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PubMed2004

Carboxylesterase 3 (EC 3.1.1.1) is a major adipocyte lipase.

Hydrolysis of triglycerides is central to energy homeostasis in white adipose tissue (WAT). Hormone-sensitive lipase (HSL) was previously felt to mediate all lipolysis in WAT. Surprisingly, HSL-deficient mice show active HSL-independent lipolysis, suggesting that other lipase(s) also mediate triglyceride hydrolysis. To clarify this, we used functional proteomics to detect non-HSL lipase(s) in mouse WAT. After cell fractionation of intraabdominal WAT, most non-HSL neutral lipase activity is localized in the 100,000 x g infranatant and fat cake fractions. By oleic acid-linked agarose chromatography of infranatant followed by elution in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid gradient, we identified two peaks of esterase activity using p-nitrophenyl butyrate as a substrate. One of the peaks contained most of the lipase activity. In the corresponding fractions, gel permeation chromatography and SDS-PAGE, followed by tandem mass spectrometric analysis of excised Coomassie Blue-stained peptides, revealed carboxylesterase 3 (triacylglycerol hydrolase (TGH); EC 3.1.1.1). TGH is also the principle lipase of WAT fat cake extracts. Partially purified WAT TGH had lipase activity as well as lesser but detectable neutral cholesteryl ester hydrolase activity. Western blotting of subcellular fractions of WAT and confocal microscopy of fibroblasts following in vitro adipocytic differentiation are consistent with a distribution of TGH to endoplasmic reticulum, cytosol, and the lipid droplet. TGH is responsible for a major part of non-HSL lipase activity in WAT in vitro and may mediate some or all HSL-independent lipolysis in adipocytes.

Adipocytes↗
PubMed1986

Meal-induced changes in lipoprotein lipase activity in brown fat and other tissues of rats.

Brown fat thermogenesis is increased after a single test meal. This study was conducted to determine whether lipoprotein lipase activity is higher in brown adipose and other tissues after a single large meal. Rats were trained to eat two large meals per day. Two hours after consuming a test meal, lipoprotein lipase activity was measured in interscapular brown adipose tissue, retroperitoneal and epididymal white adipose tissue, gastrocnemius and soleus skeletal muscles and heart. After a high carbohydrate test meal, lipoprotein lipase activity in white adipose tissue pads was higher (P less than 0.05) and that in brown adipose tissue was lower (P less than 0.05) than in these tissues from the meal-deprived group. Muscle lipoprotein lipase did not change significantly. A high fat test meal did not significantly alter lipoprotein lipase activity in brown adipose tissue, white adipose tissue, gastrocnemius or soleus when compared to the meal-deprived control, but heart lipoprotein lipase activity was significantly elevated. These findings indicate that after a single test meal lipoprotein lipase activity in brown adipose tissue is not higher than that from the meal-deprived group and therefore, lipoprotein lipase may not play a rate-limiting role in moving free fatty acids into this tissue in the postprandial state.

Adipose Tissue, Brown↗
PubMed1996

The crystal structure of lipase II from Rhizopus niveus at 2.2 A resolution.

The crystal and molecular structure of Lipase II from Rhizopus niveus was analyzed using X-ray single crystal diffraction data at a resolution of 2.2 A. The structure was refined to an R-factor of 0.19 for all available data. This lipase was purified and crystallized as Lipase I, which contains two polypeptide chains combined through non-covalent interaction. However, during crystal growth, Lipase I was converted to Lipase II, which consists of a single polypeptide chain of 269 amino acid residues, by limited proteolysis. The structure of Lipase II shows a typical alpha/beta hydrolase fold containing the so-called nucleophilic elbow. The catalytic center of this enzyme is analogous to those of other neutral lipases and serine proteases. This catalytic center is sheltered by an alpha-helix lid, which appears in neutral lipases, opening the active site at the oil-water interface.

Amino Acid Sequence↗
PubMed1996

Substitutions of Ser for Asn-163 and Pro for Leu-264 are important for stabilization of lipase from Pseudomonas aeruginosa.

