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We investigated the paraffinic hydrocarbon composition of cell suspensions of Euphorbia characias L. grown in different regimes of carbon and energy supply. Heterotropic (H), photomixotrophic (PM) and photoautotrophic (PA) cell suspensions were compared to leaves. Alkane compositions varied greatly both quantitatively and qualitatively according to the mode of culture. PM and H cells had indentical gaussian-like hydrocarbon distributions, with maxima around C 27 or C 28 compounds, while PA cells and leaf hydrocarbons were enriched in odd carbon chain-length compounds, especially C 29 , C 31 and C 33 In long-term cultivated suspensions, total hydrocarbon amounts of PM and PA cells were about 5% and 6% that of leaf, respectively, and PA suspensions presented a major enrichment of all odd carbon chain-length compounds. Minor differences were observed between the growth phases of PM as well as PA cells. On the other hand, major differences appeared between these two modes of culture and were amplified along subcultures, indicating that the paraffinic hydrocarbon composition was directly related to the mode of culture. We discuss the possible origins of these alkane patterns.

We present a general formulation of a model that can be used to analyze reaction profiles in systems in which a single enzyme catalyzes several sequential reactions with the same molecular backbone. The analysis of these so-called “repeated-attack systems” allows estimation of the specificities that the enzyme has for the various intermediate substrates that appear in the reaction mixture, relative to the specificity that it has for the initial substrate. Our analytical method has the important advantage that it is not affected by competitive or uncompetitive inhibition, nor by denaturation of the enzyme during the reaction. We carry out case studies in three different systems, the lipase-catalyzed alcoholysis of triacylglycerols, the phytase-catalyzed removal of phosphate groups from phytic acid and the β-amylase-catalyzed removal of maltose units from maltoheptaose. Our model fits well to all reaction profiles in which the phenomenon of processivity does not occur. It can therefore be used as a general tool for characterizing the relative specificities of “repeated-attack enzymes”.

The unripe fruit of babaco (Vasconcellea 9 heilbornii; syn. Carica pentagona) contains a latex, similar to that in Carica papaya, which exhibits lipolytic activity. Herein, the regioselectivity, stereoselectivity and typoselectivity in both hydrolysis and acyltransfer reactions of babaco latex lipases were studied and compared to those of Carica papaya latex. In hydrolysis, both biocatalysts are 1,3-regioselective with ratios for 1,2-2,3-diacylglycerols/1,3- diacylglycerol of 6.5 and 21 for babaco and papaya, respectively. In contrast, papaya latex had a slight sn- 3 stereopreference. Babaco latex displayed a higher activity on triacylglycerols with short chain and unsaturated fatty acids.

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units·mg−1 at 37 °C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

The effects of various detergents and pH on the interfacial binding and activity of two fungal lipases from Yarrowia lipolytica (YLLIP2) and Thermomyces lanuginosus (TLL) were investigated using trioctanoin emulsions as well as monomolecular films spread at the air–water interface. Contrary to TLL, YLLIP2 was found to be more sensitive than TLL to interfacial denaturation but it was protected by detergent monomers and lowering the temperature. At pH 7.0, both the interfacial binding and the activities on trioctanoin of YLLIP2 and TLL were inhibited by sodium taurodeoxycholate (NaTDC). At pH 6.0, however, YLLIP2 remained active on trioctanoin in the presence of NaTDC, whereas TLL did not. YLLIP2 activity on trioctanoin was associated with strong interfacial binding of the enzyme to trioctanoin emulsion, whereas TLL was mostly detected in the water phase. The combined effects of bile salts and pH on lipase activity were therefore enzyme-dependent. YLLIP2 binds more strongly than TLL at oil–water interfaces at low pH when detergents are present. These findings are particularly important for lipase applications, in particular for enzyme replacement therapy in patients with pancreatic enzyme insufficiency since high detergent concentrations and highly variable pH values can be encountered in the GI tract.

Access to the active site of human pancreatic lipase (HPL) is controlled by a surface loop (the lid) that undergoes a conformational change in the presence of amphiphiles and lipid substrate. The question of how and when the lid opens still remains to be elucidated, however. A paramagnetic probe was covalently bound to the lid via the D249C mutation, and electron paramagnetic resonance (EPR) spectroscopy was used to monitor the conformational change in solution. Two EPR spectral components, corresponding to distinct mobilities of the probe, were attributed to the closed and open conformations of the HPL lid, based on experiments performed with the E600 inhibitor. The open conformation of the lid was observed in solution at supramicellar bile salt concentrations. Colipase alone did not induce lid opening but increased the relative proportions of the open conformation in the presence of bile salts. The opening of the lid was found to be a reversible process. Using various colipase to lipase molar ratios, a correlation between the proportion of the open conformation and the catalytic activity of HPL was observed.

