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R Leduc

Publications and source records attributed to R Leduc.

At least 55 records · Page 3Linked to original sources
PubMed1993

Characterization of a secreted form of human furin endoprotease.

Human furin, a member of a recently discovered family of cellular endoproteases, has been identified as a membrane bound protein localized in the Golgi apparatus. Here, we report the presence of a secreted form of furin in the media of cells infected with a vaccinia virus recombinant containing the furin gene. Using the fluorogenic substrate boc-Arg-Val-Arg-Arg-MCA, endoproteolytic activity was detected in the media of infected BSC40 cells. Immunoprecipitations of [35S]-labeled proteins from infected cells revealed that the media contained a lower molecular form of furin than the cellular furin or than a previously characterized soluble furin mutant, hFUR713t. By using the direct linear plot representation of the Michaelis-Menten equation the results demonstrate that the soluble furin exhibited similar kinetics to the hFUR713t enzyme. Thus, our results suggest that membrane-bound furin undergoes post-translational processing to produce a soluble form of the enzyme that can be secreted.

Amino Acid Sequence↗
PubMed1993

Presence of furin mRNA in cultured bovine endothelial cells and possible involvement of furin in the processing of the endothelin precursor.

Recently it was documented that furin, a calcium-dependent serine endoprotease, cleaves many protein precursors at pairs of basic amino acids, thus liberating the biologically active peptides. The endothelin precursors follow a biosynthetic pathway similar to these proteins, where the precursor is initially processed to the intermediate, big endothelin (big ET) before its conversion to the endothelin (ET) peptide. Analysis of the amino acid sequence of the endothelin pro-proteins shows that they are susceptible to processing by endoproteases that cleave at pairs of basic amino acids. For example, human endothelin-1 (ET-1) precursor possesses a typical furin cleavage site motif (Arg-X-Lys/Arg-Arg) at the following residues: Arg32-Ser33-Lys34-Arg35 and Arg72-Ser73-Lys74-Arg75. We have isolated mRNA from cultured bovine endothelial cells and, using a human furin cRNA probe, shown that a furin mRNA of 4.5 kb is present in these cells. We propose that furin, a novel endoprotease belonging to the mammalian subtilisin family of serine proteases, may be implicated in the processing of pro-endothelin precursors, liberating big ET.

Actins↗
PubMed1992

Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic cleavage.

Human furin is a calcium-dependent serine endoprotease that can efficiently cleave many precursor proteins on the carboxyl side of the consensus cleavage sequence, -Arg-X-Lys/Arg-Arg-, both in vivo and in vitro. Analysis of furin proteins in extracts of cells infected with a vaccinia recombinant expressing human furin show that the enzyme is present as two prominent forms of 90 and 96 kDa. Because the structurally related bacterial subtilisins require endoproteolytic removal of the NH2-terminal pro-region by an autocatalytic intramolecular cleavage, we speculated that the size heterogeneity in the furin doublet similarly may result from a proteolytic removal of an NH2-terminal pro-region. Here we report identification of the 90-kDa furin NH2 terminus and, based on the reported sequence of the furin cDNA, demonstrate that this furin protein is derived from a larger precursor by an endoproteolytic cleavage on the COOH-terminal side of a consensus furin cleavage site, -Arg-Thr-Lys-Arg107-. Expression of mutant furin molecules containing an altered cleavage site (Arg104----Ala or Arg107----Gly) resulted in the production of only the 96-kDa furin protein. Assays of furin-dependent cleavage of a protein substrate in vitro showed that proteolytic activity was associated with the 90-kDa and not the 96-kDa furin protein, demonstrating that removal of the NH2-terminal pro-region is required for furin activity. Expression of a third furin construct containing a mutation of the active site aspartate (Asp153----Asn) similarly resulted in the expression of only the 96-kDa protein, suggesting that furin activation occurs by an autoproteolytic cleavage. Finally, the production of 90-kDa furin from either site-directed furin mutant could not be potentiated by overexpressing active furin, suggesting that the autoproteolytic activation was an intramolecular event.

Amino Acid Sequence↗
PubMed1992

Aerosols characterization in a forested site in Québec, Canada.

Three AERAS low pressure 11 stage cascade impactors with rotatable collecting plates (LPCR) were installed at the Duchesnay forest station near Québec City and four low pressure inertial collectors (LPIC) were installed in the forest. The analysis of the aerosol deposits was performed by static fast atom beam mass spectroscopy (FABMS); SIMS and ESCA analyses were also made. The morphological characterization and the elemental volumetric composition of the aerosols were established by a scanning electron microscope equipped with an X-ray dispersive energy analyser. Micro-weighting of the aluminium substrate before and after the sampling by the AERAS impactor enabled the study of the mass-size-distribution of the aerosols according to their aerodynamic equivalent diameter. Example of the results obtained are presented in relation to air parcels' trajectories. This study has shown that it is possible to characterize the aerosols according to the origin and trajectories of the air mass with which they travel; sources along the tracks have been shown to contribute to the aerosol loading of the air masses.

