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PubMed2000

Cloning, sequencing and expression in Escherichia coli of a thermophilic lipase from Bacillus thermoleovorans ID-1.

A thermophilic lipase of Bacillus thermoleovorans ID-1 was cloned and sequenced. The lipase gene codes 416 amino acid residues and contains the conserved pentapeptide Ala-X-Ser-X-Gly as other Bacillus lipase genes. The optimum temperature of the lipase is 75 degrees C, which is higher than other known Bacillus lipases. For expression in Escherichia coli, the lipase gene was subcloned in pET-22b(+) vector with a strong T7 promoter. Lipase activity was approximately 1.4-fold greater than under the native promoter.

Amino Acid Sequence↗
PubMed2004

Faecal elastase-1 test is superior to faecal lipase test in the assessment of exocrine pancreatic function in cystic fibrosis.

BACKGROUND AND AIMS: Direct tests are characterized by the highest sensitivity and specificity. However, their practical use, especially in children, is limited. Among the indirect tests, the highest sensitivity and specificity was documented for faecal elastase-1 test, yet the value of faecal lipase test in cystic fibrosis (CF) has not been defined. Therefore, the aim of the present study was to compare the sensitivity and the specificity of the faecal lipase test to the faecal elastase-1 test in the assessment of exocrine pancreatic function in children with CF. METHODS: The study comprised 90 CF patients and 95 healthy subjects (HS). In all subjects, faecal elastase-1 concentrations (ELISA) and lipase activities (ELISA) were measured. The presence of pancreatic insufficiency was documented by the determination of faecal fat excretion in 78 pancreatic insufficient and by the secretin-cholecystokinin test in 12 CF patients without steatorrhoea. Sensitivity and specificity of the faecal elastase-1 test and faecal lipase test were analysed and, in 50 HS, sample-to-sample and day-to-day variations were determined. RESULTS: With cut-off levels providing the same specificity for both tests (95.8%), the sensitivity of the faecal elastase-1 test (91.1%) was significantly higher (p < 0.0036) than that of the faecal lipase test (76.7%). Sample-to-sample (mean +/- SEM: 13.2 +/- 1.2% vs 23.4 +/- 2.2%) and day-to-day variations (mean +/- SEM: 16.3 +/- 1.2% vs 32.5 +/- 2.6%) were significantly lower (p < 0.0001) for elastase-1 than for lipase measurements. CONCLUSION: Among indirect tests, faecal elastase-1 test is superior to faecal lipase test in the assessment of exocrine pancreatic function in cystic fibrosis.

Adolescent↗
PubMed2004

Comparison of amylase and lipase activities in serum and plasma of dogs.

BACKGROUND: Amylase and lipase activities are most often determined in serum, although heparinized plasma is more convenient to obtain and is used for many routine biochemical analyses. OBJECTIVE: The purpose of this study was to compare amylase and lipase activities in serum and plasma of dogs and to determine whether either specimen type is acceptable for analysis. METHODS: Serum and heparinized plasma were obtained from 101 randomly selected dogs and analyzed in parallel for alpha-amylase and lipase. Results were compared using Passing-Bablock regression, Bland-Altman difference plots, and correlation analysis. RESULTS: There was a high correlation between the results obtained from serum and those from plasma. Regressions (with 95% confidence intervals in parentheses) were as follows: lipase(plasma) = 0.984 (0.976/0.995) Chi lipase(serum) - 0.9 (2.9/0.7) (r =.999); a-amylase(plasma) = 1.003 (0.977/1.032) Chi alpha-amylase(serum) - 1.9 ( 20.7/23.3) (r =.991). Mean differences (serum - plasma) were 8 U/L and 4 U/L for lipase and alpha-amylase, respectively. Classification of results as normal or abnormal did not differ according to specimen type. CONCLUSION: In dogs, lipase and alpha-amylase activities can be determined with the same level of accuracy in serum and in heparinized plasma.

Amylases↗
PubMed1999

High-level formation of active Pseudomonas cepacia lipase after heterologous expression of the encoding gene and its modified chaperone in Escherichia coli and rapid in vitro refolding.