The lipase gene from Pseudomonas aeruginosa was randomly mutated by error-prone PCR to obtain thermostable mutants, followed by screening for thermostable mutant lipases. Out of about 2,600 transformants, four thermostable clones were obtained. Their nucleotide sequences showed that they had two or three amino acid substitutions. Analysis of the thermal stabilization of these mutant lipases indicated that Asn-163 to Ser and Leu-264 to Pro mutations were essential for the increased stability of the lipase. We expressed a mutant lipase (StLipA-5) having only the Asn-163 to Ser mutation and another (StLipA-6) having only the Leu-264 to Pro mutation in P. aeruginosa PAO1161, purified them, and then confirmed that the temperature which causes a 50% decrease in the activity of the non-treated enzyme on treatment for 30 min was increased by 1.5 and 3 degrees C, respectively, compared to the wild-type enzyme. However, the thermal stability of the mutant lipase (StLipA-7) having both mutations was increased only by 2.5 degrees C. These mutant lipases were stabilized through a decrease in activation entropy. Kinetic studies showed that the Kcat/K(m) values of StLipA-5, StLipA-6, and StLipA-7 were decreased by 14.4, 52.9, and 26.0%, respectively. Interestingly, the pH-stabilities of StLipA-6 and StLipA-7 were also increased, especially at alkaline pH. Based on these results, the tertiary structure and mechanism of stabilization of the lipase were discussed.

Aspartic Acid↗
PubMed1986

Purification and general properties of a metal-insensitive lipase from Rhizopus japonicus NR 400.

Lipase [triacylglycerol lipase, EC 3.1.1.3] has been purified to homogeneity from Rhizopus japonicus NR 400 by chromatography on hydroxylapatite, octyl-Sepharose and Sephacryl S-200. It showed a molecular weight of about 30,000 by SDS-PAGE and a specific activity of 68,900 units/mg protein. The enzyme catalyzed the hydrolysis of tricapryn and tricaprylin rapidly in comparison with other triglycerides. This lipase had an optimum pH of around 5, and albumin enhanced its activity between pH 3 and 8. The composition of fatty acids liberated from linseed oil by the lipase was similar to that in the case of pancreatic lipase. The lipase activity was not affected by the addition of 1 mM metal ions or bile salts. Stimulation of the lipase activity was observed upon addition of albumin to the reaction mixture. Immunotitration experiments were also performed with antibodies raised against the purified lipase.

Albumins↗
PubMed1998

Random mutagenesis on the Pseudomonas lipase activator protein, LipB: exploring amino acid residues required for its function.

LipB, lipase activator protein from Pseudomonas aeruginosa TE3285, specifically recovers the enzymatic activity of denatured inactive lipase. To find important amino acid residues of LipB in this reactivation, random mutagenesis using error-prone PCR was performed on a gene encoding the functional region of LipB. The resultant DNA library was introduced into the lipase expression system using Escherichia coli, and LipB mutants lacking lipase activity were selected by two screening procedures. First, on agar plates containing tributyrin as a substrate for lipase, single colonies lacking active lipase secretion were selected as clones missing the active LipB. Second, to exclude nonsense and frameshift mutants, the molecular size of LipB in the given clones was confirmed by Western blotting. From the selected mutants, of which multiple residues are replaced, five single-residue substituted mutants were newly prepared. Consequently, Y99C, Y99H, S102R and R115C mutants formed no detectable complex with the lipase and lost the in vitro reactivation activity. In the case of Y99C and R115C, their single cysteine residue formed the intermolecular disulfide bridge. Thus, Tyr99 and Arg115 are likely to exist on the molecular surface of LipB, and are candidates for residues that make direct interaction with the denatured lipase in the reactivation process.

Bacterial Proteins↗
PubMed1998

Stability of porcine and microbial lipases to conditions that approximate the proventriculus of young birds.

