Human gastric lipase. A kinetic study with dicaprin monolayers

Gargouri, Youssef; Piéroni, G; Ferrato, Francine; Verger, Robert

doi:10.1111/j.1432-1033.1987.tb13588.x

Cited by 32 publications

(16 citation statements)

References 16 publications

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“…Assays on tributyrin (Fluka) and soybean oil (commercial grade) emulsified in gum arabic were carried out with the bulk pH-stat method under standard conditions as described by Gargouri et al [5]. The kinetics of the hydrolysis of 1,2-didecanoyl-sn-glycerol films by HGL [21] were recorded, with or without incubation of the enzyme with each mAb, using the barostat technique previously described by Verger and de Haas [22].…”

Section: Enzymic Activity Assaysmentioning

confidence: 99%

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

Aoubala

Daniel²,

et al. 1993

European Journal of Biochemistry

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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 IgGl class with a K 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.In humans, the hydrolysis of dietary triacylglycerols begins in the stomach and is catalyzed by one major enzyme; human gastric lipase (HGL) [l-31. HGL is a glycoprotein with a molecular mass of around 50 kDa, which is located in the chief cells of fundic mucosa [4]. In-vitro studies have shown that HGL hydrolyses both short-chain and long-chain triacylglycerols at comparable rates [5]. The enzyme is highly stable and active under acidic pH conditions. The optimal pH for HGL activity is 5.4 in the case of long-chain and 6 in the case of short-chain triacylglycerols.The amino acid sequence of HGL, deduced from the cDNA, consists of 379 residues [6] and shows a conservative pentapeptide (Gly-Xaa-Ser-Xaa-Gly) common to all the known lipases. . In order to further characterize these functional domains of HGL, an immunological approach was adopted here using mAb against HGL as probes for the spatial identification of the domains.In the present paper, we describe the production and functional characterization of five mAb against HGL. MATERIALS AND METHODS ProteinsHGL was purified in the laboratory from human gastric juice, using procedures described previously [15]. The protein had a specific activity of 1000 U . mg-' with tributyrin as substrate and showed a single band upon SDS/gel electrophoresis [16]. The bovine serum albumin and anti-mouseIgG -peroxidase conjugate used were from Sigma. Preparation of mAbTwo young female BALB/c mice were immunized with HGL as follows. The first and the second immunizations were carried out with 100 pg HGL emulsified in complete Freund's adjuvant, injected subcutaneously with a 10-day interval. Two weeks after the second injection, the mice were bled and the serum tested in a direct binding ELISA test. The mouse with the highest titre was selected and 100 pg pure antigen were again injected. 10 days later, three fusion-priming intraperitoneal injections of 50 pg HGL, each in 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 (buffer A) were given on three subsequent days. Spleen cells were obtained 1 day after the last injection and fused with non-secreting mouse myeloma

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Section: Enzymic Activity Assaysmentioning

confidence: 99%

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

Aoubala

Daniel²,

et al. 1993

European Journal of Biochemistry

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“…For the sake of comparison, results obtained using HGL at pH 3.0 [25] and PPL at pH 8.0, using dicaprin as substrate, are also given in Fig. 1.…”

Section: Variations In Gastric Lipase Activity With Surface Pressurementioning

confidence: 99%

Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)

Ransac

Gargouri

Moreau

et al. 1991

European Journal of Biochemistry

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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 lo5 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.Gastric lipase differs from pancreatic lipase in several major respects. Under acidic pH conditions, gastric lipase have been found to be remarkably stable and active, whereas pancreatic lipase irreversibly loses its lipolytic capacity. The optimum pH for gastric lipase activity is around 5.4 [l, 21, compared to 8-9 in.the case of pancreatic lipase [3]. The partial hydrolysis of alimentary triacylglycerols which occurs at the pH levels prevailing in the stomach rapidly triggers pancreatic lipase activity in the intestine [4].We have shown that some amphiphiles (bile salts, alimentary proteins and phospholipids), which inhibit pure PPL [3], activate gastric lipase, since they can prevent the irreversible denaturation of the latter enzymes at substrate/water interfaces [l, S].Human gastric lipase (HGL) and rabbit gastric lipase (RGL) are enzymes which have been found to be inactivated by classical sulfhydryl reagents [5,S'-dithiobis(2-nitrobenzoic acid), NbS, ; 4,4'-dipyridyl disulfide) and by a hydrophobic disulfide compound: dodecyl-dithio-S-(2-nitrobenzoic acid) (12: 0-S-SNb) [6, 71. More recently, we reported that ajoene, derived from ethanol extracts of garlic, specifically inactivated HGL and RGL and had no effect on porcine pancreatic lipase With emulsified systems, it is not possible to control the 'interfacial quality' and to easily assess the distribution of soluble versus adsorbed amphiphilic molecules. This prompted us to use the monolayer technique, based upon surface-pressure decrease due to lipid-film hydrolysis [16 -181. This technique is applicable to those cases where the lipid forms a stable monomolecular film at the air/water interface and where reaction products are freely soluble and diffuse away rapidly into the aqueous phase.We studied, for the first time, the covalent inhibition of lipases by the monolayer technique. We report the inactivati...

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“…At present, however, the mechanism by which free fatty acids inhibit gastric lipase action in the stomach is unknown. It can be suggested from the literature that long chain free fatty acids prevent further gastric lipase lipolysis by modifying the physicochemical properties of the lipid/water interface, especially the interfacial tension or the surface pressure (2,13,16,24,25); they could prevent the interfacial binding of gastric lipase or promote its release from the droplet surface (2), or they could limit the number of triacylglycerol molecules located at the droplet surface by steric hindrance (13). However, thus far no study has provided direct evidence for the mechanisms involved.…”

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confidence: 99%

Mechanisms of Inhibition of Triacylglycerol Hydrolysis by Human Gastric Lipase

Pafumi¹,

Lairon²,

Porte³

et al. 2002

Journal of Biological Chemistry

193

124

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In the human stomach, gastric lipase hydrolyzes only 10 to 30% of ingested triacylglycerols because of an inhibition process induced by the long chain free fatty acids generated, which are mostly protonated at gastric pH. The aim of this work was to elucidate the mechanisms by which free fatty acids inhibit further hydrolysis. In vitro experiments examined gastric lipolysis of differently sized phospholipid-triolein emulsions by human gastric juice or purified human gastric lipase, under close to physiological conditions. The lipolysis process was further investigated by scanning electron microscopy, and gastric lipase and free fatty acid movement during lipolysis were followed by fluorescence microscopy. The results demonstrate that: 1) free fatty acids generated during lipolysis partition between the surface and core of lipid droplets with a molar phase distribution coefficient of 7.4 at pH 5.40; 2) the long chain free fatty acids have an inhibitory effect only when generated during lipolysis; 3) inhibition of gastric lipolysis can be delayed by the use of lipid emulsions composed of small-size lipid droplets; 4) the release of free fatty acids during lipolysis induces a marked increase in droplet surface area, leading to the formation of novel particles at the lipid droplet surface; and 5) the gastric lipase is trapped in these free fatty acid-rich particles during their formation. In conclusion, we propose a model in which the sequential physicochemical events occurring during gastric lipolysis lead to the inhibition of further triacylglycerol lipolysis.

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Human gastric lipase. A kinetic study with dicaprin monolayers

Cited by 32 publications

References 16 publications

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

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

Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl‐dithio‐5‐(2‐nitrobenzoic acid)

Mechanisms of Inhibition of Triacylglycerol Hydrolysis by Human Gastric Lipase

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