The lipase from Pseudomonas cepacia ATCC 21808 (recently reclassified as Burkholderia cepacia) is widely used by organic chemists for enantioselective synthesis and is manufactured from recombinant P. cepacia harboring on a plasmid the clustered genes for lipase and its chaperone. High levels of expression of inactive lipase (40%) in Escherichia coli were achieved with pCYTEXP1 under the control of the strong, temperature-inducible lambdaPRL promoter. However, no overexpression of the lipase chaperone was achieved in E. coli. Thus, chemical refolding of inactive lipase in the absence of its chaperone yielded only 25 U/mg, compared to 3,470 U of the purified lipase secreted by recombinant P. cepacia per mg. Sequence analysis of the chaperone revealed a high GC content (>90%) in the 5' region of the gene and the presence of a putative membrane anchor at the N terminus. Hence, the 5' region of the gene was replaced by a synthetic fragment, and the putative membrane anchor was removed by deletion of the first 34 or 70 N-terminal amino acids. Only truncation of the gene led to overexpression of the chaperone (up to 60%) in E. coli. With this chaperone, it was possible to obtain for the first time in a simple refolding procedure a highly active Pseudomonas lipase (classes I and II) expressed in E. coli with a specific activity of up to 4,850 U/mg and a yield of 314,000 U/g of E. coli wet cells.

Amino Acid Sequence↗
PubMed1966

Inhibition of microbial lipases by fatty acids.

Addition of lard or sodium oleate to the medium used for lipase production by Pseudomonas fragi resulted in a decreased accumulation of lipase in the culture supernatant fluid without affecting cell growth. The production and activity of lipase was inhibited by lard, sodium oleate, and the salts of other unsaturated fatty acids. Some divalent cations, Tweens, lecithin, and bovine serum prevented oleate inhibition, but did not reverse it. Similar inhibitory actions were observed with Geotrichum candidum lipase, but not with a staphylococcal lipase or pancreatic lipase. A protective effect by protein in crude enzyme preparations is indicated. The ability of oleate to lower surface tension does not appear to be related to its ability to inhibit lipase.

Chromatography, Gas↗
PubMed1995

Lipopolysaccharide regulation of lipoprotein lipase expression in murine macrophages.

The enzyme lipoprotein lipase is expressed in a number of cell types and plays a central role in lipid metabolism. Multiple factors regulate its expression in a tissue-specific manner. In murine macrophages, lipopolysaccharide inhibits lipoprotein lipase enzyme activity. The current work examines this process in the established J774 macrophage line and primary peritoneal macrophages from endotoxin-sensitive (C3HeB/Fej) and endotoxin-resistant (C3H/Hej) murine strains. Lipopolysaccharide inhibition of macrophage lipoprotein lipase occurred at the enzyme and mRNA levels in a time- and concentration-dependent manner. Cells from endotoxin-resistant animals maintained their expression of lipoprotein lipase following treatment with lipopolysaccharide. Results of gel retention assays showed that lipopolysaccharide treatment of the J774 macrophages altered the level of nuclear proteins recognizing and binding the lipoprotein lipase promoter DNA. Nuclear extracts from resting J774 cells contained proteins which bound specifically to the octamer motif and to the CAAT box within the lipoprotein lipase promoter. Exposure of the J774 cells to lipopolysaccharide for 16 h increased the level of protein-octamer DNA complexes. Similar responses were obtained in endotoxin-sensitive, but not endotoxin-resistant, primary macrophages following in vitro treatment with lipopolysaccharide. This finding suggests that transcriptional events may contribute to the lipopolysaccharide regulation of macrophage lipoprotein lipase expression.

