Hydrolysis of mixed monomolecular films of tricaprylin/dilauroylphosphatidylcholine by lipase and phospholipase A2.

The purpose of this article was to describe the kinetics of the enzymatic action of one or more enzymes on mixture of substrates organized in 2D structures in order to mimic some situations existing in biological or industrial systems. Hydrolysis of the mixed monomolecular films of tricaprylin/dilauroylphosphatidylcholine (TC8/DiC12PC) by Thermomyces lanuginosus lipase (TLL) and phospholipase A2 (PLA2) was studied by measuring the decrease of the surface area and change of the surface potential at barostatic conditions. The decrease of the surface area detects the transition of the substrate into reaction products and their solubilization while the change of the surface potential detects the contribution of dipole moment of the molecules remaining at the interface during the hydrolysis. The kinetic models, describing the interfacial hydrolysis allowed us to estimate the values of the global kinetic constants for TC8 and DiC12PC hydrolysis, respectively. The role of interaction between all participants of the catalytic act in that complex catalytic system is shown. The catalytic activity of TLL and PLA2 is affected by the molecular environment in TC8/DiC12PC mixed monolayers.

Comparative study of lipolysis by PLA2 of DOPC substrates organized as monolayers, bilayer vesicles and nanocapsules.

The water-soluble lipolytic enzymes act at the interface of insoluble lipid substrates, where the catalytical step is coupled with various interfacial phenomena as enzyme penetration, solubilization of reaction products, loss of mechanical stability of organized assemblies of phospholipids molecule, etc. One biologically relevant example is the enzymatic hydrolysis of DOPC by PLA(2), which results in cleavage of phospholipids molecules into water insoluble lipolytic products, namely oleic acid and lysophospholipid. In general, the enzymatic activity depends on the substrate organization and molecular environment of the catalytic reaction. The lipolysis by phospholipase A(2) of dioleoylphosphatidylcholine substrates organized as monolayer, bilayers vesicles and lipid nanocapsules was studied by measuring the decrease of the surface area at constant surface pressure or increase of the surface pressure at constant area at air-water interface. A kinetic model describing the coupling of the catalytic act with corresponding interfacial phenomena was developed. By using the kinetic model the values for the global hydrolytic kinetic constants were obtained. The obtained value for the monolayer is five orders of magnitude higher than this obtained with small unilamellar vesicles and six orders of magnitude higher then those obtained with lipid nanocapsules. The comparison shows that the enzymatic catalytic act occurring in the lipid environment of the monolayer is more efficacious than at the vesicle and nanocapsules interfaces.

Action of Humicola lanuginosa lipase on mixed monomolecular films of tricaprylin and polyethylene glycol stearate.

The hydrolysis catalyzed by Humicola lanuginosa lipase (HLL) of pure tricaprylin (TC) or stearate of polyethylene glycol 1500 (PEG-St) as well as their mixtures spread as monomolecular films were studied. The catalytic transformation of the two substrates TC or PEG-St into their respective reaction products was detected by measuring simultaneously the decrease in the film area and the surface potential using the "zero order" trough at constant surface pressure. A kinetic model describing the enzymatic hydrolysis was developed. The surface concentrations of the two substrates and their respective reaction products as well as the values of the global kinetic constants of hydrolysis were determined. The experimentally obtained global kinetic constants of the catalytic action of HLL against TC and PEG-St present in mixed monolayers of TC/PEG-St are approximately the same as in the case of pure monolayers. These obtained results give some indications that the activity of enzyme is not significantly affected by the different molecular environments in the mixed monolayers.

Novel chromatographic resolution of chiral diacylglycerols and analysis of the stereoselective hydrolysis of triacylglycerols by lipases.

In the present study, we propose a general and accessible method for the resolution of enantiomeric 1,2-sn- and 2,3-sn-diacylglycerols based on derivatization by isocyanates, which can be easily used routinely by biochemists to evaluate the stereopreferences of lipases in a time course of triacylglycerol (TAG) hydrolysis. Diacylglycerol (DAG) enantiomers were transformed into carbamates using achiral and commercially available reagents. Excellent separation and resolution factors were obtained for diacylglycerols present in lipolysis reaction mixtures. This analytical method was then applied to investigate the stereoselectivity of three model lipases (porcine pancreatic lipase, PPL; lipase from Rhizomucor miehei, MML; and recombinant dog gastric lipase, rDGL) in the time course of hydrolysis of prochiral triolein as a substrate. From the measurements of the diglyceride enantiomeric excess it was confirmed that PPL was not stereospecific (position sn-1 vs sn-3 of triolein), whereas MML and rDGL preferentially hydrolyzed the ester bond at position sn-1 and sn-3, respectively. The enantiomeric excess of DAGs was not constant with time, decreasing with the course of hydrolysis. This was due to the fact that DAGs can be products of the stereospecific hydrolysis of TAGs and substrates for stereospecific hydrolysis into monoacylglycerols.

