Biomedical subjects

R Verger

Publications and source records attributed to R Verger.

At least 91 records · Page 5Linked to original sources

PubMed1994

Interactions between beta-cyclodextrin and insoluble glyceride monomolecular films at the argon/water interface: application to lipase kinetics.

A study of the desorption rate of insoluble monomolecular films of oleic acid (OA), monoolein (MO), 1,2-diolein (1,2-DO), 1,3-diolein (1,3-DO) and triolein (TO) at the argon/water interface by water-soluble beta-cyclodextrin (beta-CD) is reported. The desorption of OA and MO involves probably the complexation of the single acyl chain with beta-CD and sequestering of the formed soluble OA/beta-CD and MO/beta-CD complexes from the argon/water interface. In the case of monolayers of multiple acyl chain molecules such as DO and TO, no detectable change in the surface pressure occurred after beta-CD injection. The surface rheological dilatational properties of the monolayers of DO and TO in the presence of beta-CD in the subphase were studied. The elasticity of the DO monolayer remained unchanged, whereas the decrease in the surface elasticity of the TO film was attributed to the formation of a water-insoluble TO/beta-CD complex. With the 'tuning fork' model, one acyl chain of TO can be included in the beta-CD cavity. The formed TO/beta-CD complex present at the interface retarded the propagation of the dilatational deformation along the plane of the monolayer. Schematic models have been proposed in an attempt to explain the different complexation of these lipids by beta-CD at the argon/water interface. In addition to the above results, the presence of beta-CD in the water subphase makes it possible for the first time to perform kinetic measurements of the lipase hydrolysis rates of long-chain glycerides forming monomolecular films.

Argon↗

PubMed1994

Evidence for a pancreatic lipase subfamily with new kinetic properties.

Several new members of the pancreatic lipase family have been reported recently, and amino acid sequence comparison reveals that this family can now be divided into three subgroups: (1) "classical" pancreatic lipases, (2) related proteins 1 (RP1), and (3) related proteins 2 (RP2) (Giller, T., et al. (1992) J. Biol. Chem. 267(23), 16509-16516). Whereas "classical" pancreatic lipases are well characterized with respect to kinetic properties, i.e., interfacial activation and dependence on colipase in the presence of bile salts, the two latter subfamilies have been poorly investigated so far. The kinetic behavior of a lipase from guinea pig pancreas differs, however, from that of "classical" lipases (Hjorth, A., et al. (1993) Biochemistry 32, 4702-4707). This enzyme is highly homologous to RP2 lipases with the exception of a deletion in the so-called lid domain that regulates access to the active center of pancreatic lipases. We have now characterized a novel lipase from coypu (Myocastor coypus) pancreas. This enzyme, also belonging to the RP2 subfamily, possesses a full-length lid domain, but its kinetic properties are very similar to those of the guinea pig enzyme: (1) a high phospholipase activity, (2) the absence of interfacial activation, and (3) the absence of a colipase effect at high bile salt concentrations. Since both guinea pig and coypu pancreas produce a classical pancreatic lipase and no measurable phospholipase A2 activity, it is suggested that RP2 enzymes act as real phospholipases under physiological conditions. In fact, all RP2 lipases from other species might share phospholipase activity and fulfill new biological functions.

Amino Acid Sequence↗

PubMed1994

The condensing effects of egg lecithin and cholesterol on triolein monolayers are inhibited by substitution of one saturated acyl chain in the triacylglycerol.

Previous work showed that the clearance from plasma of chylomicron-like emulsions injected intravenously was affected by the acyl chains of the constituent triacylglycerols. Compared with emulsions containing triolein (OOO) as the only triacylglycerol, clearances were decreased by a single saturated chain in emulsions containing 1,3-dioleoyl-2-stearoyl-sn-glycerol (OSO), 1,2-dioleoyl-3-stearoyl-sn-glycerol (OOS) or 1-stearoyl-2,3-dioleoyl-sn-glycerol (SOO). The differences in clearance may reflect physical differences at the oil-water interface related to chain interactions of the triacylglycerol structures with other lipid components. In the present work lipid monomolecular films at the air-water interface were used to establish the capacity of OOO to interact with the pure synthetic triacylglycerols OOS and SOO, and the capacity of OOS and SOO to co-exist in monolayers of lecithin and of cholesterol was compared with OOO. Substituting one oleoyl chain by a stearoyl chain induced a 20% condensation in monomolecular films of the pure triacylglycerols. Mixtures of OOO with either pure egg yolk phosphatidylcholine or cholesterol also showed substantial condensing effects. In contrast substituting one oleoyl chain by a stearoyl chain substantially lessened the condensing effects. At surface pressures above the collapse pressure of the pure triacylglycerols, substantially more OOO than OOS or SOO was retained in mixed monolayers with phosphatidylcholine. These differences could underlie the effects on metabolism of saturated chains in emulsion triacylglycerols.