The early light-induced proteins (ELIPs) belong to the multigenic family of pigment-binding light-harvesting complexes. ELIPs accumulate transiently and are believed to play a protective role in plants exposed to high levels of light. Constitutive expression of the ELIP2 gene in Arabidopsis resulted in a marked reduction of the pigment content of the chloroplasts, both in mature leaves and during greening of etiolated seedlings. The chlorophyll loss was associated with a decrease in the number of photosystems in the thylakoid membranes, but the photosystems present were fully assembled and functional. A detailed analysis of the chlorophyll-synthesizing pathway indicated that ELIP2 accumulation downregulated the level and activity of two important regulatory steps: 5-aminolevulinate synthesis and Mg-protoporphyrin IX (Mg-Proto IX) chelatase activity. The contents of glutamyl tRNA reductase and Mg chelatase subunits CHLH and CHLI were lowered in response to ELIP2 accumulation. In contrast, ferrochelatase activity was not affected and the inhibition of Heme synthesis was null or very moderate. As a result of reduced metabolic flow from 5-aminolevulinic acid, the steady state levels of various chlorophyll precursors (from protoporphyrin IX to protochlorophyllide) were strongly reduced in the ELIP2 overexpressors. Taken together, our results indicate that the physiological function of ELIPs could be related to the regulation of chlorophyll concentration in thylakoids. This seems to occur through an inhibition of the entire chlorophyll biosynthesis pathway from the initial precursor of tetrapyrroles, 5-aminolevulinic acid. We suggest that ELIPs work as chlorophyll sensors that modulate chlorophyll synthesis to prevent accumulation of free chlorophyll, and hence prevent photooxidative stress.

Intragastric fat digestion was investigated by analyzing the products of lipolysis and the gastric lipase (HGL) levels of premature infants fed with a formula enriched with medium chain triglycerides (MCT) and those of infants fed with human milk. Infants were fed using a gastric tube and the gastric contents were aspirated twice a day for 5 d, before and at various times after gavage feeding. HGL levels were measured using the pHstat technique. After extraction, lipids were separated and quantified using thin-layer chromatography coupled to a flame ionization detector. Fatty acid methyl esters were analyzed by gas chromatography. HGL concentration increased during digestion, reaching 77.4 +/- 43.1 [mu]g/mL (around 75% of those recorded in adults). Mean HGL output was 115 +/- 43 [mu]g for 3 h and the overall intragastric lipolysis was 6.1 +/- 2.6%. Although the formula was enriched with octanoic and decanoic acid, the main fatty acids released in the stomach were palmitic (C16:0, 17.03 +/- 0.23% wt/wt) and oleic (C18:1 n-9, 28.23 +/- 1.26% wt/wt) acid. Similar results were obtained with infants fed with human milk. MCT supplementation has no quantitative or qualitative effects on the intragastric lipolysis, which is not higher in premature infant than in adults. (C) International Pediatrics Research Foundation, Inc.

Hormone-sensitive lipase (HSL) contributes importantly to the mobilization of fatty acids from the triacylglycerols stored in adipocytes, which provide the main source of energy in mammals. On the basis of amino acid sequence alignments and three-dimensional structures, this enzyme was previously found to be a suitable template for defining a family of serine carboxylester hydrolases. In this study, the HSL family members are characterized rather on the basis of their inhibition by 5-methoxy-3-(4-phenoxyphenyl)-3H-[1,3,4]oxadiazol-2-one (compound 7600). This compound inhibits mammalian HSL as well as other HSL family members, such as EST2 from the thermophilic eubacterium Alicyclobacillus acidocaldarius and AFEST from the hyperthermophilic archaeon Archaeoglobus fulgidus. Various carboxylester hydrolases that are not members of the HSL family were found not to be inhibited by compound 7600 under the same experimental conditions. These include nonlipolytic hydrolases such as Torpedo californica acetylcholinesterase and pig liver esterase, as well as lipolytic hydrolases such as human pancreatic lipase, dog gastric lipase, Thermomyces lanuginosus lipase, and Bacillus subtilis LipA. When vinyl esters were used as substrates, the residual activity of HSL, AFEST, and EST2 decreased with an increase in compound 7600 concentration in the incubation mixture. The inhibitor concentration at which the enzyme activity decreased to 50% after incubation for 5 min was 70, 20, and 15 nM with HSL, AFEST, and EST2, respectively. Treating EST2 and AFEST with the inhibitor resulted in an increase in the molecular mass, as established by performing matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis. This increase in the molecular mass, which corresponds approximately to the molecular mass of the inhibitor, indicates that a covalent enzyme-inhibitor complex has been formed. Surface-enhanced laser desorption ionization time-of-flight mass spectrometry analysis of a trypsin digest of AFEST treated with the inhibitor or not treated showed the occurrence of an increase in the molecular masses of the "GESAGG"-containing peptide, which is compatible with the formation of a covalent complex with the inhibitor.

The LIP2 lipase from the yeast Yarrowia lipolytica (YLLIP2) was obtained from two genetically modified strains with multi-copies of the lip2 gene and further purified using gel filtration and cation exchange chromatography. Four YLLIP2 isoforms were identified and subjected to N-terminal amino-acid sequencing and mass spectrometry analysis. These isoforms differed in their glycosylation patterns and their molecular masses ranged from 36,874 to 38,481 Da, whereas the polypeptide mass was 33,385 Da. YLLIP2 substrate specificity was investigated using short (tributyrin), medium (trioctanoin) and long (olive oil) chain triglyceride substrates at various pH and bile salt concentrations, and compared with those of human gastric and pancreatic lipases. YLLIP2 was not inhibited by bile salts at micellar concentrations with any of the substrates tested, and maximum specific activities were found to be 10,760+/-115 U/mg on tributyrin, 16,920+/-480 U/mg on trioctanoin and 12,260+/-700 U/mg on olive oil at pH 6.0. YLLIP2 was found to be fairly stable and still active on long chain triglycerides (1590+/-430 U/mg) at pH 4.0, in the presence of bile salts. It is therefore a good candidate for use in enzyme replacement therapy as a means of treating pancreatic exocrine insufficiency.

