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R Coulombe

Publications and source records attributed to R Coulombe.

At least 19 recordsLinked to original sources
PubMed2001

Oxygen access to the active site of cholesterol oxidase through a narrow channel is gated by an Arg-Glu pair.

Cholesterol oxidase is a monomeric flavoenzyme that catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one. Two forms of the enzyme are known, one containing the cofactor non-covalently bound to the protein and one in which the cofactor is covalently linked to a histidine residue. The x-ray structure of the enzyme from Brevibacterium sterolicum containing covalently bound FAD has been determined and refined to 1.7-A resolution. The active site consists of a cavity sealed off from the exterior of the protein. A model for the steroid substrate, cholesterol, can be positioned in the pocket revealing the structural factors that result in different substrate binding affinities between the two known forms of the enzyme. The structure suggests that Glu(475), located at the active site cavity, may act as the base for both the oxidation and the isomerization steps of the catalytic reaction. A water-filled channel extending toward the flavin moiety, inside the substrate-binding cavity, may act as the entry point for molecular oxygen for the oxidative half-reaction. An arginine and a glutamate residue at the active site, found in two conformations are proposed to control oxygen access to the cavity from the channel. These concerted side chain movements provide an explanation for the biphasic mode of reaction with dioxygen and the ping-pong kinetic mechanism exhibited by the enzyme.

Arginine↗
PubMed2000

The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site.

The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 A resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 A long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO-AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals significant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis.

Amino Acid Oxidoreductases↗
PubMed2000

Crystal structure of the NADP+-dependent aldehyde dehydrogenase from Vibrio harveyi: structural implications for cofactor specificity and affinity.

Aldehyde dehydrogenase from the bioluminescent bacterium, Vibrio harveyi, catalyses the oxidation of long-chain aliphatic aldehydes to acids. The enzyme is unique compared with other forms of aldehyde dehydrogenase in that it exhibits a very high specificity and affinity for the cofactor NADP(+). Structural studies of this enzyme and comparisons with other forms of aldehyde dehydrogenase provide the basis for understanding the molecular features that dictate these unique properties and will enhance our understanding of the mechanism of catalysis for this class of enzyme. The X-ray structure of aldehyde dehydrogenase from V. harveyi has been solved to 2.5-A resolution as a partial complex with the cofactor NADP(+) and to 2. 1-A resolution as a fully bound 'holo' complex. The cofactor preference exhibited by different forms of the enzyme is predominantly determined by the electrostatic environment surrounding the 2'-hydroxy or the 2'-phosphate groups of the adenosine ribose moiety of NAD(+) or NADP(+), respectively. In the NADP(+)-dependent structures the presence of a threonine and a lysine contribute to the cofactor specificity. In the V. harveyi enzyme an arginine residue (Arg-210) contributes to the high cofactor affinity through a pi stacking interaction with the adenine ring system of the cofactor. Further differences between the V. harveyi enzyme and other aldehyde dehydrogenases are seen in the active site, in particular a histidine residue which is structurally conserved with phosphorylating glyceraldehyde-3-phosphate dehydrogenase. This may suggest an alternative mechanism for activation of the reactive cysteine residue for nucleophilic attack.

Aldehyde Dehydrogenase↗
PubMed1998

Structural basis for specificity of papain-like cysteine protease proregions toward their cognate enzymes.

Synthetic peptides corresponding to the proregions of papain-like cysteine proteases have been shown to be good and selective inhibitors of their parental enzymes. The molecular basis for their selectivity, quite remarkable in some cases, is not fully understood. The recent determination of the crystal structures of three distinct papain-like cysteine protease zymogens allows detailed structural comparisons to be made. The reasons for the specificity shown by each proregion toward its cognate enzyme are explained in terms of the three-dimensional structure of the proregion and the interface between the mature enzyme and the proregion. These comparisons reveal that insertion and substitution of amino acids within the proregion cause major rearrangement of sidechains on the enzyme/proregion interface, allowing detailed surface and charge recognition.

Amino Acid Sequence↗
PubMed1996

Structure of human procathepsin L reveals the molecular basis of inhibition by the prosegment.

