Purification, characterization and kinetic properties of the rabbit gastric lipase

Moreau, Hervé; Gargouri, Youssef; Lecat, Daniel; Junien, Jean‐Louis; Verger, Robert

doi:10.1016/0005-2760(88)90036-7

Cited by 86 publications

(39 citation statements)

References 28 publications

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“…The DGL coding sequence was fused by performing restriction enzyme-based cloning to the polymerase chain reaction-amplified N-terminal signal sequence of the rabbit gastric lipase precursor composed of its first 22 amino acids (6,25). The resulting sequence was placed under the control of the ␥-zein promoter of maize (26), which confers a seed expression, and the nopaline synthase terminator of Agrobacterium tumefaciens (27) in the pUC18 plasmid (Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of the Open Form of Dog Gastric Lipase in Complex with a Phosphonate Inhibitor

Roussel

Miled

Berti-Dupuis

et al. 2002

Journal of Biological Chemistry

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114

132

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Fat digestion in humans and some mammals such as dogs requires the successive intervention of two lipases: gastric lipase, which is stable and active despite the highly acidic stomach environment, followed by the classical pancreatic lipase secreted into the duodenum. We previously solved the structure of recombinant human gastric lipase (HGL) at 3.0-Å resolution in its closed form; this was the first structure to be described within the mammalian acid lipase family. Among the mammalian lipases, the preduodenal and lysosomal lipases belong to a family of enzymes that are able to withstand acidic conditions, under which they continue to be active. This family shows no sequence homology with any other known lipase families. The preduodenal lipases form a group of closely related enzymes originating from the stomach, the tongue, or the pharynx, and none of them requires any specific protein cofactor for its activity (1-3).The state of malnutrition of patients suffering from exocrine pancreatic insufficiency, which is often associated with cystic fibrosis, can be improved by prescribing dietary supplements prepared from pancreatic extracts of animal origin. The use of gastric lipases may lead to considerable progress in the treatment of exocrine pancreatic insufficiency (4) because of their resistance to the gastric medium and their high lipolytic activity at the low pH values observed in the duodenum A screening program (1) and biochemical studies (5-7) led to the conclusion that dog gastric lipase (DGL), 1 a glycoprotein with a molecular mass of 49 kDa, was the most active enzyme when tested on long-chain triacylglycerols. These lipids are the main components of human dietary fats. The first attempts to produce an active recombinant DGL (r-DGL) in procaryotes (4) were unsuccessful. Joliff et al. (8) described the expression and secretion of an active r-DGL in the baculovirus insect cell system. However, with a view to therapeutic applications, r-DGL production by a safe eukaryotic system of expression such as transgenic plants provides an attractive alternative. Transgenic plants offer considerable advantages, such as large production yields and the fact that they entail no risk of contamination by pathogens infectious to humans, whereas they contain the cell machinery mediating eukaryotic protein modifications. The first transgenic plants producing r-DGL were constructed in tobacco (9) and then in maize by Meristem therapeutics in collaboration with the Institut de Recherche Jouveinal/Parke-Davis during the last decade.All the triglyceride lipases for which the three-dimensional structures have been determined so far belong to the serine esterase class (10 -14). The active serine is part of a catalytic triad, which is similar to that observed in the case of serine proteases. The three-dimensional structure of the Candida rugosa lipase, which is inhibited by two enantiomers of a phosphonate inhibitor, provided the structural basis for establishing the chiral preferences of lipases (15). Covalent adducts of Rhizomu...

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Section: Methodsmentioning

confidence: 99%

Crystal Structure of the Open Form of Dog Gastric Lipase in Complex with a Phosphonate Inhibitor

Roussel

Miled

Berti-Dupuis

et al. 2002

Journal of Biological Chemistry

Self Cite

114

132

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“…The amino acid sequence of human gastric lipase, as well as the counterparts from calf [6] (and Bernbiick et al, unpublished results) and from rat [13] starts with three hydrophobic residues followed by lysine in position four. Interestingly the rabbit lipase lacks the first three hydrophobic residues, Lys4 being the first residue [4]. Thus, Lys4 is conserved in all (pre)gastric lipases so far investigated.…”