Reorganization of lipid nanocapsules at air-water interface 3. Action of hydrolytic enzymes HLL and pancreatic PLA2.

The action of the hydrolytic enzymes humicola lanuginosa lipase (HLL) and pancreatic phospholipase A2 (PLA2) on monolayers formed from lipid nanocapsules (LNC) and model monolayers containing their components, Labrafac, Solutol and Lipoid, is studied by simultaneous measuring the changes in the film area and the surface potential in the "zero order" trough at constant surface pressure (pi). The kinetic models describing the hydrolysis by HLL of the Labrafac, Solutol and their mixtures have been proposed. By using the developed theoretical approach together with the experimental results the surface concentrations of the substrates, hydrolysis products and values of the global kinetic constants were obtained. The comparison between the global kinetic constants in the case of HLL hydrolysis of pure Labrafac, Solutol monolayers and those of the model mixed Labrafac/Solutol monolayers, shows that the rates of hydrolysis are of the same order of magnitude, i.e. an additively of the HLL enzyme action is observed. The composition of the mixed Labrafac/Solutol monolayer, formed after the interfacial LNC destabilization, was estimated.

Discrimination between closed and open forms of lipases using electrophoretic techniques.

The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases--human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase--by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.

In vitro lipolysis by human pancreatic lipase is specifically abolished by its inactive forms.

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.

Surface fluorescence resonance energy transfer studies on interfacial adsorption of Thermomyces (humicola) lanuginosa lipase, using monomolecular films of cis-parinaric acid.

The fluorescence resonance energy transfer (FRET) technique was adapted to study the process whereby lipase is adsorbed to monomolecular lipid films spread at the air-water interface. When cis-parinaric acid (cis-PnA) was spread over an aqueous subphase before the injection of sodium taurodeoxycholate (NaTDC) and Thermomyces lanuginosa lipase (TLL), no FRET was observed. Under these conditions, no adsorption of TLL was detected using an ELISA. In contrast, FRET occurred when cis-PnA was spread over an aqueous subphase containing NaTDC and TLL. The FRET signals observed were attributed to the interactions between the adsorbed TLL and the cis-PnA monomolecular films. Comparisons between the fluorescence emission spectra corresponding to the bulk phase and the aspirated film, in the presence and absence of TLL, showed that cis-PnA was undetectable in the bulk phase. We concluded that the FRET originated from the interface and not from the bulk phase. Using surface FRET, we estimated that the surface excess of the catalytically inactive mutant, TLL(S146A), was 1.6 higher than that present in the wild-type TLL. This finding is in agreement with independent measurements of the surface excess of TLL and TLL(S146A) on monomolecular films of cis-PnA.

Kinetic studies of Rhizopus oryzae lipase using monomolecular film technique.

Rhizopus oryzae lipase (ROL) was found to be a true lipase. This enzyme presents the interfacial activation phenomenon. The N-terminal amino acid sequence of ROL was compared to those of rhizopus lipases. Purified ROL possesses the same N-terminal sequence as the mature Rhizopus niveus lipase (RNL). This sequence was found in the last 28 amino acids of the propeptide sequence derived from the cDNA of Rhizopus delemar lipase (RDL). Using the baro-stat method, we have measured the hydrolysis rate of dicaprin films by ROL as a function of surface pressure. Our results show that Rhizopus oryzae lipase is markedly stereoselective of the sn-3 position of the 2,3 enantiomer of dicaprin. Polyclonal antibodies (PAB) directed against ROL have been produced and purified by immunoaffinity. The effects of these PAB on the interfacial behavior of ROL were determined. The immunoblot analysis with polyclonal antibodies anti-ROL (PAB anti-ROL) and various lipases shows a cross-immunoreactivity between the lipase from the rhizopus family (Rhizopus delemar lipase and Rhizopus arrhizus lipase).

