Biomedical subjects

M Lenfant

Publications and source records attributed to M Lenfant.

At least 19 recordsLinked to original sources

PubMed2000

Coumarin-Ser-Asp-Lys-Pro-OH, a fluorescent substrate for determination of angiotensin-converting enzyme activity via high-performance liquid chromatography.

N-Acetyl-Ser-Asp-Lys-Pro-OH (AcSDKP-OH), a negative regulator of hematopoietic stem cell proliferation, is shown to be a physiological substrate of angiotensin I-converting enzyme (ACE), a zinc-dipeptidyl carboxypeptidase, involved in cardiovascular homeostasis. Recently, a study carried out on captopril-treated volunteers revealed that the kinetics of [3H]AcSDKP-OH hydrolysis in vitro in the plasma of donors correlates closely to the plasmatic ratio angiotensin II/angiotensin I, which characterized the conversion activity of ACE. This prompted us to design a fluorescent substrate, 2-[7-(dimethylamino)-2-oxo-2H-chromen-4-yl]acetyl-SDKP-OH, or coumarin-SDKP-OH, which could be an alternative to the radiolabeled analogue used in that study, allowing an easier and more rapid determination of enzyme activity. We report here the synthesis and the determination of the kinetics constants of this fluorescent derivative compared with those of [3H]AcSDKP-OH with human plasma ACE (133 and 125 microM, respectively), which are in the same range as those of the physiological substrate angiotensin I. Furthermore, the hydrolysis of the fluorescent substrate shows the same sensitivity toward chloride concentration as the natural substrate, demonstrating its specificity for N-domain hydrolysis. This fluorescent derivative was used to develop a sensitive assay for the determination of ACE activity in human plasma.

Chromatography, High Pressure Liquid↗

PubMed1998

Lisinopril, an angiotensin I-converting enzyme inhibitor, prevents entry of murine hematopoietic stem cells into the cell cycle after irradiation in vivo.

The hemoregulatory peptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) has been shown in vivo to inhibit the cycling of murine hematopoietic stem cells triggered into S-phase by either cytotoxic drug administration or irradiation. This property, further confirmed using in vitro models, demonstrates that the peptide has an in vivo protective effect on the hematopoietic system. AcSDKP has been shown to be a physiological substrate of angiotensin I-converting enzyme (ACE), which catabolizes the peptide through a dipeptidasic activity. Thus, oral administration of ACE inhibitor to humans has led to an increase in the plasma AcSDKP concentration. In the present paper, we report on the in vivo effect of lisinopril, an ACE inhibitor, on the proliferative status of murine hematopoietic stem cells triggered into S-phase by irradiation. Administration of lisinopril (10 mg/kg) 1 hour after irradiation led to a 90 to 100% inhibition of murine plasma ACE activity as observed during the first 4 hours postirradiation. This inhibition was correlated with a 600% increase in the endogenous plasma AcSDKP level and a total suppression at 24 hours of entry of the hematopoietic stem cell into the cell cycle. We discuss the possible role of ACE in the regulation of hematopoietic stem cell proliferation through control of the AcSDKP concentration.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1996

Acute angiotensin-converting enzyme inhibition increases the plasma level of the natural stem cell regulator N-acetyl-seryl-aspartyl-lysyl-proline.

Angiotensin I-converting enzyme (ACE) has two homologous active NH2- and COOH-terminal domains and displays activity toward a broad range of substrates. The tetrapeptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) has been shown to be hydrolyzed in vitro by ACE and to be a preferential substrate for its NH2-terminal active site. This peptide is a regulatory factor of hematopoiesis which reversibly stem cells and normal early progenitors into S-phase. We found that a single oral dose of 50 mg of the ACE inhibitor, captopril, when administered to eight healthy subjects in a double-blind, crossover, placebo-controlled study, massively increased the plasma level of Ac-SDKP. ACE inhibition by captopril induced a 90-99% inhibition of in vitro [3H]Ac-SDKP hydrolysis and a long-lasting 5.5-fold (range: 4-8.5-fold) increase in the plasma levels of Ac-SDKP. These results demonstrate that Ac-SDKP is the first natural peptide hydrolyzed by the NH2-terminal domain of ACE not only in vitro but also in vivo, confirming that both catalytic sites of ACE are physiologically active. Our data suggest that ACE may also be implicated in the process of hematopoietic stem cell regulation, by permanently degrading this natural circulating inhibitor of cell entry into S-phase.