Human pancreatic lipase-related protein 2 (HPLRP2) was previously found to be secreted by the exocrine pancreas. HPLRP2 shows a high level of activity on galactolipids, and might be involved in the digestion of these common vegetable lipids. Specific antibodies were raised in rabbits using a synthetic HPLRP2 peptide selected for its weak amino acid homology with the corresponding peptides of classical human pancreatic lipase (HPL) and human pancreatic lipase-related protein 1 (HPLRP1). ELISA and Western blotting data showed that these antibodies did not react with HPL or HPLRP1. Various tissues from the digestive tract were subjected to Western blotting analysis with the specific anti-peptide HPLRP2 antibody and the expression of HPLRP2 was detected in the pancreas and colon. An ELISA was developed for specifically measuring the HPLRP2 levels in pure pancreatic juice. This procedure was performed using the anti-peptide HPLRP2 antibody as the captor antibody and a biotinylated anti-HPLRP2 polyclonal antibody as the detector antibody. The lowest HPLRP2 quantification limit was found to be 50 microg/L and the reference range for the present assay was 50 microg-500 microg/L. HPL and HPLRP2 levels were measured using specific ELISAs in pancreatic juice from patients with and without pancreatic disorders. Patients with chronic calcifying pancreatitis (CCP) had significantly lower levels of both HPL and HPLRP2 than the controls subjects. The mean HPLRP2 to HPL ratio was estimated to be 28.30% (w/w) and 23.96% (w/w) in controls subjects and CCP patients, respectively, and the difference was not significant. The levels of HPL and HPLRP2 are therefore similarly reduced in both healthy patients and CCP patients.

The mesocarp of mature oil palm fruit undergoes intensive triglycerides hydrolysis upon abscission and bruising. This generates such a high amount of free fatty acids that the oil might become unfit for human consumption without appropriate refining. The lipase (EC 3.1.1.3) involved in the breakdown of the oil is not stable after homogenization of the tissue in aqueous buffers. In this study, we have devised a solvent-based procedure that allowed us to obtain fractions with stable lipase activity. Using these fractions, we have determined the optimal conditions for assaying mesocarp lipase activity. The activity was highest at a temperature of 35 degrees C and a pH of 9. The lipase was found to be strictly calcium dependent. The specific activity of the lipase measured in optimal conditions was found to be 33 mumol fatty acids released min(-1) mg(-1) protein using olive oil as substrate. The mesocarp contains about 190 U of lipase g(-1) fresh weight. This activity was found to be inhibited by the lipase inhibitor tetrahydrolipstatin (THL), suggesting that the lipase is a serine hydrolase.

Many enzymes are active at interfaces in the living world (such as in the signaling processes at the surface of cell membranes, digestion of dietary lipids, starch and cellulose degradation, etc.), but fundamental enzymology remains largely focused on the interactions between enzymes and soluble substrates. The biochemical and kinetic characterization of lipolytic enzymes has opened up new paths of research in the field of interfacial enzymology. Lipases are water-soluble enzymes hydrolyzing insoluble triglyceride substrates, and studies on these enzymes have led to the development of specific interfacial kinetic models. Structure–function studies on lipases have thrown light on the interfacial recognition sites present in the molecular structure of these enzymes, the conformational changes occurring in the presence of lipids and amphiphiles, and the stability of the enzymes present at interfaces. The pH-dependent activity, substrate specificity and inhibition of these enzymes can all result from both “classical” interactions between a substrate or inhibitor and the active site, as well as from the adsorption of the enzymes at the surface of aggregated substrate particles such as oil drops, lipid bilayers or monomolecular lipid films. The adsorption step can provide an alternative target for improving substrate specificity and developing specific enzyme inhibitors. Several data obtained with gastric lipase, classical pancreatic lipase, pancreatic lipase-related protein 2 and phosphatidylserine-specific phospholipase A1 were chosen here to illustrate these specific features of interfacial enzymology.

In a previous study, we demonstrated that the beta5'-loop in the C-terminal domain of human pancreatic triglyceride lipase (hPTL) makes a major contribution in the function of hPTL (Chahinian et al. (2002) Biochemistry 41, 13725-13735). In the present study, we characterized the contribution of three residues in the beta5'-loop, Val-407, Ile-408, and Leu-412, to the function of hPTL. By substituting charged residues, aspartate or lysine, in these positions, we altered the hydrophilic to lipophilic ratio of the beta5'-loop. Each of the mutants was expressed, purified, and characterized for activity and binding with both monolayers and emulsions and for binding to colipase. Experiments with monolayers and with emulsions suggested that the interaction of hPTL with a phospholipid monolayer differs from the interaction of the hPTL-colipase complex with a dicaprin monolayer or a triglyceride emulsion (i.e. neutral lipids). Val-407, Ile-408, and Leu-412 make major contributions to interactions with monolayers, whereas only Val-407 and Ile-408 appear essential for activity on triglyceride emulsions in the presence of bile salt micelles. In solutions of taurodeoxycholate at micellar concentrations, a major effect of the beta5'-loop mutations is to change the interaction between hPTL and colipase. These observations support a major contribution of residues in the beta5'-loop in the function of hPTL and suggest that a third partner, bile salt micelles or the lipid interface or both, influence the binding of colipase and hPTL through interactions with the beta5'-loop.

