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R Verger

Publications and source records attributed to R Verger.

At least 109 records · Page 6Linked to original sources
PubMed1992

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.

Animals↗
PubMed1992

Competitive inhibition of lipolytic enzymes. VII. The interaction of pancreatic phospholipase A2 with micellar lipid/water interfaces of competitive inhibitors.

In a recent series of kinetic studies (De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257 and references therein) we have demonstrated that synthetic (R)-phospholipid analogues containing a 2-acylaminogroup instead of the 2-acyloxy function found in natural phospholipids, behave as strong competitive inhibitors of porcine pancreatic phospholipase A2 (PLA2). We also showed that these analogues strongly bind to the active site of the enzyme but only after their incorporation into a micellar substrate/water interface. In the present study we investigated the interaction of native PLA2 and of an inactive PLA2 in which the active site residue His-48 has been modified by alkylation with 1-bromo-2-octanone, with pure micelles of several of these inhibitors in both enantiomeric forms by means of ultraviolet difference absorption spectroscopy. Our results show that the first interaction step between native or modified enzyme and micellar lipid/water interfaces probably consists of a low-affinity Langmuir-type adsorption characterized by signals arising from the perturbation of the single Trp-3 residue. Once present at the interface the native enzyme is able to bind, in a second step, a single inhibitor molecule of the (R)-configuration in its active site, whereas the (S)-enantiomer is not bound in the active site. The overall dissociation constant of the interfacial phospholipase-inhibitor complex is three orders of magnitude lower for micelles composed of the (R)-isomer than those of the (S)-isomer. The modified PLA2 still adsorbs to micellar lipid/water interfaces but cannot bind either of the two enantiomers into its active site and similar dissociation constants were found for lipid-protein complexes with micelles of either the (R) or the (S) inhibitors. After blanking the ultraviolet signals due to the perturbation of Trp-3 in the initial adsorption step of the enzyme to a micellar surface of a non-inhibitory phospholipid analogue, the progressive binding of a single (R)-inhibitor molecule into the active site could be followed quantitatively by a tyrosine perturbation. These titrations yielded numerical values for the dissociation constants in the interface and provide a possible explanation for the large difference in overall dissociation constants of the complexes between enzyme and micelles of (R)-and (S)-inhibitors. With the use of PLA2 mutants in which each time a single tyrosine was replaced by phenylalanine, the tyrosine residues involved in binding of the monomeric inhibitor molecule were identified as Tyr-69 and Tyr-52.

Animals↗
PubMed1992

Inactivation of human pancreatic lipase by 5-dodecyldithio-2-nitrobenzoic acid.

Both thiol groups of native human pancreatic lipase can react with the new hydrophobic sulfhydryl reagent 5-dodecyldithio-2-nitrobenzoic acid (Dod-S-NbS) in the absence of a denaturing agent. Here we describe for the first time the covalent and stoichiometric modification of the inaccessible SHII group of native pancreatic lipase, using a 16-fold molar excess of this hydrophobic sulfhydryl reagent. A direct correlation was found to exist between the covalent modification of this SHII group and the loss of lipase activity. The question has not yet been answered, however, as to how Dod-S-NbS reaches the SHII-containing residue, whereas classical hydrophilic sulfhydryl reagents are unable to do so. This difference in reactivity may be attributable to the hydrophobic character of Dod-S-NbS and its potential capacity to form aggregates inducing a conformational change in the lipase molecule.

Binding Sites↗
PubMed1992

Rat platelet phospholipase A2. Kinetic characterization using the monomolecular film technique.

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.

Animals↗
PubMed1992

Competitive inhibition of lipolytic enzymes. V. A monolayer study using enantiomeric acylamino analogues of phospholipids as potent competitive inhibitors of porcine pancreatic phospholipase A2.

For the first time, we have shown that a stereospecific interaction occurs between porcine pancreatic phospholipase A2 and a monomolecular film of amidophospholipid used as inhibitor. Direct binding experiments, using radiolabelled phospholipase A2, showed that 13 times more enzyme was bound to phospholipid films of the L series by comparison with films of the D series. These results were confirmed by indirect binding studies using re-spreading experiments. Kinetic studies of the porcine pancreatic PLA2, using enantiomeric acyl-amino phospholipid analogues, have shown that: (1) inhibitors of the L series are more potent than inhibitors of the D series, (2) inhibitors having a negative charge are more potent than zwitterionic inhibitors, (3) inhibitory power values are greater when evaluated in micellar system than in a the monolayer system, (4) the inhibitory power increases continuously with surface pressure.

Animals↗
PubMed1991

Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl-dithio-5-(2-nitrobenzoic acid). A kinetic study with 1,2-didecanoyl-sn-glycerol monolayers.

