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S Ransac

Publications and source records attributed to S Ransac.

At least 19 recordsLinked to original sources
PubMed2001

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.

Air↗
PubMed2001

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.

Adult↗
PubMed2000

The nucleoside diphosphate kinase activity of DRnm23 is not required for inhibition of differentiation and induction of apoptosis in 32Dcl3 myeloid precursor cells.

DRnm23 belongs to a multigene family which includes nm23-H1, the first bona fide metastasis suppressor gene, nm23-H2, nm23-H4, and nm23-H5. Like nm23-H1, nm23-H2, and nm23-H4, DRnm23 possesses nucleoside diphosphate kinase (NDPK) activity. Upon overexpression in myeloid precursor 32Dcl3 cells, DRnm23 inhibits granulocytic differentiation and promotes apoptosis. Two specific mutants of DRnm23 (H134Q and S136P), at residues required for the NDPK activity, inhibit differentiation and promote apoptosis of 32Dcl3 cells. By contrast, substitution of serine 61 with proline (S61P) or deletion of the RGD domain (DeltaRGD) abrogates the effects of wild-type DRnm23. Like wild-type DRnm23, all four mutants show a predominantly mitochondrial subcellular localization. These studies indicate that the enzymatic activity of DRnm23 is not required for the effects observed in 32Dcl3 cells. Moreover, the inability of the S61P and DeltaRGD DRnm23 mutants to inhibit differentiation and promote apoptosis may be due to defective protein-protein interactions at the mitochondria, the predominant site of DRnm23 subcellular localization.

Animals↗
PubMed2000

Quaternary structure of nucleoside diphosphate kinases.

Nucleoside (NDP) diphosphate kinases are oligomeric enzymes. Most are hexameric, but some bacterial enzymes are tetrameric. Hexamers and tetramers are constructed by assembling identical dimers. The hexameric structure is important for protein stability, as demonstrated by studies with natural mutants (the Killer-of-prune mutant of Drosophila NDP kinase and the S120G mutant of the human NDP kinase A in neuroblastomas) and with mutants obtained by site-directed mutagenesis. It is also essential for enzymic activity. The function of the tetrameric structure is unclear.

Amino Acid Sequence↗
PubMed2000

Neuroblastoma specific effects of DR-nm23 and its mutant forms on differentiation and apoptosis.

DR-nm23 belongs to a gene family which includes nm23-H1, originally identified as a candidate metastasis suppressor gene. Nm23 genes are expressed in different tumor types where their levels have been alternatively associated with reduced or increased metastatic potential. Nm23-H1, -H2, DR-nm23 and nm23-H4 all possess NDP kinase activity. Overexpression of DR-nm23 inhibits differentiation and promotes apoptosis in hematopoietic cells. By contrast, it induces morphological and biochemical changes associated with neural differentiation in neuroblastoma cells. In this study, we show that mutations in the catalytic domain and in the serine 61 phosphorylation site, possibly required for protein-protein interactions, impair the ability of DR-nm23 to induce neural differentiation. Moreover, neuroblastoma cells overexpressing wild-type or mutant DR-nm23 are less sensitive to apoptosis triggered by serum withdrawal. By subcellular fractionation, wild-type and mutant DR-nm23 localize in the cytoplasm and prevalently in the mitochondrial fraction. In co-immunoprecipitation experiments, wild-type DR-nm23 binds other members of nm23 family, but mutations in the catalytic and in the RGD domains and in serine 61 inhibit the formation of hetero-multimers. Thus, the integrity of the NDP kinase activity and the presence of a serine residue in position 61 seem essential for the ability of DR-nm23 to trigger differentiation and to bind other Nm23 proteins, but not for the anti-apoptotic effect in neuroblastoma cells. These studies underline the tissue specificity of the biological effects induced by DR-nm23 expression.

Animals↗
PubMed1999

Inhibition of human gastric and pancreatic lipases by chiral alkylphosphonates. A kinetic study with 1,2-didecanoyl-sn-glycerol monolayer.

Enantiomerically pure alkylphosphonate compounds RR'P(O)PNP (R = CnH2n + 1, R' = OY with Y = Cn'H2n' + 1 with n = n' or n not equal to n'; PNP = p-nitrophenoxy) noted (RY), mimicking the transition state occurring during the carboxyester hydrolysis were synthesized and investigated as potential inhibitors of human gastric lipase (HGL) and human pancreatic lipase (HPL). The inhibitory properties of each enantiomer have been tested with the monomolecular films technique in addition to an enyzme linked immunosorbent assay (ELISA) in order to estimate simultaneously the residual enzymatic activity as well as the interfacial lipase binding. With both lipases, no obvious correlation between the inhibitor molar fraction (alpha 50) leading to half inhibition, and the chain length, R or Y was observed. (R11Y16)s were the best inhibitor of HPL and (R10Y11)s were the best inhibitors of HGL. We observed a highly enantioselective discrimination, both with the pure enantiomeric alkylphosphonate inhibitors as well as a scalemic mixture. We also showed, for the first time, that this enantioselective recognition can occur either during the catalytic step or during the initial interfacial adsorption step of the lipases. These experimental results were analyzed with two kinetic models of covalent as well as pseudo-competitive inhibition of lipolytic enzymes by two enantiomeric inhibitors.