Extracellular lipases were obtained from solid (SSF) and submerged (SmF) thermotolerant Rhizopus homothallicus fungus cell cultures and purified to homogeneity. The two enzymes are monomers having a molecular mass of 29.5 kDa and an identical protein structure, since the N-terminal sequences and peptide maps were identical. However, some of their properties are different, namely the specific activity on trioctanoin (8600 U/mg with SmF and 10,700 U/mg with SSF), the temperature at which maximum activity occurs (30 degrees C with SmF and 40 degrees C with SSF) and the thermal stability (half-lives at 50 degrees C of 0.44 h with SmF and 0.72 h with SSF). These differences between the kinetic properties suggest that when they were tested, one or both fungal lipases might still have been associated with non-proteic compounds originating from the culture medium. (c) 2006 Elsevier Inc. All rights reserved.

Le lait maternel est l’aliment ideal pour le nouveau-ne (OMS). Quand l’allaitement n’est pas possible ou souhaite, des formules infantiles peuvent etre administrees. Alors que la composition nutritionnelle des formules infantiles a ete optimisee, peu de travaux se sont interesses a la structure de ces formules.[br/] Quel est l’impact de la structure des emulsions (formules infantiles, lait maternel) sur leurs cinetiques de digestion (lipolyse et proteolyse) et de destructuration?[br/] Quels sont les parametres cles affectant la digestion gastrique chez le nouveau-ne ?[br/] [br/] Les traitements technologiques modifient la structure des emulsions laitieres (agregation proteique, surface specifique des gouttelettes). Cette structure influe sur la destructuration de ces matrices et sur les cinetiques de lipolyse et proteolyse au cours de la digestion gastrique. La modulation des cinetiques d’hydrolyse en phase gastrique peut impacter sur les vitesses d’absorption des nutriments et ainsi moduler les reponses metaboliques. Il serait necessaire de developper des traitements technologiques doux pour la fabrication des formules infantiles, avec un impact minimum sur le globule gras, pour mimer au mieux la destructuration et l’hydrolyse des matrices natives.

The scorpion hepatopancreas consists of digestive diverticula and interstitial tissue. A digestive diverticulum is composed of two differentiated cell types: the secretory zymogene-like cells and the digestive cells which are the most abundant. The scorpion digestive lipase (SDL) has been previously purified from scorpion hepatopancreas, but its cellular localization has not yet been established. Polyclonal antibodies specific to SDL were prepared and used in immunofluorescence and immunogold techniques to determine the cellular location of SDL. Our results clearly established that SDL was detected intracellularly in specific vesicles tentatively named (SDL+) granules of the digestive cells. No immunolabelling was observed in secretory zymogene-like cells. This immunocytolocalization indicates that lipid digestion might occur in specific granules inside the digestive cells, as suggested by previous studies on the scorpion digestive process.

Epitope mapping was performed on human pancreatic lipase (HPL) using the SPOTscan method. A set of 146 short (12 amino acid residues) synthetic overlapping peptides covering the entire amino acid sequence of HPL were used to systematically assess the immunoreactivity of antisera raised in rabbits against native HPL, HPL without a lid (HPL(-lid)) and HPL covalently inhibited by diethyl p-nitrophenyl phosphate (DP-HPL). In the latter form of HPL, the lid domain controlling the access to the active site was assumed to exist in the open conformation. All the anti-lipase sera were tested in a direct ELISA, anti-HPL serum showing the greatest antibody titer. Although from the structural point of view, the differences between the various forms of HPL were restricted to the lid domain, differences in the antigenic properties of HPL were observed with the SPOTscan method, and the anti-DP-HPL antibodies showed the strongest reactivity. Most of the peptide stretches recognized included amino acid residues which are accessible at the surface of the lipase, except for those located near the active site. Two small peptides (T173-P180, V199-A207) were identified in the vicinity of the active site, their antipeptide antibodies were produced and their reactivity towards the various forms of HPL was tested in a double sandwich ELISA. No reactivity was observed under these conditions. Two antipeptide antibodies directed against two other selected peptides, P208-V221 (belonging to the beta9 loop) and I245-F258 (belonging to the lid domain) were prepared and found to react much more strongly with DP-HPL than with HPL or HPL(-lid) in a double sandwich ELISA. These antibodies should provide useful tools for monitoring the conformational changes taking place during the opening of the HPL lid domain.

Thirty years ago, it was reported that a linear relationship does not exist between the amounts of human pancreatic lipase secreted in chronic pancreatitis and the degree of steatorrhea, which was considered to appear only after more than 90% of the pancreatic secretory capacity had been lost. From these observations, it was generally thought that the lipolytic potential of the pancreas is much higher than required. In recent years, however, it has been noted that: 1) the level of inhibition of digestive lipases and gastrointestinal lipolysis by the lipase inhibitor orlistat were almost linearly correlated with the amount of excreted fat; 2) in minipigs with experimentally-induced pancreatic exocrine insufficiency, the amounts of enteric-coated pancreatic extracts needed for restoring fat digestion to normal levels were estimated to be much higher than those usually administered; 3) human pancreatic lipase specific activity on meal triglycerides is 3 orders of magnitude lower than the very high specific activity usually measured under experimental in vitro conditions which are far from physiological conditions; 4) in patients with reduced human pancreatic lipase secretion, gastric lipase plays a significant role in fat digestion. This last observation might explain the absence of a linear relationship between human pancreatic lipase secretion in chronic pancreatitis and steatorrhea. From the low specific activity displayed by human pancreatic lipase on meal triglycerides, one can better understand why more lipase than expected is needed, why fat digestion lasts for more than a few minutes and, finally, why there is not such an excess secretory capacity for lipase as had been previously thought.

