Biomedical subjects

N Miled

Publications and source records attributed to N Miled.

6 recordsLinked to original sources

PubMed2006

Cloning and molecular modelling of turkey pancreatic lipase: structural explanation of the increased interaction power with lipidic interface.

Starting from total pancreatic mRNAs, turkey pancreatic lipase (TPL) cDNA was synthesized by RT-PCR and cloned into the PGEM-T vector. Amino acid sequence of the TPL is compared to that of human pancreatic lipase (HPL). A 3-D structure model of TPL was built using the 3-D structure of HPL as template, given the high amino acid sequence homology between the two lipases. Based on this model, the enhanced interaction power of TPL, as compared to that of HPL, into a phosphatidylcholine monolayer film, could be explained. We concluded that an increase in the exposed hydrophobic residues on the surface of TPL would be responsible for an enhanced interaction with a lipidic interface.

Amino Acid Sequence↗

PubMed2005

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.

Animals↗

PubMed2003

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.

Anthracenes↗

PubMed2003

Importance of the lid and cap domains for the catalytic activity of gastric lipases.

Human gastric lipase (HGL) is an enzyme secreted by the stomach, which is stable and active despite the highly acidic environment. It has been clearly established that this enzyme is responsible for 30% of the fat digestion processes occurring in human. This globular protein belongs to the alpha/beta hydrolase fold family and its catalytic serine is deeply buried under a domain called the extrusion domain, which is composed of a 'cap' domain and a segment consisting of 58 residues, which can be defined as a lid. The exact roles played by the cap and the lid domains during the catalytic step have not yet been elucidated. We have recently solved the crystal structure of the open form of the dog gastric lipase in complex with a covalent inhibitor. The detergent molecule and the inhibitor were mimicking a triglyceride substrate that would interact with residues belonging to both the cap and the lid domains. In this study, we have investigated the role of the cap and the lid domains, using site-directed mutagenesis procedures. We have produced truncated mutants lacking the lid and the cap. After expressing these mutants and purifying them, their activity was found to have decreased drastically in comparison with the wild type HGL. The lid and the cap domains play an important role in the catalytic reaction mechanism. Based on these results and the structural data (open form of DGL), we have pointed out the cap and the lid residues involved in the binding with the lipidic substrate.

Antibodies, Monoclonal↗

PubMed2000

A conformational transition between an open and closed form of human pancreatic lipase revealed by a monoclonal antibody.

The interfacial activation of human pancreatic lipase (HPL) probably involves the motion of a lid covering the active site of the enzyme. Here we observed that the presence of either bile salts or a small proportion of water-miscible organic solvents (called activator compounds) considerably enhances the enzymatic activity of HPL on a monomeric solution of tripropionin. This finding suggests that the activator compounds may favor the opening of the lid. This hypothesis was checked by comparing the immunoreactivity of HPL and HPL with a mini-lid (HPL(-lid)) towards anti-HPL monoclonal antibodies (mAbs), in the presence and absence of the activator compounds. A single conformational mAb (248-31) fails to immunoprecipitate HPL in the presence of activator compounds and HPL covalently inhibited with diethyl p-nitrophenyl phosphate (DP.HPL). This loss of recognition of HPL by mAb 248-31 was probably due to the motion of the lid, since HPL(-lid) was always recognized in the presence or absence of activator compounds. Furthermore, two other mAbs (81-23 and 146-40) immunoprecipitated HPL similarly whether or not the activator compounds were present. MAb 248-31 therefore specifically recognizes HPL in the closed but not the open conformation.

Antibodies, Monoclonal↗

PubMed2000

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.

Amino Acid Sequence↗