Animals↗
PubMed1986

Purification of extracellular lipase from Pseudomonas aeruginosa.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) was excreted by Pseudomonas aeruginosa PAC1R during the late logarithmic growth phase. Characterization of cell-free culture supernatants by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of significant amounts of lipopolysaccharide, part of which seemed to be tightly bound to lipase. After concentration of culture supernatants by ultrafiltration, lipase-lipopolysaccharide complexes were dissociated by treatment with EDTA-Tris buffer and subsequent sonication in the presence of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The solubilized lipase was purified by isoelectric focusing in an agarose gel containing the same detergent; the lipase activity appeared in a single peak corresponding to a distinct band in the silver-stained gel. The isoelectric point was 5.8. Analysis of purified lipase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and scanning revealed an apparent molecular weight of 29,000 and a specific activity of 760 mu kat/mg of protein. Estimations based on these data showed that a single P. aeruginosa cell excreted about 200 molecules of lipase, each having a molecular activity of 2.2 X 10(4) per s.

Bacterial Proteins↗
PubMed1994

The lipA gene of Serratia marcescens which encodes an extracellular lipase having no N-terminal signal peptide.

The lipA gene encoding an extracellular lipase was cloned from the wild-type strain of Serratia marcescens Sr41. Nucleotide sequencing showed a major open reading frame encoding a 64.9-kDa protein of 613 amino acid residues; the deduced amino acid sequence contains a lipase consensus sequence, GXSXG. The lipase had 66 and 56% homologies with the lipases of Pseudomonas fluorescens B52 and P. fluorescens SIK W1, respectively, but did not show any overall homology with lipases from other origins. The Escherichia coli cells carrying the S. marcescens lipA gene did not secrete the lipase into the medium. The S. marcescens lipase had no conventional N-terminal signal sequence but was also not subjected to any processing at both the N-terminal and C-terminal regions. A specific short region similar to the regions of secretory proteins having no N-terminal signal peptide was observed in the amino acid sequence. Expression of the lipA gene in S. marcescens was affected by the carbon source and the addition of Tween 80.

Amino Acid Sequence↗
PubMed1990

Dibutyryl cAMP-induced increases in triacylglycerol lipase activity in developing L8 myotube cultures.

Triacylglycerol (TG) lipase activity, with an alkaline pH optimum, has been identified in the cellular fraction of L8 myotube cultures. This TG lipase activity was stimulated by serum and inhibited by NaCl and protamine sulfate. These characteristics have been classically described for lipoprotein lipase. It was possible to increase the activity of this TG lipase three- to five-fold by incubating the cells with dibutyryl cAMP. Maximal enzyme activity was observed 16 h following the addition of 10-100 microM dibutyryl cAMP to the cultured cells. Enzyme activity returned to control levels 24 h after removal of the nucleotide from the culture medium. Serum-sensitive alkaline TG lipase activity was also identified in five other myotube preparations of cultured muscle cells. The highest levels of activity were found in rat skeletal muscle primary, H9, and L6 cell types. The finding that dibutyryl cAMP is an effective inducer of alkaline TG lipase activity provides us with a valuable model to investigate mechanisms regulating synthesis, compartmentalization, and transport of lipoprotein lipase in muscle.

Animals↗
PubMed1991

Restoration of lipoprotein lipase activity in insulin-deficient rats by insulin infusion is tissue-specific.

The activity of lipoprotein lipase was measured in white and brown adipose tissues, red vastus lateralis muscle, and heart of rats that have been insulin deficient (streptozotocin, 75 mg.kg-1) for 2 weeks, and that have then received implants of insulin-delivering minipumps (17 U.kg-1.day-1) for 1 or 4 days. Normal glycemia was restored in insulin-deficient animals after 4 days of insulin treatment. Hypertriglyceridemia, but not hypercholesterolemia, was reversed after 4 days of insulin infusion. After 2 weeks of insulin deficiency, fasting lipoprotein lipase activity was lowered in all tissues studied. In white adipose tissue, lipoprotein lipase decreased to 50% of control values. After a single day of insulin infusion, even if tissue weight has not yet been greatly affected, total activity was completely restored to control levels. Enzyme activity in brown adipose tissue was also depressed in deficient animals, and insulin infusion was followed by a slow recovery of activity, to a level intermediate between those of control and insulin-deficient groups. Insulin status had milder effects on lipoprotein lipase activity in vastus lateralis muscle than in the adipose tissues. Deficient rats displayed 60% less activity than controls, and 4 days of hormone infusion only partially restored enzyme activity. There was a large loss of lipoprotein lipase in the heart following 2 weeks of insulin depletion, which was not counteracted by hormone infusion. Thus the speed and extent of recovery of lipoprotein lipase activity following hormone replacement in insulin-deficient animals varied widely among tissues. These findings suggest that insulin is part of the factors that determine the tissue specificity of lipoprotein lipase regulation.