Hormone-sensitive lipase (HSL) contributes importantly to the hydrolysis of cholesteryl ester in steroidogenic tissues, releasing the cholesterol required for adrenal steroidogenesis. HSL has broad substrate specificity, because it hydrolyzes triacylglycerols (TAGs), diacylglycerols, monoacylglycerols, and cholesteryl esters. In this study, we developed a specific cholesterol esterase assay using cholesterol oleate (CO) dispersed in phosphatidylcholine and gum arabic by sonication. To continuously monitor the hydrolysis of CO by HSL, we used the pH-stat technique. For the sake of comparison, the hydrolysis of CO dispersion was also tested using other cholesteryl ester-hydrolyzing enzymes. The specific activities measured on CO were found to be 18, 100, 27, and 3 micromol/min/mg for HSL, cholesterol esterase from Pseudomonas species, Candida rugosa lipase-3, and cholesterol esterase from bovine pancreas, respectively. The activity of HSL on CO is approximately 4- to 5-fold higher than on long-chain TAGs. In contrast, with all other enzymes tested, the rates of TAG hydrolysis were higher than those of CO hydrolysis. The relatively higher turnover of HSL on CO observed in vitro adds further molecular insight on the physiological importance of HSL in cholesteryl ester catabolism in vivo. Thus, HSL could be considered more as a cholesteryl ester hydrolase than as a TAG lipase.

The storage of frozen semen for artificial insemination is usually performed in the presence of egg yolk or skimmed milk as protective agents. In goats, the use of skimmed milk extenders requires, however, that most of the seminal plasma is removed before dilution of spermatozoa because it is deleterious for their survival. It has been previously demonstrated that a lipase (BUSgp60) secreted by the accessory bulbourethral gland was responsible for the cellular death of goat spermatozoa, through the lipolysis of residual milk lipids and the release of toxic free fatty acids. This lipase was purified from the whole seminal plasma of goat and was found to display both lipase and phospholipase A activities, this latter activity representing the main phospholipase activity detected in goat seminal plasma. Based on its N-terminal amino acid sequence, identical to that of BUSgP60 purified from bulbourethral gland secretion, and the design of degenerated oligonucleotides, the lipase was cloned from total mRNA isolated from bulbourethral gland. DNA sequencing confirmed it was the goat pancreatic-lipase-related protein 2 (GoPLRP2). The physiological role of GoPLRP2 is still unknown but this enzyme might be associated with the reproductive activity of goats. A significant increase in lipase secretion was observed every year in August and the level of lipase activity in the semen remained high till December, i.e., during the breeding season. A parallel increase in the plasmatic levels of testosterone suggested that GoPLRP2 expression might be regulated by sexual hormones. The lipase activity level measured in goat seminal plasma, which could reach 1000 U/ml during the breeding season, was one of the highest lipase activity measured in natural sources, including gastric and pancreatic juices.

Human pancreatic lipase-related protein 2 (HPLRP2) was found to be expressed in the pancreas, but its biochemical properties were not investigated in detail. A recombinant HPLRP2 was produced in insect cells and the yeast Pichia pastoris and purified by cation exchange chromatography. Its substrate specificity was investigated using pH-stat and monomolecular film techniques and various lipid substrates (triglycerides, diglycerides, phospholipids, and galactolipids). Lipase activity of HPLRP2 on trioctanoin was inhibited by bile salts and poorly restored by adding colipase. In vivo, HPLRP2 therefore seems unlikely to show any lipase activity on dietary fat. In human pancreatic lipase (HPL), residues R256, D257, Y267, and K268 are involved in the stabilization of the open conformation of the lid domain, which interacts with colipase. These residues are not conserved in HPLRP2. When the corresponding mutations (R256G, D257G, Y267F, and K268E) are introduced into HPL, the effects of colipase are drastically reduced in the presence of bile salts. This may explain why colipase has such weak effects on HPLRP2. HPLRP2 displayed a very low level of activity on phospholipid micelles and monomolecular films. Its activity on monogalactosyldiglyceride monomolecular film, which was much higher, was similar to the activity of guinea pig pancreatic lipase related-protein 2, which shows the highest galactolipase activity ever measured. The physiological role of HPLRP2 suggested by the present results is the digestion of galactolipids, the most abundant lipids occurring in plant cells, and therefore, in the vegetables that are part of the human diet.

Human pancreatic lipase-related protein 2 (HPLRP2) was identified for the first time in pancreatic juice using specific anti-peptide antibodies and purified to homogeneity. Antibodies were raised in the rabbit using a synthetic peptide from the HPLRP2 protein sequence deduced from cDNA. Western blotting analysis showed that these antibodies did not react with classical human pancreatic lipase (HPL) or human pancreatic lipase-related protein 1 (HPLRP1) but cross-reacted with native rat PLRP2 (RPLRP2), as well as with recombinant rat and guinea-pig PLRP2 (GPLRP2). Immunoaffinity chromatography was performed on immobilized anti-recombinant HPLRP2 polyclonal antibodies to purify native HPLRP2 after conventional chromatographic steps including gel filtration and chromatrography on an anion-exchanger. The substrate specificity of HPLRP2 was investigated using various triglycerides, phospholipids and galactolipids as substrates. The lipase activity on triglycerides was inhibited by bile salts and weakly restored by colipase. The phospholipase activity of HPLRP2 on phospholipid micelles was very low. A significant level of galactolipase activity was measured using monogalactosyldiglyceride monomolecular films. These data suggest that the main physiological function of HPLRP2 is the hydrolysis of galactolipids, which are the main lipids present in vegetable food.

