Mustapha Aoubala

Purification of human gastric lipase by immunoaffinity and quantification of this enzyme in the duodenal contents using a new ELISA procedure

Human gastric lipase (HGL) is the first lipolytic enzyme involved in the digestion of dietary lipids along the gastrointestinal tract. We describe an improved procedure for isolating the enzyme using immunoaffinity chromatography in combination with ion-exchange chromatography. The purified enzyme, showing a single band on SDS-PAGE, expressed a specific activity of 1000 U/mg using tributyrin as the substrate. We also describe a specific enzyme-linked immunosorbent assay (ELISA) procedure for measuring duodenal HGL levels. The ELISA was performed using an anti-HGL polyclonal antibody (pAb) as the captor antibody and a biotinylated monoclonal antibody (mAb) as the detector antibody. With the double sandwich ELISA technique, HGL in the range of 1-60 ng/ml was measured in less than 5 h. Identical HGL concentrations were obtained using the above ELISA procedure when compared to those based on the enzymatic activity using the potentiometric method (correlation coefficient: r = 0.95). No significant interference from other duodenal components was observed, as proved by the quantitative HGL determinations performed on intestinal samples.

Tryptic cleavage of gastric lipases: location of the single disulfide bridge.

Human (HGL) and rabbit (RGL) gastric lipases were cleaved by trypsin and the resulting peptides were characterized. Exposure of HGL to trypsin led to the production of three identified fragments (H1, H2 and H3) resulting from cleavage sites at Lys-4 and Arg-229. Fragments H2 (Lys-4-Arg-229) and H3 (Glu-230-Lys-379) were derived from fragment H1 (Lys-4-Lys-379). The single disulfide bridge (Cys-236-Cys-244) of the molecule is localized in fragment H3. Out of the three cysteine residues conserved in all known gastric lipases, the free sulfhydryl group (Cys-227) was localized in fragment H2. Immunoblots, carried out with the tryptic fragments of HGL and anti-HGL mAbs, revealed that five inhibitory mAbs immunoreacted selectively with the N-terminal fragment H2, whereas two other non inhibitory mAbs immunoreacted exclusively with the C-terminal fragment H3. Trypsin also cleaved RGL at two sites (Arg-55 and Arg-229) leading to four identifiable fragments (R1, R2, R3 and R4). One cleavage site (Arg-229) was found to be identical in both RGL and HGL. We propose that this latter site is localized between the two domains of native gastric lipases.

A study on human gastric lipase and complexes with monoclonal antibodies using the monomolecular film technique

Abstract Five monoclonal antibodies (MAbs) directed against human gastric lipase (HGL) were used as probes to characterize and locate the functional sites (catalytic and lipid binding domains) on the surface of the enzyme. Taking advantage of the low surface activity of IgG molecules, the capacity of the MAbs to inhibit the penetration of native and sulfhydryl (SH)-modified HGL into lipid monolayers was investigated using the monomolecular film technique. A schematic representation of the topography of the epitopes recognized by the MAbs in relation to the catalytic and lipid binding sites of the enzyme is proposed. The region containing the free SH group essential for lipase activity is identified.

Immunological techniques for the characterization of digestive lipases.

Publisher Summary To gain a better understanding of the physiologic functions and structural characteristics of the digestive lipases, human pancreatic lipase (HPL) and human grastric lipase (HGL), monoclonal antibodies (mAbs) may provide useful tools. Such antibodies would also be useful for identification and purification of the enzymes from biological fluids. This chapter describes the preparation and possible application of polyclonal (pAb) and mAbs to HGL and HPL obtained from gastric and pancreatic juices, respectively. HGL is purified to homogeneity from gastric juice from volunteers with the sequential use of chromatographies on Mono S and Mono Q. The mAbs are characterized in terms of affinity to their antigen, capacity to inhibit lipase activity or to bind lipids, epitope mapping, and cross-reactivity with other gastric or pancreatic lipases. These antibodies constitute useful tools for the study of mammalian acidic and pancreatic lipases.

Lipases and esterases: reassessment of their classification in the light of their three dimensional structure

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