Chromatography, High Pressure Liquid↗
PubMed1994

Crystallization and preliminary X-ray analysis of a lipase from Bacillus subtilis.

Single crystals of the lipase from Bacillus subtilis have been obtained using a mixture of polyethylene glycol 4000 and sodium sulphate solution as the precipitant. The crystals grow at room temperature in two to three weeks in the presence of n-octyl-beta-D-glucoside. They belong to the monoclinic space group C2 with a = 121.20 A, b = 93.19 A, c = 80.96 A, and beta = 110.67 degrees, with four protein molecules per asymmetric unit. The crystals diffract to at least 2.5 A resolution and are suitable for an X-ray structure analysis.

Bacillus subtilis↗
PubMed1994

The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis.

Within the BRIDGE T-project on lipases we investigate the structure-function relationships of the lipases from Bacillus subtilis and Pseudomonas aeruginosa. Construction of an overproducing Bacillus strain allowed the purification of > 100 mg lipase from 30 l culture supernatant. After testing a large variety of crystallization conditions, the Bacillus lipase gave crystals of reasonable quality in PEG-4000 (38-45%), Na2SO4 and octyl-beta-glucoside at 22 degrees C, pH 9.0. A 2.5 A dataset has been obtained which is complete from 15 to 2.5 A resolution. P.aeruginosa wild-type strain PAC1R was fermented using conditions of maximum lipase production. More than 90% of the lipase was cell bound and could be solubilized by treatment of the cells with Triton X-100. This permitted the purification of approximately 50 mg lipase. So far, no crystals of sufficient quality were obtained. Comparison of the model we built for the Pseudomonas lipase, on the basis of sequences and structures of various hydrolases which were found to possess a common folding pattern (alpha/beta hydrolase fold), with the X-ray structure of the P.glumae lipase revealed that it is possible to correctly build the structure of the core of a protein even in the absence of obvious sequence homology with a protein of known 3-D structure.

Amino Acid Sequence↗
PubMed1994

Bacterial lipases.

Many different bacterial species produce lipases which hydrolyze esters of glycerol with preferably long-chain fatty acids. They act at the interface generated by a hydrophobic lipid substrate in a hydrophilic aqueous medium. A characteristic property of lipases is called interfacial activation, meaning a sharp increase in lipase activity observed when the substrate starts to form an emulsion, thereby presenting to the enzyme an interfacial area. As a consequence, the kinetics of a lipase reaction do not follow the classical Michaelis-Menten model. With only a few exceptions, bacterial lipases are able to completely hydrolyze a triacylglycerol substrate although a certain preference for primary ester bonds has been observed. Numerous lipase assay methods are available using coloured or fluorescent substrates which allow spectroscopic and fluorimetric detection of lipase activity. Another important assay is based on titration of fatty acids released from the substrate. Newly developed methods allow to exactly determine lipase activity via controlled surface pressure or by means of a computer-controlled oil drop tensiometer. The synthesis and secretion of lipases by bacteria is influenced by a variety of environmental factors like ions, carbon sources, or presence of non-metabolizable polysaccharides. The secretion pathway is known for Pseudomonas lipases with P. aeruginosa lipase using a two-step mechanism and P. fluorescens lipase using a one-step mechanism. Additionally, some Pseudomonas lipases need specific chaperone-like proteins assisting their correct folding in the periplasm. These lipase-specific foldases (Lif-proteins) which show a high degree of amino acid sequence homology among different Pseudomonas species are coded for by genes located immediately downstream the lipase structural genes. A comparison of different bacterial lipases on the basis of primary structure revealed only very limited sequence homology. However, determination of the three-dimensional structure of the P. glumae lipase indicated that at least some of the bacterial lipases will presumably reveal a conserved folding pattern called the alpha/beta-hydrolase fold, which has been described for other microbial and human lipases. The catalytic site of lipases is buried inside the protein and contains a serine-protease-like catalytic triad consisting of the amino acids serine, histidine, and aspartate (or glutamate). The Ser-residue is located in a strictly conserved beta-epsilon Ser-alpha motif. The active site is covered by a lid-like alpha-helical structure which moves away upon contact of the lipase with its substrate, thereby exposing hydrophobic residues at the protein's surface mediating the contact between protein and substrate.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1993

Topological characterization and modeling of the 3D structure of lipase from Pseudomonas aeruginosa.