Administration, Oral↗

PubMed1996

Catabolism of the hemoregulatory peptide N-Acetyl-Ser-Asp-Lys-Pro: a new insight into the physiological role of the angiotensin-I-converting enzyme N-active site.

The tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro (AcSDKP) was first isolated from bone marrow extracts and shown to be involved in the negative control of hematopoiesis by preventing the recruitment of primitive stem cells into S-phase. In vitro studies on AcSDKP catabolism in human plasma revealed that AcSDKP was cleaved by plasmatic angiotensin-I converting enzyme (ACE). The evaluation of the respective involvement of the two active sites of ACE in AcSDKP degradation in vitro revealed that the N-active site was preferentially involved in this catabolism. Moreover, an in vivo study on healthy volunteers of the catalytic efficiency of ACE towards AcSDKP after administration of Captopril demonstrated that AcSDKP was a physiological substrate of ACE. AcSDKP might represent the first natural specific substrate of the N-active site of the enzyme. These results pose the question of a potential role of ACE in the control of hematopoiesis as well as possible applications of ACE inhibitors to cope with dysfunctions in which AcSDKP might exert physiological control.

Binding Sites↗

PubMed1995

The hemoregulatory peptide N-acetyl-Ser-Asp-Lys-Pro is a natural and specific substrate of the N-terminal active site of human angiotensin-converting enzyme.

Angiotensin I-converting enzyme (ACE) is a zinc-dipeptidyl carboxypeptidase, which contains two similar domains, each possessing a functional active site. Respective involvement of each active site in the degradation of the circulating peptide N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP), a negative regulator of hematopoietic stem cell proliferation, was studied by using wild-type recombinant ACE and two full-length mutants containing a single functional site. Both the N- and C-active sites of ACE exhibit dipeptidyl activity toward AcSDKP, with Km values of 31 and 39 microM, respectively. However, the N-active site hydrolyzes the peptide 50 times faster compared with the C-active site, with kcat/Km values of 0.5 and 0.01 microM-1.s-1, respectively. The predominant role of the N-active site in AcSDKP hydrolysis was confirmed by the inhibition of hydrolysis using a monoclonal antibody specifically directed against the N-active site. The N-domain specificity for AcSDKP will aid the identification of specific inhibitors for this domain. This is the first report of a highly specific substrate for the N-active site of ACE, with kinetic constants in the range of physiological substrates, suggesting that ACE might be involved via its N-terminal active site in the in vivo regulation of the local concentration of this hemoregulatory peptide.

Amino Acid Sequence↗

PubMed1994

Interaction of G-actin with thymosin beta 4 and its variants thymosin beta 9 and thymosin beta met9.

Thymosin beta 4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin beta 9 and thymosin beta met9 are minor variants of thymosin beta 4. The possible function of these peptides has been investigated by comparing the actin binding properties of these beta-thymosins. Thymosin beta 9 and thymosin beta met9 were found to inhibit polymerization of ATP-actin with identical KDs of 0.7-0.8 microM (as compared to 2 +/- 0.3 microM for thymosin beta 4); like thymosin beta 4, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin beta 4 and thymosin beta met9 with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin beta 4 to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin beta 4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of approximately 1.5 microM-1 s-1 and 2 s-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin beta 4 function in vivo is discussed.

Actins↗

PubMed1993

Involvement of human plasma angiotensin I-converting enzyme in the degradation of the haemoregulatory peptide N-acetyl-seryl-aspartyl-lysyl-proline.

The degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the haematopoietic stem cell, by enzymes present in human plasma, has been investigated. Radiolabelled AcSD[4-3H]KP ([3H]AcSDKP, 1 mM) was completely metabolized in human plasma with a half-life of 80 min, leading exclusively to the formation of radiolabelled lysine. The cleavage of AcSDKP was insensitive to classical proteinase inhibitors including leupeptin, but sensitive to metalloprotease inhibitors. The degradation was completely blocked by specific inhibitors of angiotensin I-converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1), showing that the first step of the hydrolysis was indeed due to ACE. In dialysed plasma, the hydrolysis proceeded at only 17% of the maximal rate, whereas addition of 20 mM NaCl led to the recovery of the initial rate observed with normal plasma. Hydrolysis of AcSDKP by commercial rabbit lung ACE generated the C-terminal dipeptide Lys-Pro. Thus, ACE cleaves AcSDKP by a dipeptidyl carboxypeptidase activity. In fact the formation of Lys-Pro was observed when AcSDKP was incubated in human plasma in the presence of HgCl2. These results suggest that ACE is involved in the first limiting step of AcSDKP degradation in human plasma. The second step seems to be under the control of a leupeptin- and E-64-insensitive, HgCl2-sensitive plasmatic enzyme.

Amino Acid Sequence↗

PubMed1993

Modulation of the interaction between G-actin and thymosin beta 4 by the ATP/ADP ratio: possible implication in the regulation of actin dynamics.

The interaction of G-actin with thymosin beta 4 (T beta 4), the major G-actin-sequestering protein in motile and proliferating cells, has been analyzed in vitro. T beta 4 is found to have a 50-fold higher affinity for MgATP-actin than for MgADP-actin. These results imply that in resting platelets and neutrophils, actin is sequestered by T beta 4 as MgATP-G-actin. Kinetic experiments and theoretical calculations demonstrate that this ATP/ADP dependence of T beta 4 affinity for G-actin can generate a mechanism of desequestration of G-actin by ADP, in the presence of physiological concentrations of T beta 4 (approximately 0.1 mM). The desequestration of G-actin by ADP is kinetically enhanced by profilin, which accelerates the dissociation of ATP from G-actin. Whether a local drop in the ATP/ADP ratio can allow local, transient desequestration and polymerization of actin either close to the plasma membrane, following platelet or neutrophil stimulation, or behind the Listeria bacterium in the host cell, while the surrounding cytoplasm contains sequestered ATP-G-actin, is an open issue raised by the present work.

Actins↗

PubMed1993

The molecular specificity of action of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in the control of hematopoietic stem cell proliferation.

The hemoregulatory tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, M(r) = 487 amu) prevents the recruitment of hematopoietic stem cells into S phase. AcSDKP does not possess inherent inhibitory activity against hematopoietic stem cells, rather it appears to act by "blocking" the action of a hematopoietic stem cell proliferation stimulator present in extracts of regenerating hematopoietic tissue. The molecular specificity of this action was investigated using a number of peptide analogues of AcSDKP and evaluating their ability to "block" the recruitment of a primitive murine hematopoietic precursor (high proliferative potential colony forming cell HPP-CFC) into S phase following incubation with a hematopoietic stem cell proliferation stimulator. The capacity of AcSDKP to compromise the action of the hematopoietic stem cell proliferation stimulator was not evident for either AcSDDKP or AcSD beta KP, two structurally distinct forms of the molecule; similar no stimulator-"blocking" activity was observed for the tripeptide Ala-Asp-Lys (ADK, M(r) = 332 amu), while the tripeptide Ser-Asp-Lys (SDK, M(r) = 348 amu) was active. It would appear that the hemoregulatory action of AcSDKP is specific and that the tripeptide sequence SDK may be a significant component of the molecule.

Amino Acid Sequence↗

PubMed1993

The tetrapeptide AcSerAspLysPro (Seraspenide), a hematopoietic inhibitor, may reduce the in vitro toxicity of 3'-azido-3'-deoxythymidine to human hematopoietic progenitors.