Background and Aims: Human pancreatic lipases (HPL) include the classical HPL, and two related proteins known as pancreatic lipase-related proteins 1 and 2 (HPLRP1 and 2). The aim of this study was to develop an ELISA for specifically quantifying the classical-HPL level in sera of patients with and without pancreatic disorders. Methods: The specific activity of various human (including classical-HPL) and microbial lipases was measured using Lipa Vitros and potentiometric (pH-stat) assays. A double sandwich ELISA was also set up, using an anticlassical-HPL polyclonal antibody and a biotinylated monoclonal antibody (mAb 146-40) specific to the classical-HPL. Sera (n = 53) were collected from patients with and without pancreatic disorders. The lipase concentration was deduced from the measured lipolytic activity and compared with the corresponding classical-HPL concentration, measured with the ELISA. Results: Both the purified HPLRP2 and 3 lipases of microbial origin were found to have a significant and unexpected lipolytic activity under the standard Lipa Vitros assay, whereas the ELISA test developed in the present study was found to be specific for the classical-HPL, due to the absence of cross-reactivity between mAb 146-40, HPLRP1 and HPLRP2. The efficiency of the ELISA was assessed in terms of its reproducibility and accuracy. The lower detection limit of classical-HPL was found to be 0.03 μg/l. A good correlation was found to exist between the lipase concentrations obtained in the ELISA, pH-stat and Lipa Vitros tests, in both the control and pathological groups. Conclusion: This is the first time a specific method of measuring classical-HPL in human serum has been proposed. Using this ELISA, we established with the 53 sera selected in the present study, that the Lipa Vitros assay as well as the pH-stat assay were mostly detecting classical pancreatic lipase. However, it is possible that other lipases such as HPLRP2 or lipases of microbial origin, present in some pathological sera, may well interfere with the Lipa Vitros assay.

In human adults, the enzymatic hydrolysis of dietary fat along the digestive tract is sequentially catalyzed by two main enzymes, human gastric lipase (HGL) and human pancreatic lipase (HPL). Both a chemically inhibited form of HPL as well as an inactive HPL mutant with a glycine residue substituted for its catalytic serine were found to be strong inactivators of HPL activity. In the presence of bile salts, this inhibition was clearly due to competition for colipase. We established that the chemically inhibited HPL, probably in its open conformation, had a much greater affinity for colipase than the closed native form of HPL. These inhibitory effects are quite substantial, because a 0.2-M excess of the chemically inhibited HPL form relative to HPL reduced the catalytic lipolytic activity by 50% in the presence of an equimolar amount of colipase.

The structural similarities between the C-terminal domain of human pancreatic lipase (C-HPL) and C2 domains suggested a similar function, the interaction with lipids. The catalytic N-terminal domain (N-HPL) and C-HPL were produced as individual proteins, and their partitioning between the water phase and the triglyceride-water interface was assessed using trioctanoin emulsions (TC8). N-HPL did not bind efficiently to TC8 and was inactive. C-HPL did bind to TC8 and to a phospholipid monolayer with a critical surface pressure of penetration similar to that of HPL (15 mN m(-1)). These experiments, performed in the absence of colipase and bile salts, support an absolute requirement of C-HPL for interfacial binding of HPL. To refine our analysis, we determined the contribution to lipid interactions of a hydrophobic loop (beta 5') in C-HPL by investigating a HPL mutant in which beta 5' loop hydrophobicity was increased by introducing the homologous lipoprotein lipase (LPL) beta 5' loop. This mutant (HPL-beta 5'LPL) penetrated into phospholipid monolayers at higher surface pressures than HPL, and its level of binding to TC8 was higher than that of HPL in the presence of serum albumin (BSA), an inhibitory protein that competes with HPL for interfacial adsorption. The beta 5' loop of LPL is therefore tailored for an optimal interaction with the surface of triglyceride-rich lipoproteins (VLDL and chylomicrons) containing phospholipids and apoproteins. These observations support a major contribution of the beta 5' loop in the interaction of LPL and HPL with their respective substrates.

Background/Aims: The aim of this study was to exactly determine the pH stability of human gastric lipase (HGL) and to investigate the mechanism underlying the inactivation of HGL which occurs in gastric juice. Methods: Samples of human gastric juice and purified HGL were incubated at various pH values ranging from 0.5 to 8.0, and the residual HGL activity was measured as a function of time using the pHstat technique. Samples of purified HGL were also incubated in the presence of human pepsin. Electrophoresis and Western blot analysis were performed on all the samples in which HGL was inactivated. Results: HGL was found to be stable in gastric juice at pH values ranging from 2.0 to 7.0, especially between pH 3.0 and 5.0 (half-inactivation time >24 h). HGL activity decreased rapidly below pH 2.0 and above pH 7.0. The inactivation half times were only 43 ± 9 and 24 ± 18 min at pH 1 and pH 8, respectively. The pH stability of purified HGL was much lower than that of HGL in gastric juice. Acid or alkaline inactivation of HGL could occur without any prior proteolytic degradation, and this inactivation was irreversible. However, proteolytic degradation of HGL by pepsin also occurred at very low pH values, probably because the acid-denatured HGL is more sensitive to proteolytic cleavage by pepsin. An ex vivo study of HGL activity in several gastric juice samples showed that the HGL activity decreased with the pH of the sample, in both basal and pentagastrin-stimulated gastric juice. Conclusion: Although HGL is not as stable as it was previously thought to be under acidic conditions, it is nevertheless the most stable acid lipase and constitutes a good candidate tool for enzyme substitution therapy.

