/30 min) affected the metabolism of both AcSDKP and angiotensin I. Thus, RXP 407 is the first ACE inhibitor that might be used to control selectively AcSDKP metabolism with no effect on blood pressure regulation.

Angiotensin I↗
PubMed2000

Increased shedding of angiotensin-converting enzyme by a mutation identified in the stalk region.

Angiotensin-converting enzyme (ACE), an enzyme that plays a major role in vasoactive peptide metabolism, is a type 1 ectoprotein, which is released from the plasma membrane by a proteolytic cleavage occurring in the stalk sequence adjacent to the membrane anchor. In this study, we have discovered the molecular mechanism underlying the marked increase of plasma ACE levels observed in three unrelated individuals. We have identified a Pro(1199) --> Leu mutation in the juxtamembrane stalk region. In vitro analysis revealed that the shedding of [Leu(1199)]ACE was enhanced compared with wild-type ACE. The solubilization process of [Leu(1199)]ACE was stimulated by phorbol esters and inhibited by compound 3, an inhibitor of ACE-secretase. The results of Western blot analysis were consistent with a cleavage at the major described site (Arg(1203)/Ser(1204)). Two-dimensional structural analysis of ACE showed that the mutated residue was critical for the positioning of a specific loop containing the cleavage site. We therefore propose that a local conformational modification caused by the Pro(1199) --> Leu mutation leads to more accessibility at the stalk region for ACE secretase and is responsible for the enhancement of the cleavage-secretion process. Our results show that different molecular mechanisms are responsible for the common genetic variation of plasma ACE and for its more rare familial elevation.

Amino Acid Sequence↗
PubMed2000

Profiles of a healthful diet and its relationship to biomarkers in a population sample from Mediterranean southern France.

OBJECTIVES: The failure of single-nutrient supplementation to prevent disease in intervention studies underlines the necessity to develop a holistic view of food intake. The objectives of this study were to devise a diet quality index (DQI) and identify biomarkers of multidimensional dietary behavior. DESIGN: A nutrition survey was conducted in Mediterranean southern France by means of a food frequency questionnaire. The DQI was based on current dietary recommendations for prevention of diet-related diseases such as cardiovascular disease and some cancers. A second DQI included tobacco use. STATISTICAL ANALYSES: performed Spearman rank correlations, cross-classifications and intraclass correlations were computed between the DQI and biomarkers. RESULTS: Of the 146 subjects, 10 had a healthful diet and 18 had a poor diet. Erythrocyte omega-3 fatty acids-eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)-beta carotene, and vitamin E concentrations were lower and cholesterol concentrations were higher in the poor diet; the difference was significant for EPA and DHA and borderline significant for vitamin E. Significant correlation was found between the DQI and vitamin E (-0.12), EPA (-0.30), and DHA (-0.28), and beta carotene (-0.17) when tobacco use was considered, but not between the DQI and cholesterol. The correlation coefficient reached 0.58 (P0.01) for a composite index based on all biomarkers except cholesterol. CONCLUSIONS: Subjects with a beta carotene levels greater thanl micromol/L, vitamin E greater than 30 micromol/L and EPA greater than 0.65% and DHA greater than 4% of fatty acids in erythrocytes were likely to have a healthful diet. Each biomarker indicated the quality of diet, but correlation was higher with a composite index.

Adult↗
PubMed2000

In vitro and in vivo inhibition of the 2 active sites of ACE by omapatrilat, a vasopeptidase inhibitor.

The vasopeptidase inhibitor omapatrilat inhibits both neutral endopeptidase and angiotensin-converting enzyme (ACE). The in vitro and in vivo inhibitory potency of omapatrilat and the specific ACE inhibitor fosinopril toward the 2 active sites of ACE (called N- and C-domains) was investigated with the use of 3 substrates: angiotensin I, which is equally cleaved by the 2 ACE domains; hippuryl-histidyl-leucine, specific synthetic substrate of the C-domain in high- salt conditions; and a newly synthesized specific substrate of the N-domain designed by acetylating the lysine residue of AcSDKP. In vitro, omapatrilat was 5 times more potent than fosinoprilat in inhibiting angiotensin I hydrolysis. Omapatrilat inhibited similarly both N- and C-domain hydrolysis, whereas fosinoprilat was slightly more specific for the N-domain. The in vivo selective inhibitory potency of single oral doses of 10 mg omapatrilat and 20 mg fosinopril were investigated in a double-blind, placebo-controlled, cross-over study in 9 mildly sodium-depleted normotensive subjects. In accordance with the in vitro results, fosinopril appeared to be more specific for the N-domain than the C-domain in vivo, since plasma and urine AcSDKP concentrations were significantly higher than those observed with omapatrilat. This study shows that it is possible to assess separately in vitro and in vivo the selectivity of ACE or ACE/neutral endopeptidase inhibitors. A differential selectivity may explain some peculiar properties observed with some ACE inhibitors.

Adolescent↗
PubMed2000

Exclusion of angiotensin I-converting enzyme as a candidate gene involved in exudative inflammatory resistance in F344/N rats.