3'-azido-3'-deoxythymidine (AZT), the main antiviral drug used in AIDS treatment, is known to induce anemia and neutropenia. These effects have been attributed to its toxicity to hematopoietic progenitors. In this report, we present a new approach to reduce AZT hematotoxicity by using an inhibitory factor of the hematopoietic stem cells, the tetrapeptide AcSerAspLysPro (AcSDKP, Seraspenide), which has been shown to increase the survival of mice subjected to high doses of chemotherapy and to block reversibly the cycling of human granulocyte-macrophage colony forming unit (CFU-GM) and burst forming unit erythroid (BFU-E) progenitors. Normal bone marrow mononuclear cells (BMMNC) from 14 subjects were incubated with or without AcSDKP (10(-10) M) for 20 h and with or without AZT (100 microM) for another 2 h. After washing, cells were plated in methylcellulose in the presence of interleukin 3 (IL-3), granulocyte-macrophage colony stimulating factor (GM-CSF) and erythropoietin (EPO). Under these conditions, the preincubation of cells with AcSDKP reduced significantly the toxicity of AZT to both BFU-E and CFU-GM at least in 3 out of 8 and 4 out of 10 cases, respectively. A careful statistical analysis of these observations indicates that AcSDKP may be an efficient factor in preserving progenitors against AZT-induced hematopoietic toxicity.

Amino Acid Sequence↗

PubMed1993

Particular ability of cytochrome P-450 CYP3A to reduce glyceryl trinitrate in rat liver microsomes: subsequent formation of nitric oxide.

Glyceryl trinitrate was denitrated in rat hepatic subcellular fractions, with formation of glyceryl dinitrates and glyceryl mononitrates. Among differently treated-rat liver microsomes, the highest microsomal activity was obtained under anaerobic conditions with microsomal preparations from dexamethasone-treated rats and NADPH. The reaction was inhibited by O2, CO, miconazole, dihydroergotamine and troleandomycin showing that it was catalyzed by cytochrome P-450 CYP3A isoforms. The formation of a transient cytochrome P-450 Fe(II)-NO complex during this reaction was shown by visible spectroscopy. The cytosolic activity was shown to be dependent on glutathione and glutathione transferase and was not inhibited by dioxygen. In the hepatic 9000 x g supernatant containing both NADPH and cytochrome P-450 and glutathione and glutathione transferase, the cytochrome P-450-dependent reaction accounts for 30-40% of the total denitration activity observed under anaerobic conditions, using 100 microM GTN.

Animals↗

PubMed1993

Optimization of cell culture conditions for the evaluation of the biological activities of the tetrapeptide N-Acetyl-Ser-Asp-Lys-Pro, a natural hemoregulatory factor.

The present study attempts to define the difficulties in evaluating the properties of the hemoregulatory peptide AcSDKP using in vitro assays. In fact, in the presence of sera, which are generally added to basic culture media, AcSDKP is catabolized by proteases present in the serum. The kinetics of AcSDKP degradation depends on the nature and on the concentration of the added serum. In in vitro conditions, the half life of this peptide can be increased by the addition of 1 microM captopril, a metalloprotease inhibitor. Thus, these points need to be considered in designing experiments to study the effects of AcSDKP.

Amino Acid Sequence↗

PubMed1993

Catabolism of the tetrapeptide N-Ac-Ser-Asp-Lys-Pro (AcSDKP), an inhibitor of hematopoietic stem cell (CFU-S) proliferation, following in vitro incubation with hematopoietic tissues from normal and leukemic mice.

The comparative degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the hematopoietic pluripotent stem cell, was investigated following incubation with plasma, bone marrow and spleen cells from normal mice and mice bearing a transplantable myeloid leukemia. Using the tetrapeptide, specifically radiolabelled in the lysyl residue, degradation of [3H]AcSDKP was followed by measurement of [3H]Lys formation resulting from its catabolism. It was shown that already after 1 h the degradation of AcSDKP in plasma from leukemic mice was higher compared to that following incubation in plasma from normal mice, whereas incubation with bone marrow cells exhibits a small difference only after 4 hours incubation. However, no increase of AcSDKP catabolic activity was observed following incubation with spleen cells from leukemic animals when compared with incubation of normal spleen cells.