In vitro experiments were conducted to characterize the activity and the stability of lipase from animal (crude porcine, CPL; lyophilized porcine, LPL), fungal (Rhizopus arrhizus, RAL; Aspergillus niger, ANL), and bacterial (two Pseudomonas spp., PL1, PL2; and Chromobacterium viscosum, CVL) sources when exposed to conditions associated with the glandular stomach. Activity was measured at pH 3 to 8, 40 C and then monitored in response to temperature (40 C), time of exposure (0 and 30 min), pH (3 and 7), and pepsin level (5, 50, and 500 U/mL). All lipases except ANL and CVL had maximum activity at pH 7 to 8. The optimal pH for ANL and CVL were 5 and 6 to 8, respectively. Exposure of lipases to 40 C and pH 7 for 30 min reduced the activity of all lipases except ANL. In contrast, 40 C increased ANL activity 2.5-fold. Although activity of all lipases was reduced by exposure to pH 3, it was nearly eliminated for CPL and LPL. Pepsin concentration had only minor effects on lipase activity and then only at high concentration. The results demonstrate that bacterial lipases (PL1, PL2, and CVL) and ANL are more stable under conditions that approximate the glandular stomach and may explain why dietary porcine lipase has been ineffective in preventing fat malabsorption in previous in vivo studies.

Animals↗
PubMed1989

Pancreatic lipase and colipase activity increase in pancreatic acinar tissue of diabetic rats.

We studied the lipase and colipase activity in pancreatic acinar tissue of insulin-deficiency and insulin-resistance obese Zucker rats (fa/fa). After injection of streptozotocin (STX 75 mg/kg) in normal Sprague-Dawley rats, the activity of lipase and colipase in pancreatic acinar tissue was increased by approximately 100%, the increase in colipase occurring 3 days later than that of lipase. At the same time, the amylase activity was decreased by 98%. Injection of alloxan (125 mg/kg) induced a similar change of pancreatic enzyme pattern, with amylase activity strongly reduced by 79% and activity of lipase and colipase increased 20.5 and 18.6%, respectively. Correction of the diabetic state with insulin (1 U/100 g/day) reversed the activity of these enzymes to their prediabetic levels. Administration of insulin (6 U/100 g/day) to normal Sprague-Dawley rats increased the activity of amylase as well as lipase and colipase, whereas injection of glucagon (0.3 mg/100 g/day) decreased the activity of amylase and colipase but had no significant effect on lipase activity. In the obese Zucker rats (fa/fa), the activity of lipase and colipase at onset of obesity (5 weeks of age) was lower than that in their lean littermates (fa/o). Thereafter the activity of the two proteins increased with age, being 40% higher in the fa/fa rat than in the fa/o rat at age 7 weeks. During the same period, amylase activity decreased. These results indicate that pancreatic lipase and colipase activity are increased following either insulin deficiency or insulin resistance in rats by a mechanism related to the changed levels of insulin.

Amylases↗
PubMed2006

MfLIP1, a gene encoding an extracellular lipase of the lipid-dependent fungus Malassezia furfur.

Malassezia furfur is a dimorphic fungus and a member of the normal cutaneous microflora of humans. However, it is also a facultative pathogen, associated with a wide range of skin diseases. One unusual feature of M. furfur is an absolute dependency on externally provided lipids which the fungus hydrolyses by lipolytic activity to release fatty acids necessary for both growth and pathogenicity. In this study, the cloning and characterization of the first gene encoding a secreted lipase of M. furfur possibly associated with this activity are reported. The gene, MfLIP1, shows high sequence similarity to other known extracellular lipases, but is not a member of a lipase gene family in M. furfur. MfLIP1 consists of 1464 bp, encoding a protein with a molecular mass of 54.3 kDa, a conserved lipase motif and an N-terminal signal peptide of 26 aa. By using a genomic library, two other genes were identified flanking MfLIP1, one of them encoding a putative secreted catalase, the other a putative amine oxidase. The cDNA of MfLIP1 was expressed in Pichia pastoris and the biochemical properties of the recombinant lipase were analysed. MfLip1 is most active at 40 degrees C and the pH optimum was found to be 5.8. The lipase hydrolysed lipids, such as Tweens, frequently used as the source of fatty acids in M. furfur media, and had minor esterase activity. Furthermore, the lipase is inhibited by different bivalent metal ions. This is the first molecular description of a secreted lipase from M. furfur.