Cathepsin L is a member of the papain superfamily of cysteine proteases and, like many other proteases, it is synthesized as an inactive proenzyme. Its prosegment shows little homology to that of procathepsin B, whose structure, the first for a cysteine protease proenzyme, has been determined recently. We report here the 3-D structure of a mutant of human procathepsin L determined at 2.2 A resolution, describe the mode of binding employed by the prosegment and discuss the molecular basis for other possible roles of the prosegment. The N-terminal part of the prosegment is globular and contains three alpha-helices with a small hydrophobic core built around aromatic side chains. This domain packs against a loop on the enzyme's surface, with the aromatic side chain from the prosegment being located in the center of this loop and providing a large contact area. The C-terminal portion of the prosegment assumes an extended conformation and follows along the substrate binding cleft toward the N-terminus of the mature enzyme. The direction of the prosegment in the substrate binding cleft is opposite to that of substrates. The previously described role of the prosegment in the interactions with membranes is supported by the structure of its N-terminal domain. The fold of the prosegment and the mechanism by which it inhibits the enzymatic activity of procathepsin L is similar to that observed in procathepsin B despite differences in length and sequence, suggesting that this mode of inhibition is common to all enzymes from the papain superfamily.

Amino Acid Sequence↗
PubMed1996

Protein crystallization in low gravity by step gradient diffusion method.

Two-step crystallization experiments were conducted in low gravity employing a liquid-liquid diffusion method in an effort to eliminate problems associated with protein crystal growth under the supersaturating conditions required for nucleation. Experiments were performed in diffusion cells formed by the sliding of blocks on orbit. Step gradient diffusion experiments consisted of first exposing protein solutions in diffusion half-wells for brief periods to initiating buffer solutions of high precipitant concentrations to induce nucleation followed by expoure of the same protein solutions to solutions of lower precepitant concentration to promote growth of induced nuclei into crystals. To avoid convective disturbances that occur when solutions of discrepant densities are interfaced at normal gravity, crystallization of hen egg-white lysozyme and rabbit skeletal muscle aldolase by step gradient diffusion was investigated in low gravity on four NASA space shuttle flights. In general, the largest ctystals of both proteins formed at the highest initiating precipitant concentration used, which is consistent with nuclei formation upon brief exposure to high precipitant concentration, and that these nuclei are competent for sustained growth at lower precipitant concentration. The two-step approach dissociates nucleation events from crystal growth allowing parameters affecting nucleation kinetics such as time, precipitant concentration and temperature of nucleation to be varied separately from conditions used for post-nucleation growth.

Animals↗
PubMed1996

Structure of rat procathepsin B: model for inhibition of cysteine protease activity by the proregion.

BACKGROUND: Cysteine proteases of the papain superfamily are synthesized as inactive precursors with a 60-110 residue N-terminal prosegment. The propeptides are potent inhibitors of their parent proteases. Although the proregion binding mode has been elucidated for all other protease classes, that of the cysteine proteases remained elusive. RESULTS: We report the three-dimensional structure of rat procathepsin B, determined at 2.8 A resolution. The 62-residue proregion does not form a globular structure on its own, but folds along the surface of mature cathepsin B. The N-terminal part of the proregion packs against a surface loop, with Trp24p (p indicating the proregion) playing a pivotal role in these interactions. Inhibition occurs by blocking access to the active site: part of the proregion enters the substrate-binding cleft in a similar manner to a natural substrate, but in a reverse orientation. CONCLUSIONS: The structure of procathepsin B provides the first insight into the mode of interaction between a mature cysteine protease from the papain superfamily and its prosegment. Maturation results in only one loop of cathepsin B changing conformation significantly, replacing contacts lost by removal of the prosegment. Contrary to many other proproteases, no rearrangement of the N terminus occurs following activation. Binding of the prosegment involves interaction with regions of the enzyme remote from the substrate-binding cleft and suggests a novel strategy for inhibitor design. The region of the prosegment where the activating cleavage occurs makes little contact with the enzyme, leading to speculation on the activation mechanism.

Animals↗
PubMed1996

Crystallization and preliminary X-ray diffraction studies of human procathepsin L.