Animals↗

PubMed1992

The mechanism of action of the tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) in the control of haematopoietic stem cell proliferation.

The mechanism of action of the haemoregulatory tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, M(r) = 487 amu), was investigated using an in vitro assay of a murine high proliferative potential colony-forming cell (HPP-CFC) which responds to proliferation regulators of the haematopoietic stem cell population. AcSDKP had no direct inhibitory effect on the number, or the proportion in S phase, of the committed granulocyte-macrophage progenitor (GM-CFC), or cycling HPP-CFC populations. However, AcSDKP blocked the action of a stimulator of haematopoietic stem cell proliferation, preventing the switching of quiescent HPP-CFC into cell cycle. It would appear that AcSDKP exerts its inhibitory haemoregulatory role indirectly, by preventing stimulator action on haematopoietic stem cells.

Amino Acid Sequence↗

PubMed1992

Multiple enzymatic pathways involved in the metabolism of glyceryl trinitrate in Phanerochaete chrysosporium.

A study of glyceryl trinitrate metabolism by a filamentous fungus, Phanerochaete chrysosporium, carried out with the 14C-labeled substrate, provides evidence for a multienzymatic system leading to di- and mononitrate derivatives. At least two independent enzymatic activities were detected in the cytosolic fraction: an aerobic glutathione S-transferase activity and an anaerobic NADPH-dependent soluble cytochrome P450-like activity. Other hemoproteins with enzymatic activities dependent upon the presence of NADPH or ferrous ions were also detected in the microsomal fraction. Electron paramagnetic resonance spectra characteristic of an interaction between a hemoprotein and nitric oxide appeared in these two subcellular fractions during the anaerobic metabolism of glyceryl trinitrate.

Anaerobiosis↗

PubMed1991

Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation.

We have demonstrated that a filamentous fungus Phanerochaete chrysosporium converts glyceryl trinitrate (GTN) into its di- and mononitrate derivatives concurrently with the formation of nitric oxide detected by electron paramagnetic resonance (EPR), and the formation of nitrite. The metabolisms of nitrite and nitrate by the fungus are evaluated and taken into account when considering GTN degradation. Lack of evidence for nitrate formation from GTN suggests that an esterase-type activity is not involved. Furthermore, the kinetics of appearance of the hemoprotein-NO and non-heme protein-NO (FeS-NO) complexes indicate that an enzymatic process producing NO directly from GTN may be involved concurrently with a glutathione transferase-like system.

Aerobiosis↗

PubMed1991

Formation of acetyl-Ser-Asp-Lys-Pro, a new regulator of the hematopoietic system, through enzymatic processing of thymosin beta 4.

The demonstration that AcSDKP, a new regulator of the hematopoietic system, is formed in the bone marrow by a one-step enzymatic maturation processing of thymosin beta 4 (T beta 4) is presented. AcSDKP and T beta 4 were both detected in bone marrow cells (BMC). Incubation of [3H]T beta 4 with either intact or lysed BMC led to the formation of [3H]AcSDKP, whereas the labeled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of T beta 4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp-N might be involved in the formation of AcSDKP through the specific cleavage of the Pro4-Asp5 peptidic bond of T beta 4. In contrast, alpha-prolyl-endopeptidase was ineffective in carrying out a similar processing.

Amino Acid Sequence↗

PubMed1990

Involvement of thymosin beta 4 and endoproteinase Asp-N in the biosynthesis of the tetrapeptide AcSerAspLysPro a regulator of the hematopoietic system.

It is shown that AcSDKP a new regulator of the hematopoietic system can be generated from thymosin beta 4 by a one-step enzymatic cleavage in vitro and in vivo. AcSDKP and T beta 4 were both detected in bone marrow cells (BMC). Incubation of [3H]T beta 4 with either intact or lysed BMC led to the formation of [3H]AcSDKP whereas the labelled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of T beta 4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp-N might be involved in the formation of AcSDKP through the specific cleavage of the 4Pro-5 Asp peptide bond of T beta 4.

Amino Acid Sequence↗