BACKGROUND: Inbred LEW/N and F344/N rats respectively, are susceptible and relatively resistant to a broad range of inflammatory/autoimmune diseases. We recently identified a quantitative trait locus (QTL) on chromosome 10 that protects the F344/N rat from carrageenan-induced exudation in a dominant fashion. Angiotensin I-converting enzyme (ACE) is one of the candidate genes located in this QTL region that plays an important role in inflammation. MATERIALS AND METHODS: RNA was extracted from both LEW/N and F344/N rat strains and used to produce full length cDNA by reverse transcription polymerase chain reaction (RT-PCR). Both strands of the PCR products were entirely sequenced to determine nucleotide differences between strains. ACE activity was measured using the synthetic substrate 3H-hippuryl-glycylglycine. ACE protein levels were determined by Western blot using a specific ACE antibody. ACE kinetic and inhibition studies were performed using specific substrates (Hip-His-Leu and Acetyl-Seryl-Aspartyl-Acetyl-Lysyl-Proline) and inhibitors (lisinopril, captopril and quinaprilat) for each C- and N-terminal active site. Finally, the dose-effects of lisinopril treatment on carrageenen-induced exudate volume and ACE activity was studied. RESULTS: In this study, we report for the first time a missense mutation in the coding region of ACE cDNA at 5' 1021 from C to T, resulting in a Leu-341 to Phe substitution, close to the N-domain active site in the F344/N rats. Full characterization of soluble and tissue ACE in both LEW/N and F344/N rat strains showed that soluble ACE levels in serum and exudate were 1.5 fold higher in the F344/N rats than those in LEW/N rats. In addition, the soluble ACE level was inversely correlated with the exudate volume. However, the specific ACE activity and its catalytic properties were identical in both strains. Furthermore, the chronic inhibition of serum and exudate ACE levels by lisinopril treatment did not affect the exudate volume in F344/N rats, indicating that several factors besides ACE were involved in the control of carrageenan-induced exudation. CONCLUSIONS: This report describes a complete molecular, biochemical, enzymatic and pharmacologic study of a missense mutation in the ACE cDNA in F344/N rats, that taken together, excludes ACE as a candidate gene involved with resistance to carrageenan-induced exudation in F344/N rats.

Amino Acid Sequence↗
PubMed1999

RXP 407, a phosphinic peptide, is a potent inhibitor of angiotensin I converting enzyme able to differentiate between its two active sites.

The human somatic angiotensin converting enzyme (ACE) contains two homologous domains, each bearing a zinc-dependent active site. All of the synthetic inhibitors of this enzyme used in clinical applications interact with these two active sites to a similar extent. Recently, several lines of evidence have suggested that the N-terminal active site of ACE might be involved in specific hydrolysis of some important physiological substrates, like Acetyl-Seryl-Aspartyl-Lysyl-Proline, a negative regulator of hematopoietic stem cell differentiation and proliferation. These findings have stimulated studies aimed at identifying new ACE inhibitors able to block only one of the two active sites of this enzyme. By screening phosphinic peptide libraries, we discovered a phosphinic peptide Ac-Asp-(L)Phepsi(PO2-CH2)(L)Ala-Ala-NH2, called RXP 407, which is able to differentiate the two ACE active sites, with a dissociation constant three orders of magnitude lower for the N-domain of the enzyme. The usefulness of a combinatorial chemistry approach to develop new lead structures is underscored by the unusual chemical structure of RXP 407, as compared with classical ACE inhibitors. As a highly potent and selective inhibitor of the N-terminal active site of wild ACE (Ki = 12 nM), RXP 407, which is metabolically stable in vivo, may lead to a new generation of ACE inhibitors able to block in vivo only a subset of the different functions regulated by ACE.

Amino Acid Sequence↗
PubMed1999

N-domain selectivity of angiotensin I-converting enzyme as assessed by structure-function studies of its highly selective substrate, N-acetyl-seryl-aspartyl-lysyl-proline.

The physiological functions of angiotensin I-converting enzyme (ACE) are not limited to its cardiovascular role. ACE constantly degrades N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a natural circulating regulator of the hematopoietic stem cell proliferation, and thereby may be involved in hematopoietic stem cell regulation. AcSDKP is hydrolyzed 50-fold faster by the N-domain active site compared to the C-domain active site. The aim of the present study was to investigate which aminoacid residues from AcSDKP are required to ensure N-domain specificity. Several peptides were designed by progressively increasing the length of the peptidic chain from a tripeptide to a pentapeptide. Kinetic studies of the wild-type ACE and of the two ACE mutants containing a single active domain (N- or C-domain) were performed using Bz (benzoyl) Asp-Lys-Pro, benzoyl-glycyl (Bz-Gly)-Asp-Lys-Pro, and Bz-Gly-Ser-Asp-Lys-Pro (with its intermediate product Bz-Gly-Ser-Asp) as substrates. The unexpected importance of an aspartic acid in the P1 position was discovered, as well as the interaction of the P2 and P3 positions in the substrate to increase or decrease N-domain specificity. Substrates longer than five residues may involve interdependence between subsites. Finally, the discovery of highly specific and novel N-domain substrates cannot be predicted from single subsite mapping, but may require other approaches such as combinatorial peptide libraries.