Chemical Phenomena↗

PubMed1994

Cutinase, a lipolytic enzyme with a preformed oxyanion hole.

Cutinases, a group of cutin degrading enzymes with molecular masses of around 22-25 kDa (Kolattukudy, 1984), are also able to efficiently hydrolyse triglycerides (De Geus et al., 1989; Lauwereys et al., 1991), but without exhibiting the interfacial activation phenomenom (Sarda et al., 1958). They belong to a class of proteins with a common structural framework, called the alpha/beta hydrolase fold (Martinez et al., 1992; Ollis et al., 1992). We describe herein the structure of cutinase covalently inhibited by diethyl-p-nitrophenyl phosphate (E600) and refined at 1.9-A resolution. Contrary to what has previously been reported with lipases (Brzozowski et al., 1991; Derewenda et al., 1992; Van Tilbeurgh et al., 1993), no significant structural rearrangement was observed here in cutinase upon the inhibitor binding. Moreover, the structure of the active site machinery, consisting of a catalytic triad (S120, H188, D175) and an oxyanion hole (Q121 and S42), was found to be identical to that of the native enzyme, whereas the oxyanion hole of Rhizomucor lipase (Brzozowski et al., 1991; Derewenda et al., 1992), like that of pancreatic lipase (van Tilbeurgh et al., 1993), is formed only upon enzyme-ligand complex formation. The fact that cutinase does not display interfacial activation cannot therefore only be due to the absence of a lid but might also be attributable to the presence of a preformed oxyanion hole.

Amino Acid Sequence↗

PubMed1994

Digestive lipases: inactivation by phosphonates.

Phosphonates mimicking the transition state which occurs during carboxyester hydrolysis were synthesized and investigated as potential inactivators of human pancreatic (HPL) and gastric (HGL) lipases. Their efficiency as inactivators was studied on the basis of the alkyl chain length, the nature of the leaving group and the influence of the ester substituent. In each case, HGL was found to be more sensitive than HPL towards these phosphonates. The released p-nitrophenol to enzyme ratio indicates that a 1:1 complex was formed. In the absence of substrate, the most powerful inactivator was O-methyl O-(p-nitrophenyl) n-pentylphosphonate (4A), which has a short alkyl chain, a small methoxy substituent and a good leaving group.

Enzyme Activation↗

PubMed1994

Structure-function relationships in naturally occurring mutants of pancreatic lipase.

From primary structure comparison, the pancreatic lipase family is now divided into three subgroups: classical pancreatic lipases, pancreatic lipase-related proteins 1 (RPI) and pancreatic lipase-related proteins 2 (RP2). Among the RP2 subfamily, the guinea-pig and coypu enzymes share kinetic properties which differ from those of classical pancreatic lipases. Both enzymes display a high phospholipase activity and are not interfacially activated using a short chain triglyceride as substrate. Their activity towards insoluble triglycerides is inhibited by micellar concentrations of bile salts and is not restored by addition of colipase. These atypical kinetic properties are discussed in the light of amino acid sequence comparison between RP2 and classical pancreatic lipases, based on the closed and open conformations of the 3-D structure of human pancreatic lipase.

Amino Acid Sequence↗

PubMed1993

Inactivation of pancreatic lipases by amphiphilic reagents 5-(dodecyldithio)-2-nitrobenzoic acid and tetrahydrolipstatin. Dependence upon partitioning between micellar and oil phases.

We have reported previously that Cys103 (SHII) of human pancreatic lipase (HPL), unlike the nonessential Cys181 (SHI), was buried and inaccessible to classical water-soluble sulfhydryl reagents. The lipolytic activity of HPL was lost after the labeling of the above two SH groups with the amphiphilic sulfhydryl reagent, 5-(dodecyldithio)-2-nitrobenzoic acid (C12-TNB), suggesting that the SHII residue may play an important role in the hydrolytic process [Gargouri, Y., Cudrey, C., Medjoub, H., & Verger, R. (1992) Eur. J. Biochem. 204, 1063-1067]. For the present experiments, we selected dog pancreatic lipase (DPL), purifying it for the first time, and recombinant guinea pig pancreatic lipase (r-GPL), which both contain a buried SHII group but no accessible SHI group. The single SHII of DPL and r-GPL reacted only with the amphiphilic SH reagent (C12-TNB), and its labeling was correlated with a rapid lipase inactivation. Although it is spatially remote from the catalytic triad, the SHII group of pancreatic lipases, when chemically labeled, was found to be responsible for the loss of their lipolytic activity. The presence of a bulky dodecyl chain, linked by a disulfide bond to the SHII, may have prevented the critical beta-5 loop (residues 76-85) movement by steric hindrance and consequently disturbed the formation of the oxyanion hole. Thus, pancreatic lipase inactivation by the amphiphilic sulfhydryl reagent can be said to be due to the prevention of a productive induced fit. Tetrahydrolipstatin (THL) is an amphiphilic inactivator reacting with the essential serine of the lipase active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Improved purification and biochemical characterization of phospholipase D from cabbage.

Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) was purified from cabbage leaves. The two step purification procedure involved hydrophobic chromatography on Octyl-Sepharose followed by a Mono-Q/FPLC-column with a total yield of 23% and a purification factor of 1000. A zymographic assay was used to detection of PL D activities at various stages of purification under non denaturing PAGE. The molecular mass was determined to be 90 kDa using the SDS/PAGE method, and 90,200 Da as calculated from the amino acid analysis. The isoelectric point of the enzyme is acidic (pI = 4.7). The amino-acid composition and 29 residues of the NH2-terminal amino-acid sequence were determined.

Amino Acid Sequence↗

PubMed1993

Purification of human gastric lipase by immunoaffinity and quantification of this enzyme in the duodenal contents using a new ELISA procedure.

Human gastric lipase (HGL) is the first lipolytic enzyme involved in the digestion of dietary lipids along the gastrointestinal tract. We describe an improved procedure for isolating the enzyme using immunoaffinity chromatography in combination with ion-exchange chromatography. The purified enzyme, showing a single band on SDS-PAGE, expressed a specific activity of 1000 U/mg using tributyrin as the substrate. We also describe a specific enzyme-linked immunosorbent assay (ELISA) procedure for measuring duodenal HGL levels. The ELISA was performed using an anti-HGL polyclonal antibody (pAb) as the captor antibody and a biotinylated monoclonal antibody (mAb) as the detector antibody. With the double sandwich ELISA technique, HGL in the range of 1-60 ng/ml was measured in less than 5 h. Identical HGL concentrations were obtained using the above ELISA procedure when compared to those based on the enzymatic activity using the potentiometric method (correlation coefficient: r = 0.95). No significant interference from other duodenal components was observed, as proved by the quantitative HGL determinations performed on intestinal samples.

Chromatography, Affinity↗

PubMed1993

Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography.

The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).

Binding Sites↗

PubMed1993

Crystallization of pancreatic procolipase and of its complex with pancreatic lipase.

The human pancreatic lipase-porcine procolipase complex has been crystallized in space group P3(2)21 (a = b = 80.3 A and c = 251 A) from a solution containing polyethylene glycol, NaCl and beta-octyl glucoside. The crystals diffract to 2.6 A on a synchrotron beam. The complex in the presence of bile salts and phospholipids crystallizes in a tetragonal space group P4(2)2(1)2 (a = b = 133.4 A, c = 92.6 A). Crystals of procolipase alone were obtained under slightly different experimental conditions (space group I432, a = b = c = 164.3 A).

Animals↗

PubMed1993

Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies.

Five monoclonal antibodies (mAb) directed against human gastric lipase (HGL) have been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. All these mAb belong to the IgG1 class with a kappa light chain. The effects of these mAb on the enzymic activity of HGL were studied and used to define three classes of antibodies, depending upon their immunoinactivation properties. As determined by ELISA and immunoinactivation studies, four overlapping epitopes were found to be part of the functional sites of the enzyme. The mAb appear to be suitable probes for studying the lipid binding and catalytic domains of HGL. The results of the ELISA additivity test were used to describe tentatively the epitopes of HGL in terms of a schematic spatial map.

Antibodies, Monoclonal↗

PubMed1993

Controlling lipase stereoselectivity via the surface pressure.

In the present study, the stereoselectivity of Rhizomucor miehei lipase, lipoprotein lipase, Candida antarctica B lipase, and human gastric lipase towards racemic dicaprin spread as a monolayer at the air-water interface was investigated. For this purpose we have developed a method with which the enantiomeric excess of the residual substrate can be measured in monomolecular films. The stereoselectivity, which is one of the main aspects of enzymic catalysis, was found to depend on the surface pressure of the substrate. With all four lipases tested, low surface pressures enhanced the stereoselectivity while decreasing the enzymes' catalytic activity.

Candida↗

PubMed1993

Stereoselective hydrolysis of triglycerides by animal and microbial lipases.