We studied the covalent inhibition of lipases by the monolayer technique. We report the inactivation of porcine pancreatic and human and rabbit gastric lipases, acting on mixed monomolecular films of dicaprin containing tetrahydrolipstatin or new hydrophobic disulfide compounds, which can be described as a 'poisoned-interface' system. A kinetic model is presented for depicting the covalent inactivation of lipolytic enzymes at a lipid/water interface. The stoichiometry of the interfacial situation can be described as follows: one lipase molecule embedded among 10(5) substrate molecules will be inactivated to half its initial velocity by the presence of 10 tetrahydrolipstatin molecules. This inactivation was independent of the surface pressure. When tested in the form of mixed films, all the disulfide compounds investigated specifically reduced the hydrolysis of 1,2-didecanoyl-sn-glycerol films by gastric lipases, but did not affect hydrolysis by pancreatic lipase. With this poisoned-interface system, tetrahydrolipstatin was found to be the most potent inactivator, whereas disulfide compounds showed a higher degree of selectivity than tetrahydrolipstatin.

Animals↗
PubMed1991

Purification and biochemical characterization of dog gastric lipase.

A lipase was found to be present in dog stomach which appeared to be more abundant in the fundic than in the pyloric mucosa. Dog gastric lipase was extracted by soaking the gastric tissue and further purified after cation exchange, anion exchange and gel-filtration using fast protein liquid chromatography. The amino-acid composition, N-terminal amino-acid sequence, substrate specificity, interfacial and kinetic behavior and inactivation by sulfhydryl reagents were determined and compared with those of human and rabbit gastric lipases. We report for the first time that a gastric lipase is 13 times more active on long-chain than on short-chain triacylglycerols at pH 4.0, reaching a maximal specific activity of 950 U/mg on Intralipide emulsion.

Amino Acid Sequence↗
PubMed1991

Inactivation of pancreatic and gastric lipases by THL and C12:0-TNB: a kinetic study with emulsified tributyrin.

THL is a potent inhibitor of pancreatic (PPL) and gastric (HGL, RGL) lipases. Inactivation occurs preferentially at the oil/water interface (method B, C). In the aqueous phase (method A), the inhibition of HGL was accelerated by the presence of bile salts. C12:0-TNB, a disulfide reagent, specifically inactivates gastric lipases and had no effect on the pancreatic lipase (in the presence of bile salts) whatever the method used. The capacity of THL and C12:0-TNB to inactivate lipases using Methods B and C was found to depend directly upon the interfacial area of the system used. Consequently, inactivation can be reduced or prevented by further addition of a water-insoluble substrate which reduces the surface density of inactivator molecules. With a heterogeneous system of this kind, typical of lipolysis, the use of a classical Michaelis-Menten model is irrelevant and hence the traditional kinetic parameters (Km, KI, Vmax) are only apparent values.

Animals↗
PubMed1991

Inactivation of gastric and pancreatic lipases by diethyl p-nitrophenyl phosphate.

Reacting gastric and pancreatic lipases with mixed diethyl p-nitrophenyl phosphate/bile salt micelles resulted in a stoichiometric inactivation of these enzymes as tested on emulsified tributyroylglycerol and trioleoylglycerol as substrates. Diethyl p-nitrophenyl phosphate treated gastric lipases were also inactive on water-soluble p-nitrophenyl acetate, whereas the modified pancreatic lipase was still able to hydrolyze this water-soluble substrate. The binding of diethyl p-nitrophenyl phosphate modified pancreatic and gastric lipases to tributyroylglycerol/water interface was comparable to that of native lipases. The essential free sulfhydryl group of gastric lipases underwent no chemical changes due to the reaction with micellar diethyl p-nitrophenyl phosphate. All in all, these results indicate that, in both gastric and pancreatic lipases, the essential serine residue which was stoichiometrically labeled by this organophosphorus reagent is involved in catalysis and not in lipid binding.

Amino Acid Sequence↗
PubMed1991

Activation of protein kinase C in lipid monolayers.

The potential of lipid monolayers spread at an air-water interface was investigated as a well defined membrane model able to support protein kinase C (PKC) association and activation. PKC association to a mixed phospholipid film (phosphatidylcholine, phosphatidylserine) could be detected by an increase of the monolayer surface pressure. This association was strikingly dependent upon the presence of submicromolar concentrations of Ca2+. The effect of Ca2+ resulted in an increase of the PKC penetration into the lipid core at a given permissive surface pressure as well as in a marked increase of the critical surface pressure (29-38 dynes/cm) above which the enzyme was excluded from the membrane. Inclusion of diacylglycerol or tetradecanoate phorbol acetate (TPA) did not modify the PKC-monolayer association in a detectable manner. PKC associated to the lipid layer exhibited the expected catalytic property and was fully activated when diacylglycerol or TPA was included in the membrane. PKC activity was highly dependent upon the surface pressure of the lipid monolayer, being optimal between 30 and 35 dynes/cm. Study of the compression isotherm of various diacylglycerol structures revealed that all potent PKC agonists exhibited an expanded liquid phase behavior with collapse pressure below 40 dynes/cm, in contrast to weak activators which showed condensed isotherms with high collapse pressure (approximately equal to 60 dynes/cm). These observations showed that the lipid monolayer system is well adapted to the study of the molecular mechanisms involved in the regulation of PKC activity at a model membrane interface. They are in line with the suggestion of a major role of Ca2+ in the association (translocation) of PKC to membrane in living cell and suggest that diacylglycerol (and TPA) might activate membrane-associated PKC through local change in the surrounding lipid phase organization.