Surface behaviour of bile salts and tetrahydrolipstatin at air/water and oil/water interfaces.

The surface behaviour of two bile salts, sodium deoxycholate (NaDC) and sodium taurodeoxycholate (NaTDC), as well as that of tetrahydrolipstatin (THL), a potent gastrointestinal lipase inhibitor, was studied at air/water and oil/water interfaces, using interfacial tensiometry methods. The surface behaviour of NaDC and NaTDC was comparable at both oil/water and air/water interfaces. A fairly compact interfacial monolayer of bile salts is formed well below the critical micellar concentration (CMC) and can help to explain the well-known effects of bile salts on the kinetic behaviour of pancreatic lipases. Using the Wilhelmy plate technique, the surface pressure-molecular area curves recorded with THL at the air/water interface showed a collapse point at a surface pressure of 24.5 mN.m(-1), corresponding to a molecular area of 70 A(2). Surprisingly, using the oil drop method, a limiting molecular area of 160 A(2) was found to exist at the oil/water interface. On the basis of the above data, space-filling models were proposed for bile salts and THL at air/water and oil/water interfaces.

Synthetic routes and lipase-inhibiting activity of long-chain alpha-keto amides.

Synthetic routes to primary and N-alkyl alpha-keto amides are presented in this paper. Primary alpha-keto amides may be prepared by using an aldehyde as starting material. Commercially available alpha-keto acids may be coupled in high yield with primary amines by the mixed carbonic anhydride method affording N-alkyl alpha-keto amides. Alternatively, N-alkyl alpha-keto amides may be prepared by coupling long-chain alpha-hydroxy acids with amino components, followed by oxidation with pyridinium dichromate or NaOCl in the presence of 4-acetamido-2,2,6,6-tetramethyl-1-piperidinyloxy free radical. The alpha-keto amide derivatives prepared according to these procedures were tested for their ability to form stable monomolecular films at the air/water interface. The inhibition of porcine pancreatic lipase by the alpha-keto amides, spread as mixed films with 1,2-dicaprin, was studied with the monolayer technique. Among the compounds tested in this study, methyl 2-[(2-ketododecanoyl)amino]hexadecanoate was shown to be the most potent inhibitor, causing a 50% decrease in lipase activity at a 0.09 molar fraction.

Effects of gum arabic on lipase interfacial binding and activity.

We investigated the surface behavior of gum Arabic (GA) as well as its effects on the lipolytic activity of human pancreatic lipase (HPL) and Humicola lanuginosa lipase (HLL), using emulsions of triacylglycerols (TAG) with various chain lengths. The effects of GA on the interfacial binding of HPL were also investigated. In the presence of 4 mM sodium taurodeoxycholate (NaTDC), GA (3% w/v, final concentration) had no effect on the HPL activity measured in the presence of colipase, whatever the type of TAG used. However, in the absence of bile salts or at low bile salt concentrations, GA inhibited the HPL activity when trioctanoin (TC8) and purified soybean oil (PSO) were used as substrates. At 3% (w/v, final concentration), GA strongly desorbed pure HPL from the TC8 interface and the classical anchoring effect of colipase was clearly observed. Both crude and dialyzed GA solutions were found to be highly tensioactive at the air-water as well as the oil-water interface using the drop technique. In conclusion, GA, or a putative contaminant present in GA, was found to be surface active and to have similar effects to those of bile salts on the interfacial binding and activity of HPL.

Inhibition of gastrointestinal lipolysis by Orlistat during digestion of test meals in healthy volunteers.

The inhibition of digestive lipases by the antiobesity drug Orlistat along with lipolysis levels and fecal fat excretion were measured in healthy humans. Orlistat was found to be a powerful gastric lipase inhibitor, achieving 46.6--91.4% enzyme inhibition and thus greatly reducing gastric lipolysis of solid and liquid meals (11--33% of respective controls). Gastric lipase inhibition by Orlistat was extremely fast (half-inhibition time < 1 min). Duodenal lipolysis was reduced significantly by Orlistat given with the solid meal (32.6--37.6% of controls) but was only slightly reduced by Orlistat given with the liquid meal (74.5--100% of controls). Human pancreatic lipase (HPL) inhibition was found to be high (51.2--82.6%), however, regardless of the meal. These paradoxical results were explained when in vitro lipolysis experiments were performed. The rates of HPL inhibition by Orlistat were found to be similar with both types of meals (half-inhibition time 5--6 min), but the preemulsified triglycerides of the liquid meal were rapidly hydrolyzed by HPL before the enzyme was significantly inhibited by Orlistat. With the solid meal, the rate of hydrolysis of the meal triglycerides by HPL was slower than the rate of HPL inhibition by Orlistat. As predicted from the previous results, the effects of Orlistat on fat excretion levels were found to be much greater with the solid (40.5--57.4% of ingested fat) than with the liquid (4.2--18.8%) test meal.