Aerosols↗
PubMed1992

Nucleotide and deduced amino acid sequence of bovine adrenal medulla chromogranin B (secretogranin I).

1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain.

Adrenal Medulla↗
PubMed1991

Kex2-like endoproteases PC2 and PC3 accurately cleave a model prohormone in mammalian cells: evidence for a common core of neuroendocrine processing enzymes.

Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrine-precursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursor-processing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processing-deficient BSC-40 cells cleaved native mouse POMC at the -Lys-Arg- sites flanking corticotropin. The -Lys-Arg- within beta-lipotropin was less efficiently cleaved to release beta-endorphin. Expression of PC2 together with PC3 resulted in efficient conversion of beta-lipotropin, as occurs in pituitary melanotrophs. Furthermore, coexpression of PC2 together with mouse POMC in bovine adrenomedullary chromaffin cells resulted in conversion of beta-lipotropin to gamma-lipotropin and beta-endorphin in the regulated secretory pathway. Finally, the processing selectivities of PC3 and PC2 expressed together in BSC-40 cells were determined by using a series of mutant mouse POMCs containing all possible pairs of basic residues at certain sites. The observed pattern of cleavage site selectivities mimicked that of the endogenous endoproteases of the insulinoma and bovine adrenomedullary chromaffin cells, suggesting that PC2 and PC3 may represent important core endoproteases in the catalysis of prohormone processing in many neuroendocrine cell types.

Adrenocorticotropic Hormone↗
PubMed1990

Effect of soil/contaminant interactions on the biodegradation of naphthalene in flooded soil under denitrifying conditions.

The mineralization of 14C-labelled naphthalene was studied in pristine and oil-contaminated soil slurry (30% solids) under denitrifying conditions using a range of concentrations from below to above the aqueous phase saturation level. Results from sorption-desorption experiments indicated that naphthalene desorption was highly irreversible and decreased with an increase in the soil organic content, thus influencing the availability for microbial consumption. Under denitrifying conditions, the mineralization of naphthalene to CO2 occurred in parallel with the consumption of nitrate and an increase in pH from 7.0 to 8.6. When the initial substrate concentration was 50 ppm (i.e. close to the aqueous phase saturation level), about 90% of the total naphthalene was mineralized within 50 days, and a maximum mineralization rate of 1.3 ppm day-1 was achieved after a lag period of approx. 18 days. When added at concentrations higher than the aqueous phase saturation level (200 and 500 ppm), similar mineralization rates (1.8 ppm day-1) occurred until about 50 ppm of the naphthalene was mineralized. After that the mineralization rates decreased logarithmically to a minimum of 0.24 ppm day-1 for the rest of the 160 days of the experiments. For both of these higher concentrations, the reaction kinetics were independent of the concentration, indicating that desorption of the substrate governs the mineralization rate. Other results indicated that pre-exposure of soil to oil contamination did not improve the degradation rates nor reduce the lag periods. This study clearly shows the potential of denitrifying conditions for the biodegradation of low molecular weight PAHs.

Biodegradation, Environmental↗
PubMed1990

Fragmentation of bovine chromogranin A by plasma kallikrein.

Chromogranin A has been reported to be processed in vivo by an as yet undefined proteinase(s) suggesting that it is a precursor of biologically active peptides such as pancreastatin. In this study, plasma kallikrein was used as a model proteinase to identify the cleavage sites exposed in bovine parathyroid chromogranin A. Purified bovine parathyroid chromogranin A was digested with human plasma kallikrein. The proteolytic fragments produced were isolated by HPLC and chemically characterized by amino acid composition and sequence analysis. The combined results indicate that the enzyme has preference for specific single Arg residues, cutting C-terminal to this amino acid, although certain pairs of basic sites were also cleaved. The characterized fragments were released in a selective manner from the whole molecule with rapid production of the fragments covering positions 1-247 and 352-358.

Amino Acid Sequence↗
PubMed1990

Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo.

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (furin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or furin with the mouse beta-nerve growth factor precursor (pro-beta-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo.

Amino Acid Sequence↗
PubMed1989

The transport of lipoprotein cholesterol into bile: a reassessment of kinetic studies in the experimental animal.