Adipose Tissue↗
PubMed1993

Acute diabetes does not reduce heparin-releasable lipoprotein lipase activity in perfused hearts from Wistar-Kyoto rats.

The induction of diabetes (3-5 days duration) in Wistar-Kyoto rats by the administration of streptozotocin (100 mg/kg) did not increase plasma concentrations of triacylglycerols or free fatty acids, and did not reduce heparin-releasable (functional) lipoprotein lipase activity in perfused hearts. By comparison, diabetic Sprague-Dawley rats were characterized as having hypertriglyceridemia and decreased heparin-releasable lipoprotein lipase activity in perfused hearts. Therefore, the diabetes-induced reduction in myocardial lipoprotein lipase activity in Sprague-Dawley rat hearts may, at least in part, be a compensatory response to the hypertriglyceridemia and increased fatty acid delivery to the myocardial cell, which is a characteristic feature of most severe, insulin-deficient models of diabetes mellitus. Although functional, endothelium-bound lipoprotein lipase activity was not reduced in diabetic perfused hearts from Wistar-Kyoto rats, cellular and heparin-releasable lipoprotein lipase activity was reduced in cardiac myocyte preparations, suggesting that other mechanisms in addition to plasma triacylglycerol must regulate lipoprotein lipase activity in the whole diabetic Wistar-Kyoto rat heart and that cardiac myocytes may not be the exclusive source of functional lipoprotein lipase in the diabetic myocardium.

Acute Disease↗
PubMed1977

Oscillatory changes in muscle lipoprotein lipase activity of fed and starved rats.

Lipoprotein lipase activity was measured at short time intervals in cardiac and skeletal muscles of normal and streptozotocin-treated diabetic rats fed ad libitum or deprived of food. In normal animals fed ad libitum, lipoprotein lipase activities of heart, diaphragm, soleus, and fast-twitch red fibers of the quadriceps muscle showed rhythmic oscillations that appeared to coincide with the nocturnal feeding habits of the animals. During the day (7 A.M. to 7 P.M.), when food consumption by the rats was greatly reduced, lipoprotein lipase activity in all muscles increased, followed by a decline to basal levels during the night. Similar oscillatory changes in lipoprotein lipase activity were observed in the muscles of diabetic rats fed ad libitum. In normal rats deprived of food, however, the oscillatory changes in muscle lipoprotein lipase activity were not abolished and persisted for at least 48 h. In diabetic rats starved during a 48-h period, the oscillatory changes in muscle lipoprotein lipase activity were markedly altered. In all animals, muscle lipoprotein lipase activities were not correlated to plasma glucagon levels.

Animals↗
PubMed1978

Rat lingual lipase: factors affecting enzyme activity and secretion.

The lingual serous glands of rat tongue secrete a potent lipase that acts in the stomach in which it initiates the digestion of dietary fat. The subcellular localization of the enzyme and factors affecting its activity and secretion were studied in adult male Sprague-Dawley rats. In a fraction rich in secretory granules, 42% of the lipase content of lingual serous glands was recovered after fractionation of homogenates of discontinuous gradients of urografin in 0.3 M sucrose. Lipase secretion was stimulated by isoprenaline: 2 h after isoprenaline administration, the lipase content of lingual serous glands was 73 +/- 5% lower than that of the control group. Accumulation of lipase began 8 h after the injection, reaching 57 +/- 7% of the initial level of the control group after 25 h. Bilateral resection of the glosso-pharyngeal nerves or bilateral sympathectomy led to a 40--50% decrease of enzyme activity in 7 days. Enzyme activity was markedly affected by the fat content of the diet. Feeding a high-fat diet (22% corn oil) for a period of 2 wk led to a 45% increase in the lipase content of lingual serous glands. The exponential rise in the lipase activity of the lingual serous glands immediately after birth could be related to the high-fat content of rat milk. The data suggest that the lingual serous glands are similar to other exocrine glands such as pancreas and parotid gland in the subcellular localization of secretory enzymes and mechanisms of enzyme secretion.

