Gerard Pieroni

Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions.

Under optimal conditions, assay for pure human gastric lipase was carried out with short- and long-chain triacylglycerol emulsions. Maximal specific activities of 1160 and 620 U/mg were obtained with tributyrin and soybean emulsion, respectively. We observed that with a tributyrin substrate, bovine serum albumin or bile salts must be added before the addition of the enzyme in order to prevent its irreversible interfacial denaturation. With long-chain triacylglycerols as substrate, a decrease with time in the rate of hydrolysis was associated with release of protonated long-chain fatty acids. The inhibitory effect of protonated fatty acids was also observed using tributyrin at pH 3.0. These observations support the conclusion that human gastric lipase shows no intrinsic specificity for short-chain triacylglycerols and that its apparent specificity is modulated by pH and presence of amphiphile in the incubation medium. Our conclusions support the view that, in the human, gastric lipolysis may play an important role in long-chain fat digestion.

Molecular cloning of a human gastric lipase and expression of the enzyme in yeast

The molecular cloning of a cDNA coding for human gastric lipase and its expression in yeast is described. A lipase present in human gastric aspirates was purified and its N-terminal amino-acid sequence was determined. This was found to be homologous with the N-terminal sequence of rat lingual lipase. A cDNA library was constructed from mRNA isolated from human stomach tissue and probed with cloned rat lingual lipase DNA. One clone, pGL17, consisting of approximately 1450 base-pairs, contained the entire coding sequence for a human gastric lipase. The amino-acid sequence from the isolated protein and the DNA sequence obtained from the cloned gene indicated that human gastric lipase consists of a 379 amino acid polypeptide with an unglycosylated Mr of 43,162. Human gastric lipase and rat lingual lipase amino-acid sequences were closely homologous but were unrelated to porcine pancreatic lipase apart from a 6 amino-acid sequence around the essential Ser-152 of porcine pancreatic lipase. A yeast expression plasmid containing the phosphoglycerate kinase promoter and terminator sequences together with the human gastric lipase gene was constructed. Yeast transformed with this vector synthesised the lipolytically active enzyme.

Importance of human gastric lipase for intestinal lipolysis: an in vitro study

Using soybean triacylglycerols emulsified with egg lecithin we have studied, in vitro, the influence of substrate prehydrolysis by human gastric lipase upon subsequent degradation by the pancreatic lipase-co-lipase system. Fatty acids liberated by pure human gastric lipase or juice trigger immediate activity of human pancreatic lipase. Gastric lipolysis appears to be of prime importance for dietary lipid digestion in human.

Interactions of lipases with lipid monolayers: Facts and questions

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

Intestinal Phospholipase, a Novel Enzyme

We evaluated phospholipase activity in the intestine of rats and other species. Phospholipase activity was assayed by a surface barostat technique or an egg yolk titration system. Mucosal activity was found only by the surface barostat technique with phosphatidylglycerol as substrate; it was not found with phosphatidylcholine as substrate in assays by either technique. In gut luminal fluid activity was found when both phosphatidylcholine and phosphatidylglycerol were used as substrate in assays by the surface barostat technique, and phosphatidylcholine as substrate yielded activity in egg yolk titration. In rats in which pancreatic juice had been diverted, mucosal and gut luminal phospholipase activity was greater than in controls, thus demonstrating that enzyme activity was not due to pancreatic phospholipase. Bacterial origin of phospholipase activity was excluded in that phospholipase activity was found in germ-free rats; gastric and salivary gland origins were excluded in that continued phospholipase activity was found in rats with gastric fistula. The physiological importance of the enzyme was established by the finding that rats with pancreatic fistula absorbed 111 mumol of phosphatidylcholine and that controls absorbed 119 mumol of a 135-mumol load. Activity was found to be three times greater in the distal than in the proximal intestine; in cryptal cells it was 10 times greater than in villus tip cells. 65% of the activity in the gut lumen was tightly bound to particulate matter. We propose that intestinal phospholipase may be important in gut bacterial control, in the digestion of vegetable matter (phosphatidylglycerol is a major phospholipid in both plants and bacteria), and in the digestion of phospholipids in the gut lumen.

Porcine pancreatic procolipase and its trypsin-activated form: lipid binding and lipase activation on monomolecular films.

Triand diglycerides passing into the small intestine are hydrolyzed to fatty acids and 2-monoglycerides by pancreatic lipase assisted by its protein cofactor colipase [ 1,2]. Both lipase and colipase are secreted by the pancreas and colipase is synthesized as a precursor form [3]. Cleavage of the N-terminal pentapeptide by trypsin yields an activated molecule. This proteolytic cleavage is assumed to take place when colipase is secreted into the duodenum in a similar way as for the zymogens of pancreatic origin. The importance of procolipase activation was first observed in a substrate system using the commercially available Intralipid emulsion (long-chain triglycerides emulsified with phospholipids) and was demonstrated as a decrease in a lag time before lipase hydrolysis proceeded at a high rate [3]. We have now studied the interactions of colipase and its proform with a monomolecular rue-1,2-dilaurin film in the presence of taurodeoxycholate and their lipase-activating properties. In this system, at high surface pressure, we have observed an all or none effect on the lipase activation by colipase and procolipase, respectively. These results support the idea that trypsin activation of procolipase yields a colipase molecule with better lipid-binding properties. This can be compared to the well-documented case of trypsin activation of prophospholipase AZ [4,5]. In view of earlier results the relevance of procolipase activation to the in vivo situation will be discussed.