Studies were undertaken to assess the contribution of lipoprotein cholesterol to bile and to determine whether already-existent hepatic free cholesterol and the free cholesterol which is newly generated from the hydrolysis of hepatic cholesteryl esters are equally available for secretion into bile or constitute metabolically separate pools. Rats with a bile fistula were injected with an intravenous bolus of high-density lipoprotein recombinants containing free [14C]cholesterol and [3H]cholesteryl esters. Results showed (1) that bile free [14C]cholesterol secretion was a constant and linear proportion of the whole liver free [14C]cholesterol pool, (2) that secretion into bile of free [3H]cholesterol was in direct proportion to the rate of hydrolysis of hepatic [3H]cholesteryl esters, and (3) that rates of biliary cholesterol secretion were very similar when secretion was calculated using the specific activity of free [14C]cholesterol and free [3H]cholesterol in the entire liver to 'label' the precursor free cholesterol pool. Furthermore, rates of secretion that were calculated using either isotope closely approximated the mass of free cholesterol that was directly measured in bile. Results thus indicate that because of equilibration and extensive dilution by the large pool of already-existent free cholesterol, the transport of isotopic cholesterol from lipoproteins cannot be used to directly assess the contribution of lipoprotein cholesterol to the cholesterol that is secreted in bile. These studies further suggest that the totality of preformed free cholesterol in the liver is in metabolic equilibrium in one single kinetic pool and that all hepatic free cholesterol is potentially available for secretion into bile.

Animals↗
PubMed1987

Chromogranin B (secretogranin I), a putative precursor of two novel pituitary peptides through processing at paired basic residues.

During the course of reversed-phase high-pressure liquid chromatography (RP-HPLC) purification of the 7B2 peptide originally isolated in our laboratory from human pituitary gland extracts, two novel peptides were identified and purified to homogeneity. The complete amino acid sequence of the first one was established in 1985 and recently found to be entirely homologous to positions 420-493 of the just published chromogranin B sequence. This peptide, denoted GAWK, could originate from chromogranin B following specific cleavage at the basic amino acids flanking both termini of GAWK. Moreover, another peptide isolated in our laboratory from the same source and denoted CCB has been discovered and its sequence is also part of the same chromogranin B molecule. Here again, this peptide, occupying positions 597-653 and located at the COOH-terminal region of chromogranin B, could derive from specific processing at basic amino acids, Arg-Lys-Lys, present at positions 594-596. In a manner reminiscent of the relationship between pancreastatin and chromogranin A, it is proposed that both GAWK and CCB are produced from chromogranin B after specific processing at basic amino acids. These data are thus in favor of a putative role of chromogranins as precursors to potentially bioactive peptides.

Amino Acid Sequence↗
PubMed1987

Influence of different molecular species of phosphatidylcholine on cholesterol transport from lipoprotein recombinants in the rat.

Studies were performed to determine to what extent phosphatidylcholines (PCs) of different composition influence the turnover of lipoprotein cholesterol. Lipoprotein recombinants with the composition and structure of spherical high density lipoproteins (HDL-R) were prepared with apoproteins, 14C-labeled unesterified cholesterol (UC), a [3H]cholesteryl ester (CE), and one of four single molecular species of PC. PCs were selected to include relatively hydrophilic species (16:1-16:1 and 16:0-18:2 PCs) and relatively hydrophobic species (18:0-18:2 and 20:1-20:1 PCs). PCs were also selected to include molecules with novel acyl group pairs (16:1-16:1 and 20:1-20:1 PCs) that would permit the whole molecule to be traced during its clearance from the serum. Rats were injected with HDL-R as an intravenous bolus, and serum, liver, and bile samples were obtained for up to 2 h. The clearance from the serum of each PC was monoexponential with the two most hydrophilic species much more rapidly cleared than either of the two less hydrophilic species. Clearance of specific PCs was not accompanied by PC remodeling (i.e. transacylations), and in the main could not be attributed to the action of lecithin-cholesterol acyltransferase (LCAT). In incubations designed to simulate in vivo conditions, no more than 15% of the disappearance of 16:1-16:1 PC, one of the most rapidly cleared PCs, was due to the action of LCAT. With 20:1-20:1 PC, one of the least rapidly cleared PCs, no LCAT activity could be detected. The clearance of radiolabeled UC was multiexponential and closely corresponded to the rate of disappearance of each PC. The clearance of radiolabeled CE was linear and, in contrast to UC, was the same with the administration of different PCs. Uptake of radiolabeled UC by the liver and excretion of radiolabeled UC into bile took place in parallel and corresponded to the rapidity of turnover of UC (and PCs) in the serum. With administration of 16:1-16:1 PC, complete equilibration of serum, liver, and bile UC was achieved by about 90 min, whereas with 20:1-20:1 PC, serum UC had not equilibrated by the end of the study. These findings demonstrate that, in the live animal, the kinetic pattern of transport of different lipids from an HDL recombinant is highly disparate, the rate of PC clearance is more rapid with molecular species of greater hydrophilic strength, and the rates of PC and UC clearance are closely coordinated and largely independent of the clearance of CE.