Human procathepsin L has been expressed in the yeast Pichia pastoris and its inactive (Cys25Ser) and unglycosylated (Thr110Ala) mutant purified, concentrated to 4 mg/ml, and crystallized by vapor diffusion against solution containing 1.4 M (Na,K)PO4 buffer, pH 7.8. Crystal size was increased by multiple macroseeding. The crystals are orthorhombic, of space group P2 1 2 1 2 1, with cell dimensions of a = 40.2 A, b = 88.4 A, and c = 94.9 A. A 2.2 A native data set was collected using synchrotron radiation. Although molecular replacement solution for the mature portion of the enzyme was easily found, the resulting maps could not be interpreted in the proregion. Heavy-atom derivative search is in progress.

Cathepsin L↗
PubMed1996

Crystal structure of two new bifunctional nonsubstrate type thrombin inhibitors complexed with human alpha-thrombin.

The crystal structures of two new thrombin inhibitors, P498 and P500, complexed with human alpha-thrombin have been determined at 2.0 A resolution and refined to crystallographic R-factors of 0.170 and 0.169, respectively. These compounds, with picomolar binding constants, belong to a family of potent bifunctional inhibitors that bind thrombin at two remote sites: the active site and the fibrinogen recognition exosite (FRE). The inhibitors incorporate a nonsubstrate type active site binding fragment: Dansyl-Arg-(D)Pipecolic acid (Dns-Arg-(D)Pip), reminiscent of the active-site directed inhibitors MD-805 and MQPA, rendering them resistant to thrombin-induced hydrolysis. The FRE binding fragment of these inhibitors corresponds to the hirudin55-65 sequence. They differ in the chemical nature of the nonpeptidyl linker bridging these two functional activities. In both cases, the active site binding fragment is well defined in the electron density. The DnsH1, ArgH2, and (D)PipH3 groups occupy the S3, S1, and S2 subsites of thrombin, respectively, in a way similar to that observed in the thrombin-MQPA complexes. Binding in the active site of thrombin is characterized by numerous van der Waals contacts and ring-ring system interactions. Unlike in the substrate-like inhibitors, ArgH2 enters the S1 specificity pocket from the P2 position and adopts a bent conformation to make an hydrogen bond to the carboxylate of Asp189. In this noncanonical position, its carbonyl points away from the oxyanion hole, which is now occupied by well-ordered solvent molecules. The linkers fit in the groove extending from the active site to the FRE. The C-terminal fragments of both inhibitors bind in the same way as analogous FRE binding elements in previously described complexes.

Amino Acid Sequence↗
PubMed1996

Novel active site in Escherichia coli fructose 1,6-bisphosphate aldolase.

The molecular architecture of the Class II E. coli fructose 1,6-bisphosphate aldolase dimer was determined to 1.6 A resolution. The subunit fold corresponds to a singly wound alpha/beta-barrel with an active site located on the beta-barrel carboxyl side of each subunit. In each subunit there are two mutually exclusive zinc metal ion binding sites, 3.2 A apart; the exclusivity is mediated by a conformational transition involving side-chain rotations by chelating histidine residues. A binding site for K+ and NH4+ activators was found near the beta-barrel centre. Although Class I and Class II aldolases catalyse identical reactions, their active sites do not share common amino acid residues, are structurally dissimilar, and from sequence comparisons appear to be evolutionary distinct.

Binding Sites↗
PubMed1996

[Training needs for educators and nurses using the individualized services plan in mental health].

With the help of the method elaborated by Herskowitz (1977), this study examines the training needs of 42 workers (19 educators and 23 nurses) using when needed, the individualized services plan in a context of external services (deinstitutionalization) in mental health and internal services (hospital). A modified version of a questionnaire developed by Hurteau (1988) adapted to the context of mental health, identifies the training needs of workers using the individualized services plan under five specific dimensions and three types of knowledge. In the light of results obtained, certain training needs appear as top priority, especially conducting meetings and methods of elaboration of individualized services plan. Moreover, workers would like to obtain training on community-oriented services offered to people with needs in mental health.

Community Mental Health Services↗
PubMed1988

Metabolism of arachidonic acid through the 5-lipoxygenase pathway in normal human peritoneal macrophages.