Fungal Proteins↗
PubMed1996

Pancreatin preparations used in the treatment of cystic fibrosis--lipase content and in vitro release.

BACKGROUND: Pancreatic extracts are essential in the treatment of the majority of cystic fibrosis patients. The clinical response to different preparations is often unpredictable and at present there is no sure method of determining the best preparation for a particular patient. METHODS: Creon, Nutrizym GR, Pancrease and the high-lipase versions, Creon 25,000, Nutrizym 22 and Pancrease HL, were investigated for lipase content and resistance to simulated gastric conditions. The rates of lipase release in response to pH change, bile salts and duodenal solids were investigated. The stability of lipase and its binding to duodenal solids were also investigated. RESULTS: Declared values for lipase content were exceeded in all preparations. All preparations were acid resistant. The release of lipase in response to pH change showed notable differences in release rates. After 20 min at pH 5.5, Creon released three times the amount of lipase compared with Pancrease, the other preparations coming within the range. Above pH 5.75, the release rates were comparable amongst the preparations. Bile salts influenced release variably whilst release in a solid-rich duodenal fluid was much slower than in buffers. The released lipase was susceptible to proteolysis and pH-dependent binding to duodenal solids; these effects may compromise lipolysis. CONCLUSIONS: These results show some factors contributing to variable clinical responses to pancreatic supplements. Improvements may result if a patient is assessed on different preparations.

Bile Acids and Salts↗
PubMed1993

Role of the lipB gene product in the folding of the secreted lipase of Pseudomonas glumae.

The LipB protein of Pseudomonas glumae is essential for the production of active extracellular lipase encoded by the lipA gene. When lipase is overproduced in P. glumae in the absence of a functional lipB gene, the enzyme accumulates intracellularly in an inactive conformation. Heterologous expression of the lipase in Pseudomonas aeruginosa, Bacillus subtilis and Escherichia coli indicated that LipB is not directly involved in the translocation of the lipase across the inner or outer membrane. However, the presence of LipB was essential for obtaining active lipase and had a profound influence on the stability of the protein to proteolytic degradation. Inactive lipase, produced in the absence of LipB could be activated in vitro by unfolding and refolding, which demonstrates that LipB activity is not responsible for an essential covalent modification of the enzyme. We propose that LipB is a lipase-specific foldase. Furthermore, proper folding of the lipase in the periplasm appears to be essential for Xcp-mediated translocation across the outer membrane.

Bacterial Proteins↗
PubMed1994

The Pseudomonas fluorescens lipase has a C-terminal secretion signal and is secreted by a three-component bacterial ABC-exporter system.

Both Pseudomonas aeruginosa and Pseudomonas fluorescens secrete a lipase into the extracellular medium. Unlike the lipase of P. aeruginosa, the lipase produced by P. fluorescens does not contain any N-terminal signal sequence. We show that the P. fluorescens lipase is secreted through the signal peptide-independent pathway of the alkaline protease that we previously identified in P. aeruginosa. Secretion of this protease (AprA) is dependent on the presence of three genes located adjacent to the aprA gene, aprD, aprE and aprF. The three secretion functions permit an efficient secretion of P. fluorescens lipase. Inactivation of one of them (AprE) prevented this secretion. In Escherichia coli, the three proteins AprD, AprE, AprF are necessary and sufficient for efficient secretion of lipase to the extracellular medium. The secretion signal is located within the C-terminal part of the lipase sequence and can promote efficient secretion of a passenger protein. Thus the P. fluorescens lipase secretion system belongs to the group of the three-component bacterial ABC-exporter systems.