Animals↗
PubMed1991

Gastric lipase and pepsinogen during the ontogenesis of rabbit gastric glands.

In rabbit stomach, gastric lipase activity level was found to increase from birth to 30 days old (weaning), and then decreased. In contrast, pepsin activity only appeared between 30 to 45 days old, and increased till to the adult level. It was observed that maturation of gastric glands in cardial mucosa was a downward elongation process from the mitotic cell pool. These mitotic cells were always found in the neck of the gastric glands, corresponding to the bottom of the gland at 6 days old and to the mid-zone of the gland in adult. Location of rabbit gastric lipase (RGL) cells in cardial glands varied with age and was found along the pit of the gastric glands at 6 days old. The extent of this cellular location decreased with age, whereas a second RGL cell zone appeared below the mitotic cell area at 18 and 30 days old. At 45 days old, the pepsinogen cells appeared in the bottom of the gland, and consequently the RGL cells were located in the mid-zone of the gastric glands, between mitotic cells (neck of the gland) and pepsinogen cells (lower part of the gland). Ultrastructural study of cardial gastric glands revealed different morphologies of the secretion granules in the cells along the gastric glands. In 6-day-old rabbits, secretory granules were found uniformly electron dense in the bottom of the glands and were RGL-labeled by the immunogold technique. In the medium part of the glands, granules appeared biphasic, with a clear and a dense part, and RGL labeling was confined to the electron-dense part.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗
PubMed1990

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique.

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).

Animals↗
PubMed1990

Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases.

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward chemically alike, but sterically nonequivalent ester groups within one single triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin or triolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by high performance liquid chromatography. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. The corresponding values with porcine pancreatic lipase were 3% in the case of trioctanoin and 8% in that of triolein. It is worth noting that rabbit gastric lipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the most striking catalytic differences observed between these two gastric lipases.

Animals↗
PubMed1990

Competitive inhibition of lipolytic enzymes. IV. Structural details of acylamino phospholipid analogues important for the potent inhibitory effects on pancreatic phospholipase A2.

1-Acyl-2(R)-acylamino phospholipids are effective competitive inhibitors of porcine pancreatic phospholipase A2 (EC 3.1.1.4, Bonsen et al. (1972) Biochim. Biophys. Acta 270, 364-382). By systematically varying the substituent at C-1 and the acyl chain length at C-2, a series of phospholipid analogues was obtained for which the inhibitory power was determined in a detergent-containing and occasionally also in a detergent-free micellar substrate system. The recently proposed kinetic model applicable to water-insoluble inhibitors (Ransac et al. (1990) Biochim. Biophys. Acta 1043, 57-66) allowed a quantitative comparison of the inhibitory power Z of the various substrate analogues. The most powerful inhibitors of the enzyme were found to possess the general 2-(R)-structure: (formula; see text) Using as substrate (R)-1,2-didodecanoylglycero-3-phosphocholine in mixed micelles with sodium taurodeoxycholate, the inhibitor molecule with m = 4 and n = 11 showed a Z-value of 15,000. This implies an affinity of the inhibitor for the active site of the enzyme higher than 4 orders of magnitude stronger as compared with the substrate molecule. Slightly higher and lower m-values resulted in a sharp drop of the inhibitory power, which suggests that the enzyme must possess a rather short, but well-defined hydrophobic binding pocket for the C-1 alkyl chain. Variation of n (keeping m = 2 constant) resulted in inhibitors with nearly equal Z-values for n = 11, 13 and 15. Most probably the binding cleft on the enzyme for the C-2 acylamino chain is longer, more losely constructed and contributing less to the overall binding energy. Several members of the 2-acylamino phospholipids are water-soluble and possess relatively high critical micelle concentrations. Their inhibitory power could be tested not only in micellar substrate dispersions but also in assay systems where both the inhibitor and substrate are molecularly dispersed. It appeared that these water-soluble phospholipid analogues are effective inhibitors of the enzyme only after incorporation into an organized substrate/water interface. In contrast, in molecularly dispersed substrate solutions the same molecules have completely lost their inhibitory power. These observations support our kinetic model of lipolysis and interfacial inhibition.