Bis-2-oxo amide triacylglycerol analogues: a novel class of potent human gastric lipase inhibitors.

A novel class of potent human gastric lipase inhibitors, bis-2-oxo amide triacylglycerol analogues, was developed. These analogues of the natural substrate of lipases were prepared starting from 1,3-diaminopropan-2-ol. They were designed to contain the 2-oxo amide functionality in place of the scissile ester bond at the sn-1 and sn-3 position, while the ester bond at the sn-2 position was either maintained or replaced by an ether bond. The derivatives synthesized were tested for their ability to form stable monomolecular films at the air/water interface by recording their surface pressure/molecular area compression isotherms. The inhibition of human pancreatic and gastric lipases by the bis-2-oxo amides was studied using the monolayer technique with mixed films of 1,2-dicaprin containing variable proportions of each inhibitor. The nature of the functional group (ester or ether), as well as the chain length, at the sn-2 position influenced the potency of the inhibition. Among the compounds tested, 2-[(2-oxohexadecanoyl)amino]-1-[[(2-oxohexadecanoyl)-amino]methyl]ethyl decanoate was the most potent inhibitor, causing a 50% decrease in HPL and HGL activities at 0.076 and 0.020 surface molar fractions, respectively.

Oil-bodies as substrates for lipolytic enzymes.

Plant seeds store triacylglycerols (TAGs) in intracellular organelles called oil-bodies or oleosomes, which consist of oil droplets covered by a coat of phospholipids and proteins. During seed germination, the TAGs of oil-bodies hydrolysed by lipases sustain the growth of the seedlings. The mechanism whereby lipases gain access to their substrate in these organelles is largely unknown. One of the questions that arises is whether the protein/phospholipid coat of oil-bodies prevents the access of lipase to the oil core. We have investigated the susceptibility of almond oil-bodies to in vitro lipolysis by various purified lipases with a broad range of biochemical properties. We have found that all the enzymes assayed were capable of releasing on their own free fatty acids from the TAG of oil-bodies. Depending on the lipase, the specific activity measured on oil-bodies using the pH-stat technique was found to range from 18 to 38% of the specific activity measured on almond oil emulsified by gum arabic. Some of these lipases are known to have a dual lipase/phospholipase activity. However, no correlation was found to exist between the ability of a lipase to readily and efficiently hydrolyse the TAG content of oil-bodies and the presence of a phospholipase activity. Kinetic studies indicate that oil-bodies behave as a substrate as other proteolipid organelles such as milk fat globules. Finally we have shown that a purified water-soluble plant lipase on its own can easily hydrolyse oil-bodies in vitro. Our results suggest that the lipolysis of oil-bodies in seedlings might occur without any pre-hydrolysis of the protein coat.

Use of the tape stripping technique for directly quantifying esterase activities in human stratum corneum.

The enzymes secreted in the intercellular spaces of stratum corneum (SC), the outermost layer of the epidermis, are thought to be involved in normal desquamation and skin barrier function. Their activity can barely be measured due to the difficulty in isolating enough biological material. Human SC layers were obtained from the forearm of healthy volunteers by the tape stripping technique. Assays for esterase activities were carried out in specially designed plates which contained the SC blotted on tape strips, using various fluorescent methylumbelliferone acyl esters as substrates. Triacylglycerol hydrolase activities were also studied by this method. By using radiolabeled triolein and fluorescent 4-methylumbelliferyl 7-oleate as substrates, true lipase activities could be detected and quantitated in SC at pH 5.5 and 7.5. These activities were shown to be strongly inhibited by tetrahydrolipstatin while this was not the case with 4-methylumbelliferyl 7-heptanoate. The method described here combines the painless tape stripping technique with a sensitive plate assay analysis. Since the whole process needs little manipulation, this method can permit rapid quantitation of multiple enzyme activities from a single strip. Therefore, it will permit the study of the involvement of enzyme activities in epidermis aging and skin pathologies.