Peritoneal macrophages (PM), obtained from 39 healthy women with normal laparoscopy findings, were stimulated with the ionophore A23187 or/and arachidonic acid (AA) both in adherence and in suspension. AA lipoxygenase metabolites were determined by reversed-phase HPLC. The major metabolites identified were 5-hydroxyeicosatetraenoic acid (5-HETE), leukotriene (LT)B4 and LTC4. The 20-hydroxy-LTB4, 20-carboxy-LTB4, and 15-HETE were not detected. Incubations of adherent PM with 2 microM A23187 induced the formation of LTB4, 110 +/- 19 pmol/10(6) cells, 5-HETE, 264 +/- 53 pmol/10(6) cells and LTC4, 192 +/- 37 pmol/10(6) cells. When incubated with 30 microM exogenous AA, adherent PM released similar amounts of 5-HETE (217 +/- 67 pmol/10(6) cells), but sevenfold less LTC4 (27 +/- 12 pmol/10(6) cells) (p less than 0.01). In these conditions LTB4 was not detectable. These results indicate that efficient LT synthesis in PM requires activation of the 5-lipoxygenase/LTA4 synthase, as demonstrated previously for blood phagocytes. When stimulated with ionophore, suspensions of Ficoll-Paque-purified PM produced the same lipoxygenase metabolites. The kinetics of accumulation of the 5-lipoxygenase/LTA4 synthase products in A23187-stimulated adherent cells varied for the various metabolites. LTB4 reached a plateau by 5 min, whereas LTC4 levels increased up to 60 min, the longest incubation time studied. Levels of 5-HETE were maximal at 5 min, and then slowly decreased with time. Thus, normal PM, in suspension or adherence, have the capacity to produce significant amounts of 5-HETE, LTB4, and LTC4. The profile of lipoxygenase products formed by the PM and the reactivity of this cell to AA and ionophore A23187 are similar to those of the human blood monocyte, but different from those of the human alveolar macrophage.

Adult↗
PubMed1988

Stoichiometric carboxyl methylation of chromogranins from bovine adrenal medullary cells.

The secretory proteins from adrenal chromaffin granules, chromogranins A, A1 and A2, were found to be excellent in vitro methyl acceptor proteins. The purified protein-carboxyl methylase (PCM) from bovine erythrocytes incorporated 660, 2540 and 7890 pmol of methyl group/10 min/mg protein in chromogranins A, A1 and A2, respectively. However, dopamine beta-hydroxylase, another secretory protein within chromaffin granules was poorly methylated. The stoichiometry of methylation for chromogranins A, A1 and A2 was 0.26, 0.89 and 1.3 mol of methyl group/mol of protein, respectively. Kinetic analysis showed that chromogranins A1 had the highest turnover rate followed by chromogranins A2 and A. These chromogranins are the first secretory proteins to be stoichiometrically methylated in vitro without prior deamidation.

Adrenal Medulla↗
PubMed1988

Protein-carboxyl methylation in adrenal medullary cells.

1. The protein-carboxyl methylating system has been studied in adrenal medullary cells either using disrupted cell components or with intact cells. Whereas the enzyme protein-carboxyl methylase (PCM) is cytosolic, the majority of its substrates is on or within chromaffin granules. With intact granules, methylation of surface proteins results in solubilization of membrane proteins. 2. Membrane PCM substrates have been identified as two proteins with apparent molecular weights of 55,000 and 32,000. Among the substrates located inside the granules, the chromogranins are excellent substrates, while dopamine beta-hydroxylase is poorly methylated. 3. Under physiological conditions, stimulation of the splanchnic nerve results in an increase in adrenal medullary protein-methyl ester formation as well as in an augmented methanol production. With adrenal medullary cells in culture, carboxyl-methylated chromogranin A is detected in mature chromaffin granules between 3 and 6 hr after labeling. Methylated chromogranins are secreted concomitantly with catecholamines following cholinergic stimulation. 4. These data coupled with those of Chelsky et al. (J. Biol. Chem. 262:4303-4309, 1987) on lamin B suggest that PCM methylates residues other than D-aspartyl and L-isoaspartyl in proteins. They further suggest that methylation may occur on nascent peptide chains before they are injected into the rough endoplasmic reticulum.

Adrenal Medulla↗
PubMed1988

Bronchoalveolar lavage cell differential on microscope glass cover. A simple and accurate technique.