Section: Discussionmentioning

confidence: 97%

Human gastric lipase

Bernbäck

Bläckberg

1989

European Journal of Biochemistry

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Human gastric lipase subjected to limited tryptic proteolysis lost its ability to hydrolyze emulsified long‐chain triacylglycerol. Activity against a water‐soluble substrate was however retained, indicating that proteolysis did not affect the active site. Sequence analysis revealed that trypsin specifically cleaved the linkage between lysine‐4 and leucine‐5. This cleavage rendered the enzyme unable to bind to emulsified triacylglycerol particles, e.g. human milk fat globules. We suggest that the N‐terminal tetrapeptide, in particular lysine‐4, is essential for the binding of human gastric lipase to lipid/water interfaces, and hence, for its physiological function.

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“…RGL and rDGL are known to display an optimal activity at an acidic pH ( 36,46 ). rHPL and PPL prefer to catalyze the hydrolysis of TG at neutral or slightly alkaline pH ( 48,49 ).…”

Section: Kinetics Of Lipases Using Phiblamentioning

confidence: 99%

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

Camacho-Ruiz¹,

Mateos-Díaz²,

Carrière

et al. 2015

Journal of Lipid Research

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subsequent catalysis sensu stricto ( 6, 7 ). Most of the lipases are water-soluble enzymes acting on water insoluble substrates (super-substrates). The two-dimensional nature of enzyme catalysis by lipases does not obey MichaelisMenten kinetics and critically depends on the quality of the interface ( 3,8,9 ). Obtaining accurate, i.e., substratespecifi c, measurements of lipase activity as well as developing reliable lipase assay systems requires taking these unique features into account.Many biotechnological applications for lipases have been described in the food, cosmetic, detergent, and pharmaceutical industries ( 3, 10, 11 ), and an interest exists for new sources of this kind of enzyme in an industrial setting.Novel lipases can be obtained either by isolating them from various natural sources or by using classical protein engineering methods and/or directed evolution procedures (12)(13)(14). All these studies require convenient, sensitive, and specifi c assays for measuring lipase activity ( 15 ). In addition, screening procedures require continuous assays and substrate stability that are compatible with high sample throughput.Considerable progress has been made in the past few years in the development of high-throughput screening (HTS) methods for carboxyl-ester hydrolases. These HTS assays have been developed using chromogenic or fl uorogenic substrates such as butyrate, octanoate, and palmitate of nitrophenol or 4-methylumbelliferone. However, all of these synthetic esters are liable to undergo nonenzymatic alkaline hydrolysis as well as hydrolysis by the nonspecifi c carboxyl-ester hydrolases often present in biological Abstract A continuous assay is proposed for the screening of acidic, neutral, or alkaline lipases using microtiter plates, emulsifi ed short-and medium-chain TGs, and a pH indicator. The lipase activity measurement is based on the decrease of the pH indicator optical density due to protonation which is caused by the release of FFAs during the hydrolysis of TGs and thus acidifi cation. Purifi ed lipases with distinct pH optima and an esterase were used to validate the method. The rate of lipolysis was found to be linear with time and proportional to the amount of enzyme added in each case. Specific activities measured with this microplate assay method were lower than those obtained by the pH-stat technique. Nevertheless, the pH-dependent profi les of enzymatic activity were similar with both assays. In addition, the substrate preference of each enzyme tested was not modifi ed and this allowed discriminating lipase and esterase activities using tributyrin (low water solubility) and tricaprylin (not water soluble) as substrates. This continuous lipase assay is compatible with a high sample throughput and can be applied for the screening of lipases and lipase inhibitors from biological samples. Lipases (TG ester hydrolases, EC 3.1.1.3) are lipolytic carboxylester hydrolases which catalyze the hydrolysis of the ester bonds of TGs to form FFAs and glycerol in some cases . They are widely distr...

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Purification, characterization and kinetic properties of the rabbit gastric lipase

Cited by 86 publications

References 28 publications

Crystal Structure of the Open Form of Dog Gastric Lipase in Complex with a Phosphonate Inhibitor

Crystal Structure of the Open Form of Dog Gastric Lipase in Complex with a Phosphonate Inhibitor

Human gastric lipase

A broad pH range indicator-based spectrophotometric assay for true lipases using tributyrin and tricaprylin

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