Animals↗
PubMed1990

Competitive inhibition of lipolytic enzymes. I. A kinetic model applicable to water-insoluble competitive inhibitors.

It is now becoming clear from the abundant lipolytic enzyme literature that any meaningful interpretation of inhibition data has to take into account the kinetics of enzyme action at the lipid/water interface. We attempt in the present paper to provide a kinetic model applicable to water-insoluble competitive inhibitors, in order to quantitatively compare the results obtained at several laboratories. We derived kinetic equations applicable to the pre-steady state as well as steady state. By measuring the inhibitory power, as described in the present paper, it is possible to obtain a normalized estimation of the relative efficiency of various potential inhibitors. Furthermore, with the kinetic treatment developed here, it is possible to make quantitative comparisons with the same inhibitor placed under various physico-chemical situations, i.e., micellar or monolayer states.

Binding, Competitive↗
PubMed1990

Competitive inhibition of lipolytic enzymes. III. Some acylamino analogues of phospholipids are potent competitive inhibitors of porcine pancreatic phospholipase A2.

Competitive inhibition of porcine pancreatic phospholipase A2 was studied in mixed-micellar systems composed of long- and medium-chain substrates, potential inhibitors and detergents. A number of positional and stereoisomeric monoacylamino, acyloxyglycerophospholipids were investigated for their inhibitory properties, using as substrates the corresponding diacyl-sn-glycero-3-phospholipids possessing the same polar headgroup and identical acyl chain lengths. Based on a kinetic model applicable to water-insoluble inhibitors (see accompanying paper I), which allows a quantitative comparison of the inhibitory power (Z) of the various phospholipid analogues, the following results were obtained: Substitution of a single acylester bond in a diacylglycerophospholipid by an acylamino group can transform the substrate molecule in a potent competitive inhibitor. This property is acquired only when this substitution occurs on the phospholipase-susceptible ester bond of the substrate. If the acylamino group replaces an ester bond which cannot be attacked by the highly positional and stereospecific phospholipase, the resulting molecule binds with similar affinity to the active site of the enzyme as the parent substrate molecule. Because of its positional and stereospecificity, this so-called inhibitory 'amide effect' suggests that these inhibitors behave as substrate-derived analogues. The inhibitory 'amide-effect' observed with several medium- and long-chain monoacyloxy-, monoacylamino-deoxyglycerophosphatides is completely lost upon specific alkaline hydrolysis of the single acylester bond. Reesterification of the free glycerol OH group in these lysoacylaminophosphoglycerides, even with an acetyl residue, restores the inhibitory properties. These observations indicate that specific binding of phospholipids to the active site of pancreatic phospholipase A2, requires the presence of two chains in substrate or inhibitor structure and suggest that those results obtained with lysophospholipids and single-chain analogues may be questionable.

Animals↗
PubMed1990

Interactions of lipases with lipid monolayers. Facts and questions.

Among the proteins, lipolytic enzymes provide a valuable model for studying protein-lipid interactions. Lipases having a catalytic action which is strictly dependent upon the presence of a lipid interface were used in the present study in order to gain better insight into protein-lipid interactions. Most of the data presented here were obtained using the monolayer technique, by recording (either independently or simultaneously) the lipolytic activity, the amount of protein adsorbed to the lipid monolayer, and the surface pressure variations following protein adsorption. Several non-enzymatic proteins were used as controls in order to determine how lipase behaviour differs from that of other proteins. At all initial surface pressures tested, with zwitterionic monolayers, a good correlation was observed between the amount of lipase bound to the monolayer and the surface pressure increase, in agreement with previous studies. Conversely, with neutral lipid monolayers the amount of lipase bound to the monolayer was not found to be surface pressure dependent. This latter behaviour observed with lipases on neutral films is not specific to lipases, since it was also observed with bovine serum albumin and beta-lactoglobulin A. Lipase activity in the presence of various proteins was investigated with monomolecular films of glycerol didecanoate, either at constant surface area or at constant surface pressure. Depending upon the nature of the lipase and the protein, inhibition of lipase activity was either observed or not. Inhibition was correlated with a decrease in lipase surface concentration. The ability of the various proteins to inhibit lipolysis is: (i) a function of their excess versus lipase in the bulk phase, and: (ii) correlated with their penetration capacity (i.e., the initial rate of surface pressure increase of a glycerol didecanoate monolayer having an initial surface pressure of 20 dyn/cm, after the injection-of the protein). Since lipase inhibition was observed with low surface densities of inhibitory proteins, a long-range effect is probably involved in the mechanism of interfacial lipase inhibition. The nature of the ionic charge added to the monolayer by the protein is not critical for determining lipase adsorption or desorption. It is hypothesized that the lack of lipase adsorption to, or desorption from, the lipid monolayer results from a change in the organization of the hydrocarbon moiety of the lipid.

Animals↗