Binding Sites↗
PubMed1999

Hydrolysis by somatic angiotensin-I converting enzyme of basic dipeptides from a cholecystokinin/gastrin and a LH-RH peptide extended at the C-terminus with gly-Arg/Lys-arg, but not from diarginyl insulin.

Endoproteolytic cleavage of protein prohormones often generates intermediates extended at the C-terminus by Arg-Arg or Lys-Arg, the removal of which by a carboxypeptidase (CPE) is normally an important step in the maturation of many peptide hormones. Recent studies in mice that lack CP activity indicate the existence of alternative tissue or plasma enzymes capable of removing C-terminal basic residues from prohormone intermediates. Using inhibitors of angiotensin I-converting enzyme (ACE) and CP, we show that both these enzymes in mouse serum can remove the basic amino acids from the C-terminus of CCK5-GRR and LH-RH-GKR, but only CP is responsible for converting diarginyl insulin to insulin. ACE activity removes C-terminal dipeptides to generate the Gly-extended peptides, whereas CP hydrolysis gives rise to CCK5-GR and LH-RH-GK, both of which are susceptible to the dipeptidyl carboxypeptidase activity of ACE. Somatic ACE has two similar protein domains (the N-domain and the C-domain), each with an active site that can display different substrate specificities. CCK5-GRR is a high-affinity substrate for both the N-domain and C-domain active sites of human sACE (Km of 9.4 microm and 9.0 microm, respectively) with the N-domain showing greater efficiency (kcat : Km ratio of 2.6 in favour of the N-domain). We conclude that somatic forms of ACE should be considered as alternatives to CPs for the removal of basic residues from some Arg/Lys-extended peptides.

Animals↗
PubMed1999

In vivo assessment of captopril selectivity of angiotensin I-converting enzyme inhibition: differential inhibition of acetyl-ser-asp-lys-pro and angiotensin I hydrolysis.

Angiotensin I-converting enzyme (ACE) is a zinc metallopeptidase that plays a major role in blood pressure regulation. The demonstration that the hemoregulatory peptide acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a natural and specific substrate of the N-active site of ACE suggests that this enzyme may have a new physiological role such as the modulation of hematopoietic stem cells. In vitro studies have shown that ACE inhibitors displayed various potencies in inhibiting the degradation of different natural or synthetic substrates of ACE, among which captopril inhibits AcSDKP hydrolysis more potently than angiotensin I hydrolysis. To look for this selectivity in vivo, we investigated the pharmacodynamic effect of increasing doses of captopril (0.01-10 mg/kg) during the 90 min after i.v. administration to spontaneously hypertensive rats. Plasma and urinary AcSDKP levels were measured. The renin-angiotensin system was evaluated by measurements of ACE activity in plasma samples, using the synthetic substrate Hip-His-Leu, by determinations of plasma renin concentrations and measurements of arterial blood pressure. The results showed that captopril (0.01-0.3 mg/kg) selectively inhibited AcSDKP hydrolysis, with limited effects on the renin-angiotensin system. AcSDKP levels in plasma and urine rose to a plateau 4 times the basal level for doses more than 0.3 mg/kg. All of the parameters reflecting the renin-angiotensin system were significantly affected at doses of 1 and 10 mg/kg. The present study therefore confirms that captopril can be used to protect hematopoietic stem cells during antitumor chemotherapy while having only a limited effect on cardiovascular homeostasis.

Angiotensin I↗
PubMed1998

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

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 kcat 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 Phe-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 Ki values in the nanomolar range, whereas lisinopril and fosinoprilat were less potent. We show that the two Drosophila ACEs are alternatively expressed in stages P1 (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.

Amino Acid Sequence↗
PubMed1997

Cleavage-secretion of angiotensin I-converting enzyme in yeast.

Angiotensin I-converting enzyme (ACE) is a type I transmembrane protein composed of two domains (N and C domains) which undergoes a post-translational proteolytic cleavage in mammalian cells to release the soluble ectodomain. The protease involved in ACE cleavage-secretion (ACE-secretase) is not well characterised and eludes isolation: the presence of a yeast homologue, thus more amenable to genetic manipulation, would facilitate its identification. We have expressed a secreted form of the ACE C domain, lacking the C-terminal membrane anchor (C domain(deltaCOOH)), and the membrane-anchored C domain (C domain) in the yeast Pichia pastoris by fusion to prepro-alpha-factor. Immunofluorescent labelling localises the ACE C domain to the periphery of yeast cells but not C domain(deltaCOOH), however, expression of both C domain and C domain(deltaCOOH) produced soluble enzymes in the culture medium. Immunocharacterisation of the two soluble forms of the C domain indicates a proteolytic cleavage of the membrane-bound C domain to produce the soluble counterpart. Thus ACE undergoes a proteolytic cleavage in yeast.

Amino Acid Sequence↗