ATP-Binding Cassette Transporters↗
PubMed1990

Lipases catalyse hydrolysis of fatty acid anhydrides.

Regio-specific and non-regio-specific lipases from mammals and microorganisms catalyse the hydrolysis of short, medium and long-chain fatty acid anhydrides. All the lipases tested in the present study can catalyse the hydrolysis of pure fatty acid anhydrides more efficiently than that of glycerol tributyrate. Molecular turnovers more than four times higher than that measured using glycerol tributyrate were calculated. The presence of 0.5% (by mass) anhydride in a triacylglyceride can double the initial rate of proton release during enzymatic hydrolysis. This should be taken into account when testing the chain specificity of a lipase for various synthetic substrates. Lipase inhibition was found to be associated very often with anhydride hydrolysis. The inhibition rates depended on the anhydride and the origin of the lipase. Inhibition of lipase activity is probably due to the formation of a poorly reversible acyl-lipase complex which differs from the classical fully reversible acyl-lipase complex at the catalytic centre.

Anhydrides↗
PubMed1994

The C-terminus of lipoprotein lipase is essential for biological function but contains no domain for glycosylphosphatidylinositol anchoring.

In this study we present evidence that the C-terminus of lipoprotein lipase contains no glycosylphosphatidylinositol addition signal and is therefore not a glycosylphosphatidylinositol-anchored protein. Furthermore, we present additional evidence that the C-terminus of lipoprotein lipase is essential for biological function. Flow cytometric analysis and enzyme-activity monitoring experiments revealed no pool of lipoprotein lipase releasable by phosphatidylinositol-specific phospholipase present on the membrane of COS cells transfected with the human lipoprotein lipase gene while, in contrast, a heparin-releasable pool could be demonstrated. [14C]Ethanolamine, a constituent of the glycosylphosphatidylinositol anchor, was not incorporated into lipoprotein lipase during metabolic labeling. C-terminal deletion mutants were constructed and expressed in COS cells to investigate the presence of glycosylphosphatidylinositol addition signal on the C-terminus of human lipoprotein lipase (LPL). The specific activities of the mutants M442 [des-(Leu443-Gly448)-LPL] and M437 [des-(Cys438-Gly448)-LPL] were 78% and 59%, respectively, less than the wild type, while the M432 mutant [des-(Ala433-Gly449)-LPL] was catalytically inactive. Determination of the stability of the mutants revealed a decreased stability of the M437, compared with wild-type, whereas M442 showed the same stability. Flow cytometric analysis showed sustained membrane expression for all mutants including the inactive M432 mutant. These results suggest that the C-terminus of lipoprotein lipase is essential for maintaining intact catalytic activity but is not involved in any posttranslational proteolytic processing, including cleavage of a glycosylphosphatidylinositol addition signal. We therefore conclude that membrane-binding of the lipase is not mediated by such anchoring.

Amino Acid Sequence↗
PubMed2005

Should serum pancreatic lipase replace serum amylase as a biomarker of acute pancreatitis?

BACKGROUND: Serum pancreatic lipase may improve the diagnosis of pancreatitis compared to serum amylase. Both enzymes have been measured simultaneously at our hospital allowing for a comparison of their diagnostic accuracy. METHODS: Seventeen thousand five hundred and thirty-one measurements of either serum amylase and or serum pancreatic lipase were made on 10 931 patients treated at a metropolitan teaching hospital between January 2001 and May 2003. Of these, 8937 were initially treated in the Emergency Department. These results were collected in a database, which was linked by the patients' medical record number to the radiology and medical records. Patients with either an elevated lipase value or a discharge diagnosis of acute pancreatitis had their radiological diagnosis reviewed along with their biochemistry and histology record. The diagnosis of acute pancreatitis was made if there was radiological evidence of peripancreatic inflammation. RESULTS: One thousand eight hundred and twenty-five patients had either elevated serum amylase and or serum pancreatic lipase. The medical records coded for pancreatitis in a further 55 whose enzymes were not elevated. Three hundred and twenty of these had radiological evidence of acute pancreatitis. Receiver operator characteristic analysis of the initial sample from patients received in the Emergency Department showed improved diagnostic accuracy for serum pancreatic lipase (area under the curve (AUC) 0.948) compared with serum amylase (AUC, 0.906, P < 0.05). A clinically useful cut-off point would be at the diagnostic threshold; 208 U/L (normal <190 U/L) for serum pancreatic lipase and 114 U/L (normal 27-100 U/L) for serum amylase where the sensitivity was 90.3 cf., 76.8% and the specificity was 93 cf., 92.6%. 18.8% of the acute pancreatitis patients did not have elevated serum amylase while only 2.9% did not have elevated serum pancreatic lipase on the first emergency department measurement. CONCLUSION: It is concluded that serum pancreatic lipase is a more accurate biomarker of acute pancreatitis than serum amylase.