Crystallization and preliminary crystallographic study of a recombinant phospholipase D from cowpea (Vigna unguiculata L. Walp).

The plant phospholipase D (PLD) is considered to be a key enzyme involved in various physiological processes such as signal transduction and membrane metabolism. Crystals of the PLD protein from Vigna unguiculata have been produced from the recombinant 768 amino-acid protein. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 157.7, b = 65.6, c = 90.2 A, beta = 111.5 degrees. There is one molecule in the asymmetric unit. Frozen crystals diffract to at least 1.94 A resolution using synchrotron radiation. A search for heavy-atom derivatives using ytterbium and tungstate is currently under way in order to solve the three-dimensional structure.

Digestive lipases: from three-dimensional structure to physiology.

Human gastric lipase (HGL) is a lipolytic enzyme that is secreted by the chief cells located in the fundic part of the stomach. HGL plays an important role in lipid digestion, since it promotes the subsequent hydrolytic action of pancreatic lipase in duodenal lumen. Physiological studies have shown that HGL is able of acting not only in the highly acid stomach environment but also in the duodenum in synergy with human pancreatic lipase (HPL). Recombinant HGL (r-HGL) was expressed in the baculovirus/insect cell system in the form of an active protein with a molecular mass of 45 kDa. The specific activities of r-HGL were found to be similar to that of the native enzyme when tested on various triacylglycerol (TG) substrates. The 3-D structure of r-HGL was the first solved within the mammalian acid lipase family. This globular enzyme (379 residues) shows a new feature, different from the other known lipases structures, which consists of a core domain having the alpha/beta hydrolase fold and a cap domain including a putative 'lid' of 30 residues covering the active site of the lipase (closed conformation). HPL is the major lipolytic enzyme involved in the digestion of dietary TG. HPL is a 50 kDa glycoprotein which is directly secreted as an active enzyme. HPL was the first mammalian lipase to be solved structurally, and it revealed the presence of two structural domains: a large N-terminal domain (residues 1-336) and a smaller C-terminal domain (residues 337-449). The large N-terminal domain belongs to the alpha/beta hydrolase fold and contains the active site. A surface loop called the lid domain (C237-C261) covers the active site in the closed conformation of the lipase. The 3-D structure of the lipase-procolipase complex illustrates how the procolipase might anchor the lipase at the interface in the presence of bile salts: procolipase binds to the C-terminal domain of HPL and exposes the hydrophobic tips of its fingers at the opposite site of its lipase-binding domain. These hydrophobic tips help to bring N-terminal domain into close conformation with the interface where the opening of the lid domain probably occurs. As a result of all these conformational changes, the open lid and the extremities of the procolipase form an impressive continuous hydrophobic plateau, extending over more than 50 A. This surface might able to interact strongly with a lipid-water interface. The biochemical, histochemical and clinical studies as well as the 3-D structures obtained will be a great help for a better understanding of the structure-function relationships of digestive lipases.

Synthesis of 2-Oxo amide triacylglycerol analogues and study of their inhibition effect on pancreatic and gastric lipases.

A general method for the synthesis of chiral 2-oxo amide triacylglycerol analogues, from (R)- or (S)-3-aminopropane-1,2-diol, was developed. These novel inhibitors of digestive lipases are analogues of the triacylglycerol molecule, a natural substrate of lipases, and they were designed to contain the 2-oxo amide functionality in place of the scissile ester bond at the sn-1 or sn-3 position and nonhydrolysable ether bonds instead of ester bonds at the other two remaining positions. The 2-oxo amide derivatives synthesised were tested for their ability to form stable monomolecular films at the air/water interface by recording their surface pressure/molecular area compression isotherms. The inhibition of porcine pancreatic and human gastric lipases by the 2-oxo amides was studied by means of the monolayer technique with mixed films of 1,2-dicaprin and with variable proportions of each inhibitor. The alpha50 values of these triacylglycerol analogues for PPL and HGL varied between 4.4 to 7.0% and 5.6 to 15.9%, respectively. The chirality at the sn-2 position of 2-oxo amide triacylglycerol analogues affected the alpha50 value for HGL, but not for PPL.

A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase.

A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of