We describe a quick and easy technique to perform cell differentials on bronchoalveolar lavage: the microscope glass cover. Lavage fluids of 72 subjects were analyzed by 3 techniques: glass cover, filter, and cytocentrifuge preparations. Seventy-seven other lavages were analyzed by glass cover and cytocentrifuge preparations alone. Data for the 72 subjects studied by all 3 techniques showed that the cell counts on glass cover and filter preparations were similar, e.g., lymphocytes, 19.2% (range, 0.5 to 94%) and 20.9% (range, 3 to 95%), respectively (Spearman's correlation coefficient, 0.98). However, on cytocentrifuge preparations, lymphocyte counts were lower (8.3%; range, zero to 87%) and macrophage counts were higher (p less than 0.005). Comparison of glass cover and cytocentrifuge preparation mixtures with varying amounts (20 to 80%) of purified blood leukocytes labeled with 51Cr (greater than or equal to 72% lymphocytes) showed that a significant amount of radioactive cells was lost during the cytocentrifuge technique in contrast to the glass cover technique. Because neutrophils represented a low proportion of lavage cells, we also evaluated cell suspensions with known neutrophil contents (10 to 70%); we found no difference in neutrophil counts obtained with the 3 techniques. Lavage data analysis of 40 young nonsmoking volunteers showed that glass cover lymphocyte count was also higher than counts on cytocentrifuge preparations: 16.5% (range, 3 to 45%) and 8.2% (range, 2.5 to 35%), respectively. In this group, the distribution of glass cover lymphocyte percentages was normal (p = 0.21, chi 2 test), and the one-tailed 95% confidence interval was 18.6 to 34.7% (mean plus 1.65 standard deviation).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1986

Decreased leukotriene B4 synthesis in smokers' alveolar macrophages in vitro.

Recent studies have shown that alveolar macrophages (AM) are able to release leukotrienes (LTs). Since cigarette smoking inhibits the cyclooxygenase pathway of arachidonic acid metabolism in the AM, we evaluated the LT production by AM from smokers and nonsmokers. AM were obtained from 35 volunteers, 16 nonsmokers, and 19 smokers. The cells were incubated under various conditions including stimulation with 30 microM arachidonic acid, 2 microM ionophore A23187, or both. Each experiment was performed in parallel using cells from a smoker and a nonsmoker. Lipoxygenase products were analyzed by reverse-phase high performance liquid chromatography. After stimulation, nonsmokers' AM produced LTB4 and 5-hydroxy-eicosatetraenoic acid (5-HETE). In incubations of AM with arachidonic acid and ionophore, the amounts of products formed were: LTB4, 317 +/- 56 pmol/10(6) cells and 5-HETE, 1,079 +/- 254, mean +/- SEM. No metabolites were generated under control conditions (no stimulation). In all incubations performed, the peptido-LTs (LTC4, LTD4, and LTE4) were undetectable. In comparison with AM from nonsmokers, those from smokers showed a 80-90% reduction of 5-HETE and LTB4 synthesis (P less than 0.05 to P less than 0.001 according to stimulatory conditions). This defective lipoxygenase metabolite production in AM from smokers was observed over a wide range of stimuli concentrations and incubation times; AM from smokers also had lower levels of intracellular (esterified) 5-HETE than nonsmokers' AM. We also studied blood polymorphonuclear leukocytes (PMNL) and no difference in the synthesis of 5-lipoxygenase products in these cells was noticed between smokers and nonsmokers. These data show that cigarette smoking causes a profound inhibition of the 5-lipoxygenase pathway in AM but not in blood PMNL.

Adult↗
PubMed1976

The effects of propranolol, practolol, and placebo on the clinical manifestations of thyrotoxicosis.

Thirty patients with thyrotoxicosis were randomly divided into 3 groups of ten which were treated for 8 weeks with either practolol, propranolol or placebo. The pills were administered in a double blind manner and the investigator had the option of doubling the dose at each visit. Propranolol and practolol were equally effective, and better than placebo, at relieving the patients general well-being as well as the tachycardia, subjective and objective tremor, skin warmness and moistness and the intensity of the thyroid bruit.

Auscultation↗