Animals↗
PubMed1984

Beta-adrenergic stimulation of lipoprotein lipase in rat brown adipose tissue during acclimation to cold.

Lipoprotein lipase activity in adult rats was investigated in animals subjected to cold and to different hormonal treatments. In contrast to changes in tissue wet weight and total protein content, which showed a lag time of about 1 day, lipoprotein lipase activity was markedly (fourfold) increased after only 4 h in the cold. Total lipoprotein lipase activity reached a plateau already after 1-3 days, whereas wet weight and protein content did not plateau until 3 wk. Neither insulin nor glucose injections could mimic the cold-induced increase in lipoprotein lipase activity seen after 4 h. However, the effect of norepinephrine injections was identical to the effect of cold. The beta-agonist isoprenaline was as effective as norepinephrine, whereas the alpha-agonist phenylephrine had no effect. The beta-antagonist propranolol inhibited the cold-induced increase in lipoprotein lipase activity. It is concluded that, in contrast to white adipose tissue, brown adipose tissue lipoprotein lipase is stimulated in vivo by a beta-adrenergic mechanism and that it is this beta-adrenergic mechanism that is responsible for the rapid recruitment of lipoprotein lipase during cold exposure.

Adipose Tissue, Brown↗
PubMed2002

Extensive investigation of patients with mild elevations of serum amylase and/or lipase is 'low yield'.

BACKGROUND: Serum amylase and lipase levels are widely used as markers of pancreatic inflammation. However, it would seem that mild elevations of amylase and lipase rarely predict significant pancreatic pathology. Pancreatic imaging tests are expensive. The gold standard, endoscopic retrograde cholangiopancreatography, carries risk of morbidity and mortality. OBJECTIVE: To determine whether extensive investigation of patients with mild, nonspecific abdominal symptoms and mild elevations of amylase and/or lipase results in a significant diagnostic yield. METHODS: Outpatient evaluations were retrospectively analyzed over 12 months. Inclusion criteria were nonspecific abdominal pain, and mild elevations (less than three times the upper limit of normal) of serum amylase or lipase, or both. Exclusion criteria included a history of chronic pancreatitis, elevation of liver tests and acute pain syndromes. RESULTS: Nineteen patients over the study period met the criteria. Of the nineteen patients, 58% had elevation of lipase alone, 21% amylase alone and 21% had elevations of both. In addition, 89.5% of the patients had nonspecific abdominal pain. After imaging with one or more of ultrasound, computed tomography, magnetic resonance cholangiopancreatography, endoscopic ultrasound and endoscopic retrograde cholangiopancreatography, small bowel follow through or hepatobiliary scanning, 78.9% patients were thought to have a normal pancreas. Of the remaining patients, 15.8% had mild or equivocal changes of chronic pancreatitis, and one patient was found to have a pancreatic tail pseudocyst. The average cost of investigation was US$2,255, taking only direct procedural costs into account. No patient was found to have malignancy. CONCLUSIONS: The majority of patients with nonspecific abdominal pain and isolated elevations of amylase and/or lipase (less than three times the upper limit of normal) had no identifiable pancreatic pathology. The diagnostic yield in patients with mild elevations of lipase alone was particularly poor. The cost effectiveness and risk-benefit ratio of extensive investigation of this group of patients warrants further study.

Abdominal Pain↗
PubMed2000

Effect of fasting on hepatic lipase activity in the liver of developing rats.