Background/Aim: Gastric emptying is a major cause of variability when studying gastrointestinal parameters as a function of time. Here, we investigate whether the parametric variability could be reduced by running experiments on a gastric emptying basis rather than on a time basis. Methods: Healthy volunteers were intubated with gastric and duodenal tubes and were given a liquid meal containing polyethylene glycol to monitor gastric emptying. Gastric pH and human gastric lipase (HGL) concentrations were measured. Their variations were plotted as a function of either time or gastric emptying (%). In both cases, mean curves of variation were established by polynomial regression. Results: When time was the variable used, the overall deviation of the experimental values from the values given by the best-fitting curve was high (χ2 = 33 for gastric pH; χ2 = 1,744 for HGL), and the individual deviations increased with time. When gastric emptying was the variable used, the overall deviation of the experimental values from the values given by the best-fitting curve was much lower (χ2 = 10 for gastric pH; χ2 = 642 for HGL). Conclusions: Expressing gastric pH or HGL concentration as a function of gastric emptying instead of time makes it possible to reduce the individual variability. This new type of data analysis may be of a general interest to observe specific variations of gastrointestinal parameters induced by drugs, hormones, and meals, and that might be masked by the large intrinsic variability induced by gastric emptying.

Photoautotrophic, photomixotrophic and heterotrophic cell suspension cultures of Euphorbia characias were analysed for fatty acid composition of their neutral lipids. Contents in exponential and stationary phases of growth were compared to those of seeds, leaves and calli from which cell suspensions were derived. Quantitatively, on a dry weight basis, neutral lipid amounts varied in the different cells from 15 to 40% of that in the leaves (5.14 mg per g dry wt) but they were very low compared to that in the seeds (330 mg per g dry wt). The highest values were observed during the exponential phase of growth with photosynthetic cell suspensions. Qualitatively, fatty acid distribution was similar in photoautotrophic cell suspensions and leaves with a high content of linolenic acid. The linolenic acid/linoleic acid ratio decreased from photoautotrophic to heterotrophic cell suspensions. In seeds, the maximal amount of linoleic acid is reached while linolenic acid almost disappears. The desaturation of C18 fatty acids is closely linked with the development of the photosynthetic apparatus and with a low growth rate.

Background & Aims: The lipolytic potential of digestive lipases in vivo has always been deduced so far from their in vitro activities under nonphysiologic conditions. In the present study, the specific activities of human gastric lipase (HGL) and pancreatic lipase (HPL) were measured on dietary triglycerides (TGs) during test meal lipolysis. Methods: Healthy human volunteers ingested a liquid or solid meal. The specific activities of HGL and HPL were estimated from the lipase and free fatty acid (FFA) outputs at the postpyloric and duodenal levels, respectively. Based on the in vivo data, lipolysis was also performed in vitro by mixing the meal either with gastric juice and subsequently with pancreatic juice and bile or with purified HGL and HPL. FFAs were measured by thin-layer chromatography, and the specific activities of HGL and HPL were expressed as micromoles of FFA per minute per milligram of lipase. Results: In vitro, the specific activities on the liquid meal TGs were 32 (gastric juice) and 34 (pure lipase) μmol · min −1 · mg −1 with HGL and 47 (pancreatic juice) and 43 (pure lipase) μmol · min −1 · mg −1 with HPL. The specific activities on the solid meal TGs were 33 (gastric juice) and 32 (pure lipase) μmol · min −1 · mg −1 with HGL and 12 (pancreatic juice) and 15 (pure lipase) μmol · min −1 · mg −1 with HPL. The in vivo values obtained were in the same range. The secretory lipase outputs were 21.6 ± 14.5 mg HGL and 253.5 ± 95.5 mg HPL with the liquid test meal and 15.2 ± 5.1 mg HGL and 202.9 ± 96.1 mg HPL with the solid test meal. Conclusions: The specific activities of HGL and HPL on meal TGs were much lower than those measured in vitro under optimized assay conditions (1300–8000). However, these low specific activities are enough for the meal TGs to be completely lipolysed, given the amounts of HGL and HPL secreted during a meal. GASTROENTEROLOGY 2000;119:949-960

Both classical pancreatic lipase (DPL) and pancreatic lipase-related protein 1 (DPLRP1) have been found to be secreted by dog exocrine pancreas. These two proteins were purified to homogeneity from canine pancreatic juice and no significant catalytic activity was observed with dog PLRP1 on any of the substrates tested: di- and tri-glycerides, phospholipids, etc. DPLRP1 was crystallized and its structure solved by molecular replacement and refined at a resolution of 2.10 A. Its structure is similar to that of the classical PL structures in the absence of any inhibitors or micelles. The lid domain that controls the access to the active site was found to have a closed conformation. An amino-acid substitution (Ala 178 Val) in the DPLRP1 may result in a steric clash with one of the acyl chains observed in the structures of a C11 alkyl phosphonate inhibitor, a transition state analogue, bound to the classical PL. This substitution was suspected of being responsible for the absence of DPLRP1 activity. The presence of Val and Ala residues in positions 178 and 180, respectively, are characteristic of all the known PLRP1, whereas Ala and Pro residues are always present in the same positions in all the other members of the PL gene family. Introducing the double mutation Val 178 Ala and Ala 180 Pro into the human pancreatic RP1 (HPLRP1) gene yielded a well expressed and folded enzyme in insect cells. This enzyme is kinetically active on triglycerides. Our findings on DPLRP1 and HPLRP1 are therefore likely to apply to all the RP1 lipases.