Lipase from Pseudomonas aeruginosa is a M(r) 29 kDa protein with a single functional disulfide bond as shown by a shift in electrophoretic mobility after treatment with dithiothreitol and iodoacetamide. Limited proteolysis of lipase with Staphylococcus aureus protease V8 resulted in cleavage after amino acid residues Asp38 and Glu46. Comparison of the lipase amino acid sequence with those of other hydrolases with known 3D structures indicated that the folding pattern might be compatible with the alpha/beta hydrolase fold, thereby allowing us to construct a 3D model which fitted the biochemical properties. The model predicts a catalytic triad consisting of Ser82, Asp229 and His251, and contains a disulfide bond connecting residues Cys183 and Cys235. Residues Asp38 and Glu46 are located at the surface of the enzyme, whereas the disulfide bond is rather inaccessible, which is in agreement with the finding that the protein needed to be partly unfolded before a reduction of the disulfide bond could take place. A striking prediction from the model was the lack of a lid-like alpha-helical loop structure covering the active site which confers to other well-characterized lipases a unique property known as interfacial activation. Experimental determination of lipase activity under conditions where the substrate existed either as monomeric solutions or aggregates confirmed the absence of interfacial activation.

Amino Acid Sequence↗
PubMed1993

Controlling lipase stereoselectivity via the surface pressure.

In the present study, the stereoselectivity of Rhizomucor miehei lipase, lipoprotein lipase, Candida antarctica B lipase, and human gastric lipase towards racemic dicaprin spread as a monolayer at the air-water interface was investigated. For this purpose we have developed a method with which the enantiomeric excess of the residual substrate can be measured in monomolecular films. The stereoselectivity, which is one of the main aspects of enzymic catalysis, was found to depend on the surface pressure of the substrate. With all four lipases tested, low surface pressures enhanced the stereoselectivity while decreasing the enzymes' catalytic activity.

Candida↗
PubMed1992

Rat platelet phospholipase A2. Kinetic characterization using the monomolecular film technique.

We have determined some kinetic parameters of rat platelet phospholipase A2, such as surface pressure dependency and substrate specificity, using the monomolecular film technique. We found that rat platelet phospholipase A2 is very specific for phospholipids having a negatively charged headgroup, no activity was detected when using zwitterionic phospholipids such as phosphatidylcholine. Furthermore, the interfacial pressure window which permits enzyme activity is very narrow as compared to pancreatic phospholipase A2. Maximal enzyme activity is found at 22 mN/m when using 1,2-dilauroylphosphatidylglycerol as substrate. Studies of the competitive inhibition of mixed films containing 2-acylaminophosphatidylglycol show that platelet phospholipase A2 is less sensitive than pancreatic and intestinal phospholipase A2. These results imply that, despite the high degree of sequence similarity, one must be very cautious in extrapolating inhibition data from one phospholipase A2 to similar enzymes from other origins.

Animals↗
PubMed1992

Competitive inhibition of lipolytic enzymes. V. A monolayer study using enantiomeric acylamino analogues of phospholipids as potent competitive inhibitors of porcine pancreatic phospholipase A2.

For the first time, we have shown that a stereospecific interaction occurs between porcine pancreatic phospholipase A2 and a monomolecular film of amidophospholipid used as inhibitor. Direct binding experiments, using radiolabelled phospholipase A2, showed that 13 times more enzyme was bound to phospholipid films of the L series by comparison with films of the D series. These results were confirmed by indirect binding studies using re-spreading experiments. Kinetic studies of the porcine pancreatic PLA2, using enantiomeric acyl-amino phospholipid analogues, have shown that: (1) inhibitors of the L series are more potent than inhibitors of the D series, (2) inhibitors having a negative charge are more potent than zwitterionic inhibitors, (3) inhibitory power values are greater when evaluated in micellar system than in a the monolayer system, (4) the inhibitory power increases continuously with surface pressure.

Animals↗
PubMed1991

Inactivation of pancreatic and gastric lipases by tetrahydrolipstatin and alkyl-dithio-5-(2-nitrobenzoic acid). A kinetic study with 1,2-didecanoyl-sn-glycerol monolayers.

We studied the covalent inhibition of lipases by the monolayer technique. We report the inactivation of porcine pancreatic and human and rabbit gastric lipases, acting on mixed monomolecular films of dicaprin containing tetrahydrolipstatin or new hydrophobic disulfide compounds, which can be described as a 'poisoned-interface' system. A kinetic model is presented for depicting the covalent inactivation of lipolytic enzymes at a lipid/water interface. The stoichiometry of the interfacial situation can be described as follows: one lipase molecule embedded among 10(5) substrate molecules will be inactivated to half its initial velocity by the presence of 10 tetrahydrolipstatin molecules. This inactivation was independent of the surface pressure. When tested in the form of mixed films, all the disulfide compounds investigated specifically reduced the hydrolysis of 1,2-didecanoyl-sn-glycerol films by gastric lipases, but did not affect hydrolysis by pancreatic lipase. With this poisoned-interface system, tetrahydrolipstatin was found to be the most potent inactivator, whereas disulfide compounds showed a higher degree of selectivity than tetrahydrolipstatin.

Animals↗