Animals↗
PubMed1987

The complete amino acid sequence of rat submaxillary gland tonin does contain the aspartic acid at the active site: confirmation by protein sequence analysis.

The revised amino acid sequence of rat submaxillary gland tonin, a serine protease, does contain the active site Asp residue. The active site of this kallikrein-related enzyme is thus made up of the same catalytic triad (Asp, Ser, and His) found in all known serine proteases. The important Asp residue has now been localized in a 16 amino acid peptide previously reported as missing in the tonin sequence. The complete amino acid sequence thus contains 235 residues corresponding to a molecular weight of 25,658, more in agreement with previously reported molecular weights. Moreover, the revised structure led (a) to the assignment of Arg, Asn, and Val residues instead of His, Asp, and Gly at positions 63, 165, and 169, respectively; (b) to the assignment of residues occupying an overlapping sequence at positions 165-171, and finally (c) to the localization of two N-glycosylation sites at positions 82 and 165. These results further document the close relationship of tonin to the ever expanding kallikrein family.

Amino Acid Sequence↗
PubMed1985

GAWK, a novel human pituitary polypeptide: isolation, immunocytochemical localization and complete amino acid sequence.

During the course of reverse-phase high pressure liquid chromatography (RP-HPLC) purification of a postulated big ACTH (1) from human pituitary gland extracts, a highly purified peptide bearing no resemblance to any known polypeptide was isolated. The complete sequence of this 74 amino acid polypeptide, called GAWK, has been determined. Search on a computer data bank on the possible homology to any known protein or fragment, using a mutation data matrix, failed to reveal any homology greater than 30%. An antibody produced against a synthetic fragment allowed us to detect several immunoreactive forms. The antisera also enabled us to localize the polypeptide, by immunocytochemistry, in the anterior lobe of the pituitary gland.

Amino Acid Sequence↗
PubMed1984

The major androgen-dependent protease in dog prostate belongs to the kallikrein family: confirmation by partial amino acid sequencing.

Canine prostate fluids and seminal plasma contain a major androgen-dependent protein which was identified as a proteolytic enzyme exhibiting an Arg-esterase activity. This protease, as characterized, is shown to be present as a two-chain structure held together by at least one disulfide bridge and composed of approximately 220 amino acids. Amino acid sequence determination of both chains has revealed a clear homology to other known amino acid sequences of serine proteases. Furthermore, the comparison of the presented 58 amino acids of the Arg-esterase with the other sequences revealed a very strong homology (larger than 50%) to members of the kallikrein family. The two chain structure could thus result from autolysis of a single chain enzyme in the 'kallikrein autolysis loop'. Amino acid composition of the canine prostatic enzyme suggests that it is related, but not identical, to pancreatic canine kallikrein.

Amino Acid Sequence↗
PubMed1984

Angiotensin II: dependence of hormone affinity on the electronegativity of a single side chain.

Structure-activity studies on rabbit aorta of angiotensin II analogues have suggested a possible relationship between the electronegativity of the aromatic side chain in position 4 (Tyr) and the observed affinity. In order to test this hypothesis, several other analogues modified in position 4 have been prepared, and all available analogues were tested in three bioassays: in vitro on rabbit aorta strip, in vivo on the rat blood pressure, and the binding assay on beef adrenocortical membranes. In all three bioassays the postulated correlation has confirmed that angiotensin II affinity depends inversely on the electronegativity of the aromatic side chain in position 4.

Adrenal Cortex↗
PubMed1982

Composite sampling in the determination of pyrethrins in fruit samples.

A residue method is described for measuring up to 6 naturally occurring pyrethrins in several varieties of fruits. Fresh samples are extracted into acetone, partitioned between water and hexane-dichloro-methane, and cleaned up by adsorption on a Florisil column followed by elution with acetone-hexane (15 + 85). Residues are detected and quantitated by gas chromatography with electron capture detection. Detection limits for single 10 g samples are about 10-30 ng/g. The method was applied to a survey of pyrethrins in fruit samples. Because of the high tolerance (1 ppm) allowed for pyrethrins, the expected low incidence, and the low detection limits of the method, composite samples (up to 5-6) of fruit were used to quickly determine residue levels. This procedure allowed 130 samples to be processed in the time normally needed for 30-40. Provided the above conditions can be met, composite sampling is proposed as an attractive approach to data gathering in pesticide residue analysis.

Chromatography, Gas↗