Acute Disease↗
PubMed1998

Effect of sham feeding and acute suppression of acid secretion on human gastric lipase secretion.

OBJECTIVE: Gastric lipase and gastric acid are secreted simultaneously. The aim of this study was to investigate whether the acid interferes with the lipase secretion. The secretion of human gastric lipase was studied during blockade of gastric acid secretion and modified sham feeding to estimate the impact of these conditions on both gastric lipase enzyme activity and immunoreactivity. METHODS: Eight healthy volunteers were intubated with a nasogastric tube. We examined gastric aspirates for the amount and activity of lipase secretion during basal conditions, after blockade of acid secretion with a proton pump inhibitor (omeprazole iv. infusion), and in response to sham feeding (chewing gum) during the blockade. RESULTS: The amount of secreted gastric lipase was unaffected by blockade of acid secretion and increased significantly after sham feeding (169.9+/-35.7 microg/15 min to 348.1+/-79.2 microg/15 min; p < 0.01). Likewise, the output of enzyme activity increased after sham feeding (0.63+/-0.09 kU/15 min to 1.52+/-0.36 kU/15 min; p < 0.03). The concentration of enzyme activity remained unchanged by blockade of acid secretion, whereas the output of enzyme activity was decreased, probably because of reduced volume secretion or denaturation and conformational changes of the enzyme. Plasma concentrations of gastrin increased in response to blockade of acid secretion (basal 9.6+/-1.4 pmol/L to 13.3+/-2.9 pmol/L; p < 0.02). CONCLUSIONS: Gastric acid secretion is not a prerequisite for gastric lipase secretion. Lipase enzyme activity, though, is sensitive to anacidic conditions.

Adult↗
PubMed1992

Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.

Amino Acid Sequence↗
PubMed1998

Development of a lipase fermentation process that uses a recombinant Pseudomonas alcaligenes strain.

Pseudomonas alcaligenes M-1 secretes an alkaline lipase, which has excellent characteristics for the removal of fatty stains under modern washing conditions. A fed-batch fermentation process based on the secretion of the alkaline lipase from P. alcaligenes was developed. Due to the inability of P. alcaligenes to grow on glucose, citric acid and soybean oil were applied as substrates in the batch phase and feed phase, respectively. The gene encoding the high-alkaline lipase from P. alcaligenes was isolated and characterized. Amplification of lipase gene copies in P. alcaligenes with the aid of low- and high-copy-number plasmids resulted in an increase of lipase expression that was apparently colinear with the gene copy number. It was found that overexpression of the lipase helper gene, lipB, produced a stimulating effect in strains with high copy numbers (> 20) of the lipase structural gene, lipA. In strains with lipA on a low-copy-number vector, the lipB gene did not show any effect, suggesting that LipB is required in a low ratio to LipA only. During scaling up of the fermentation process to 100 m3, severe losses in lipase productivity were observed. Simulations have identified an increased level of dissolved carbon dioxide as the most probable cause for the scale-up losses. A large-scale fermentation protocol with a reduced dissolved carbon dioxide concentration resulted in a substantial elimination of the scale-up loss.

Amino Acid Sequence↗