In the present paper, a study on the stereoselectivity of 25 lipases of animal and microbial origin towards homogeneous prochiral triglycerides is presented. All the lipases tested catalyse the hydrolysis of the chemically alike but sterically nonequivalent ester groups in trioctanoin and triolein with different degrees of stereobias, depending on the fatty acyl chain length of the substrate (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990). Hydrolysis of the sn-2 ester group is catalysed by very few lipases and only Candida antarctica A shows a clear preference for this position. Most of the lipases investigated (12 with trioctanoin and 16 with triolein) showed a preference for the sn-1 position. Using trioctanoin as substrate we observed a total stereoselectivity for position sn-1 with Pseudomonas sp. and Pseudomonas aeruginosa and for position sn-3 with Candida antarctica B. This was not the case with triolein as substrate. Among the 23 lipases studied here and the other two lipases described previously (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990), 17 show a higher stereoselectivity with trioctanoin than with triolein. With guinea pig pancreatic lipase and with three mold lipases (Geotrichum candidum M, Geotrichum candidum A, and Candida antarctica B), the preference switches from sn-3 to sn-1 when the acyl chain length increases from eight to 18 carbon atoms. The main conclusion to emerge from the present study is that the specific stereopreference of each lipase for a given substrate under given lipolytic conditions can be said to be its fingerprint.

Animals↗

PubMed1993

Secretion and contribution to lipolysis of gastric and pancreatic lipases during a test meal in humans.

BACKGROUND: The aim of this study was to quantitatively evaluate the relative contributions to in vivo lipolysis of gastric and pancreatic lipases. METHODS: Gastric and pancreatic lipase secretions were measured, and their respective levels were determined in duodenal fluid during the digestion of a liquid test meal in healthy volunteers. Gastric lipase activity was clearly distinguished from that of pancreatic lipase by using both a specific enzymatic assay and an enzyme-linked immunosorbent assay. Lipolysis products were monitored throughout the digestion period. RESULTS: On a weight basis, the ratio of pancreatic lipase to gastric lipase total secretory outputs was found to be around four after 3 hours of digestion. The level of gastric hydrolysis was calculated to be 10% +/- 1% of the acyl chains released from the meal triglycerides. Gastric lipase remained active in the duodenum where it might still hydrolyze 7.5% of the triglyceride acyl chains. CONCLUSIONS: Globally during the whole digestion period, gastric lipase might hydrolyze 17.5% of the triglyceride acyl chains. In other words, gastric lipase might hydrolyze 1 acyl chain of 4, which need to be hydrolyzed for a complete intestinal absorption of monoglycerides and free fatty acids resulting from the degradation of two triglyceride molecules.

Adult↗

PubMed1993

Gastric and pancreatic lipase levels during a test meal in dogs.

The levels of gastric and pancreatic lipases in the duodenum and the jejunum were measured during the digestion of a test meal in dogs. Using both a specific enzymatic titration and an enzyme-linked immunosorbent assay, it is shown for the first time that gastric lipase remains active in the duodenal and jejunal contents. An experimental device was set up for measuring the secretions and the intestinal flows of lipases during the digestion of a liquid test meal. In a dog equipped with gastric and duodenal cannulae, the secretion of gastric lipase was stimulated by food ingestion, reaching 3.0 +/- 0.3 mg/h (three times the basal secretion rate) during the 1st h of digestion. The total secretory outputs of gastric and pancreatic lipases recorded over a 3-h period of digestion were 7.2 +/- 1.2 mg and 18.7 +/- 1.2 mg, respectively.

Animals↗

PubMed1992

Structure of the pancreatic lipase-procolipase complex.

Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.

Colipases↗

PubMed1992

Competitive inhibition of lipolytic enzymes. VIII: Inhibitor-induced aggregation of porcine pancreatic phospholipase A2.

Several 2-acylaminophospholipid analogues have been demonstrated to behave as potent competitive inhibitors of porcine pancreatic phospholipase A2 (De Haas, G.H., Dijkman, R., Ransac, S. and Verger, R. (1990) Biochim. Biophys. Acta 1046, 249-257). Their inhibitory power appeared to be strictly controlled by the stereoconfiguration around the chiral C-2 atom and effective inhibition of the enzyme was observed only when incorporated into a micellar substrate-water interface. In the present study various direct binding techniques were applied to investigate the interaction of the enzyme with pure micelles of the stereoisomeric forms of 2-tetradecyl-amino-hexanol-1-phosphocholine (R-C14-PN and S-C14-PN). Upon equilibrium gel filtration of the enzyme (monomeric molecular mass = 14 kDa) on calibrated Superdex columns running in micellar solutions of R-C14-PN, the phospholipase eluted as a lipid-protein complex of 74 kDa. Under identical conditions, micellar solutions of S-C14-PN did not give rise to high-molecular mass aggregates and the enzyme eluted at its normal 14 kDa position. Light scattering experiments, ultrasedimentation and time-resolved fluorescence spectroscopy studies confirmed the formation of a high-molecular mass aggregate between enzyme and R-C14-PN micelles. The ultimate complex was shown to consist of four protein and about ten inhibitor molecules. Using time-resolved fluorescence spectroscopy the interaction was studied between the active site of phospholipase A2 and R-C14-PN molecules, both incorporated in an inert lipid matrix.

Animals↗