Regulation by the “interfacial quality” of some biological activities

Abstract A few examples are given of regulation by the “interfacial quality” of some biological activities. Experiments with monolayers have the unique advantage that the arrangement and packing of the molecules can be easily measured and controlled. The first part is devoted to proteins which do not degrade lipids. Soluble cardiotoxins are generally injected into the subphase below a preformed lipid monolayer and measurements are taken either at constant surface area or at constant surface pressure. These experiments can give information on the penetration capacity of the protein into the interface and its lipid specificity, with direct access to the area of the protein segment interacting with lipids. Most intrinsic membrane proteins are insoluble in water. In the absence of detergent they aggregate and display no affinity for lipid interfaces. These proteins can be spread from an organic solvent solution but with the risk of being denatured. In order to circumvent this difficulty a method for spreading an aqueous suspension of lipoproteins or natural membrane vesicles was developed. This spreading method allows the formation of lipoprotein films retaining biological activities and native membrane constituents. In the second part, the use of lipid monolayers as substrates for lipolytic enzymes is reviewed. The monolayer technique permits an accurate study to be made of the influence of surface pressure and protein cofactors on the hydrolysis velocity and lag time in lipolysis. Two examples are developed: first, the assistance provided by colipase during the penetration of phospholipid films by pancreatic lipase; second the activation by apolipoprotein CII of phospholipid-monolayer hydrolysis by lipoprotein lipase. The monolayer technique is ideally suited to the study of the mode of action of lipolytic enzymes on monolayers of controlled “interfacial quality”.

Importance of sulfhydryl group for rabbit gastric lipase activity

We have shown recently that rabbit gastric lipase (RGL) purified from gastric tissue presents catalytic properties comparable with those of human gastric lipase (HGL). We report here that only one sulfhydryl group was modified per molecule of native RGL after incubation at pH 8.0 with 5,5′‐dithiobis(2‐nitrobenzoic acid) (NbS2) for 4 h or 4,4′‐dithiopyridine (4‐PDS) for 60 min. With both reagents, a direct correlation was found between the modification of one sulfhydryl group per enzyme molecule and loss of RGL activity. Incubation of RGL with the new hydrophobic sulfhydryl reagent, dodecyldithio‐5‐(2‐nitrobenzoic acid) (C12‐NbS), at 30‐fold molar excess, at pH 3.0, 5.0 and 8.0, induced immediate and complete inactivation of RGL. Unlike NbS2 and 4‐PDS, C12‐NbS almost instantaneously stopped the course of tributyrin hydrolysis by RGL, in contrast to porcine pancreatic lipase (PPL). RGL can be included with HGL in the group of sulfhydryl enzymes.

Lipolysis and heterogeneous catalysis. A new concept for expressing the substrate concentration

A new concept is proposed for quantifying the substrate concentration during heterogeneous catalysis of the kind which occurs during lipolysis. The number of molecules of protein (enzyme) adsorbable to the lipid substrate interface per unit of volume was evaluated and defined as a volumetric concentration of protein (enzyme) binding site (PEBS). Using porcine pancreatic lipase (EC 3.1.1.3) as a model enzyme, the maximal PEBS concentration was measured under various assay conditions by determining the saturation of the lipid substrate with the enzyme. Abacuses correlating the lipid substrate concentration (M) with the PEBS concentration (M) under each experimental conditions were used to express the kinetic data in terms of a volumetric concentration of PEBS. Comparisons could thus be made between data obtained with various enzymes and lipid interfaces because they were expressed with the same unit. In the case of pancreatic lipase, using triolein and tributyrylglycerol as substrates,Km values of 2.7 and 7.5 nM PEBS were obtained, respectively, andKD values ranging around 9 nM PEBS were also obtained from Scatchard plots. In addition, the average superficial density of PEBS was found to be 10×1011 molecules·cm−2, which is a value commonly obtained with structural proteins and enzymes adsorbed to an acylglyceride-water interface, this finding supports the idea that the PEBS concept represents the room in which the protein molecule adsorbs at the lipidic interface.

Stereoselective lipase-catalysed acylation of terpenic allylic alcohols by fatty acid anhydrides.

Abstract Lipase from pancreatic powder preferentially catalyses the acylation of the E isomer of terpenic allylic alcohols using fatty acid anhydrides as the acylating agent. The course of the reaction can be described by a first-order equation.