The effect of fasting on hepatic lipase was studied during postnatal development in the rat. It was found that fasting produced a significant decrease in hepatic lipase only in neonatal (1-day-old) and adult (60-day-old) rats. We studied the effect of fasting on the distribution of hepatic lipase between extracellular (heparin-releasable) and intracellular (liver-retained or residual) compartments in perfused livers, and on the secretion of hepatic lipase by isolated hepatocytes. Fasting had similar effects in neonates and adults: it decreased both the heparin-releasing and the residual activities in perfused livers, and also decreased the rate of hepatic lipase secretion by isolated hepatocytes. Finally, the effect of fasting on hepatic lipase mRNA relative abundance in developing rat livers was determined. No difference was observed among the groups studied. It is concluded that the mechanisms involved in the effect of fasting on hepatic lipase appear to be similar in neonates and adult animals and may involve the post-translational processing of the enzyme.

Aging↗
PubMed1988

The ontogeny of serum immunoreactive pancreatic lipase and cationic trypsinogen in the premature human infant.

We evaluated the development of the exocrine pancreas in 16 healthy preterm infants (29.3 +/- 1.6 weeks). The infants were fed breast milk with formula supplements (n = 8) or formula alone (n = 8). Growth was monitored weekly for 12 weeks then at 3, 6, 9, 12 months. At the same intervals sera were determined for pancreatic lipase and cationic trypsinogen. In addition, cord blood samples were analysed from another 33 preterm (27.6 +/- 5.2 weeks) and 75 healthy full-term infants. Serum pancreatic lipase in the cord blood of term (3.7 +/- 0.4 micrograms/l) and preterm infants (1.8 +/- 0.2 micrograms/l) was significantly below values reported for older children (10.5 +/- 0.9 micrograms/l; p less than 0.001). In the preterm infant, serum lipase was also significantly lower than values obtained at term (p less than 0.001). At birth, serum trypsinogen for preterm (16.8 +/- 1.3 micrograms/l) and term infants (23.3 +/- 1.9 micrograms/l) were below those for older children (31.4 +/- 3.7 micrograms/l; p less than 0.05). Over the first 3 weeks of life, serum lipase and trypsinogen increased significantly. From 3 weeks to 12 months of age, serum trypsinogen values remained unchanged, but serum lipase increased dramatically after 10 weeks of age. Thus, at 6 and 12 months of age, the preterm infants had significantly higher serum lipase values than infants of the same age born at term. These two pancreatic enzymes appear to show independent age-related maturation in infants born before term. The rate of maturation of lipase appears to be accelerated by exposure to the extrauterine environment.

Aging↗
PubMed1991

Lipase and pepsin activities in the stomach mucosa of the suckling dog.

The origin of preduodenal lipases was investigated in the suckling dog. In this species, gastric lipase is the main (or only) preduodenal lipase (activity range 1.03-8.32 U/mg protein), lingual-lipase activity being absent or amounting to traces only (0.77-2.3 mU/mg tissue). The localization of lipase activity in the stomach was mapped and compared to that of pepsin. The data show that the highest lipase activity was found in biopsy specimens of gastric mucosa from the cardia area (5.32 +/- 1.29-8.32 +/- 0.93 U/mg protein), and the lowest in the antrum (1.03 +/- 0.16-1.94 +/- 0.43 U/mg protein). Activity was also significantly higher in the mucosa along the greater rather than the lesser curvature of the stomach. Pepsin activity was highest in the cardia and gastric body areas (26.2 +/- 0.89-89 +/- 23.61 and 26.83 +/- 15.98-69.51 +/- 9.82, respectively). Contrary to lipase activity, considerable pepsin activity was present in the antrum (18.17 +/- 4.12-23.07 +/- 5.60 U/mg protein). No difference in pepsin activities was found in the greater as compared to the lesser curvature. The data show similar origin and tissue distribution of gastric digestive enzymes in the suckling dog and human infant. The role of the newborn dog as an animal model for fat digestion in the human infant is discussed.

Animals↗