Epitope mapping was performed using four anti-HPL monoclonal antibodies (mAb's 81-23, 146-40, 315-25, and 320-24) directed against human pancreatic lipase (HPL). Three HPL mutants produced in insect cells were tested for this purpose: (i) N-HPL, which consists of only the N-terminal domain of HPL, (ii) HPL(-lid), in which a short loop consisting of 5 amino acid residues replaces the full-length 23-residue lid domain present in HPL, and (iii) N-GPLRP2/C-HPL chimera, a chimeric mutant consisting of the N-terminal domain of the guinea pig pancreatic lipase related protein 2 (GPLRP2) fused to the C-terminal domain of HPL. The C-terminal domain of HPL (C-HPL) was prepared in a pure form after performing chymotryptic digestion of HPL. The mAb 146-40 recognizes HPL, HPL(-lid), and N-HPL but not GPLRP2, N-GPLRP2/C-HPL chimera, or the C-HPL. The antibody mAb 146-40 therefore specifically recognizes the N-terminal domain of HPL, and the epitope recognized does not include the amphiphilic lid. On the other hand, mAb's 81-23, 315-25, and 320-24 react specifically to the C-terminal domain of HPL, since they recognize HPL, HPL(-lid), the N-GPLRP2/C-HPL chimera, and the C-HPL but not N-HPL or GPLRP2. It was further established that these three mAb's recognize the same conformational epitope, the structure of which is stabilized by the N-terminal domain in the presence of SDS at concentrations greater than its critical micellar concentration. This conformational epitope was found to be located in the vicinity of Met 397 and Arg 414. These two residues delineate a highly exposed peptide stretch extending from the HPL C-terminal domain, which includes a hydrophobic surface loop (beta5'). Kinetic studies on the HPL/mAb's complexes showed that the lipase activity was much lower in these complexes than in HPL. The results of the present study suggest for the first time that the beta5' loop from the C-terminal domain may be involved in the interaction of HPL with a lipid/water interface.

Publisher Summary Lipases are carboxylic ester hydrolases and have been termed glycerolester-hydrolase in the international system of classification. They greatly differ with respect to both of their origins and their kinetic properties. They can catalyze in vitro the hydrolysis, or synthesis, of a wide range of different carboxylic esters; however, they all show a higher specific activity toward glyceridic substrates. Under physiological conditions, because natural triacylglycerols are water insoluble, lipases that are generally soluble in water, catalyze the hydrolysis of carboxylic ester bonds at lipid/water interfaces. Some lipases, such as gastric lipases, rapidly become denatured at an interface with a pure tributyrin or tributanoylglycerol emulsion. Consequently, it is impossible to assess experimentally what interfacial activation may have occurred. The three-dimensional structures of lipases suggested that the interfacial activation phenomenon might be due to the presence of an amphiphilic surface loop covering the active site of the enzyme in solution, just like a lid. When contact occurs with a lipid/water interface, this lid might undergo a conformational rearrangement as the result of which the active site becomes accessible.

The four main classes of biological substances are carbohydrates, proteins, nucleic acids and lipids. The first three of these substances have been clearly defined on the basis of their structural features, whereas the property which is common to all lipids is a physicochemical one. Lipids are in fact a group of structurally heterogeneous molecules which are all insoluble in water but soluble in apolar and slightly polar solvents such as ether, chloroform and benzene.

In this chapter we review recent molecular knowledge on two structurally related mammalian triglyceride lipases which have evolved from a common ancestral gene. The common property of the lipase family members is that they interact with non-polar substances. Pancreatic lipase hydrolyzes triglycerides in the small intestine in the presence of many dietary components, other digestive enzymes and high concentrations of detergents (bile salts). Lipoprotein lipase acts at the vascular side of the blood vessels where it hydrolyses triglycerides and some phospholipids of the circulating plasma lipoproteins. A third member of the gene family, hepatic lipase, is found in the liver of mammals. Also, this lipase is involved in lipoprotein metabolism. The three lipases are distantly related to some non-catalytic yolk proteins from Drosophila (Persson et al., 1989; Kirchgessner et al., 1989; Hide et al., 1992) and to a phospholipase A1 from hornet venom (Soldatova et al., 1993).

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.

Apart from their general biological significance, lipolytic enzymes play an increasingly important role in biotechnology and medicine.

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.

The bile salt-stimulated lipase or carboxyl ester hydrolase (CEH) is a non-specific enzyme secreted by the exocrine pancreas and mammary glands. Recently we demonstrated that PEG esters were good substrates for CEH as it exhibited the highest specific activity ever recorded for this enzyme on PEG-8 monocaprylate. The aim of this study was to develop a specific and sensitive method for assaying CEH in biological samples in which several lipases are contained. Eight different PEG-n mono and dicaprylates with ethylene oxide units ranging from n = 6 to 32 were tested using the pH-stat technique. PEG-20 dicaprylate was the best substrate to discriminate CEH from other digestive lipases. Since pancreatic lipase related-protein 2 (PLRP2) was also found to display a significant activity on PEG-20 dicaprylate, experiments were designed to optimize the specificity of the assay for CEH. The main parameters for increasing CEH activity, while reducing that of PLRP2, were the pH and the concentration of bile salts. A linear relationship between the enzyme activity and the mass of CEH was established. The specificity of this assay was validated using gastrointestinal fluids containing CEH, PLRP2, gastric and pancreatic lipases.