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PubMed1987

Lipase kinetics at the triacylglycerol-water interface using surface tension measurements.

Two methods, the so-called "oil drop" and "Teflon plunger" methods, were designed to monitor lipase hydrolysis of natural long-chain triacylglycerols through the variation with time of the oil-water interfacial tension. The first part of this work is devoted to the development of these two techniques using pure, well-characterized porcine pancreatic lipase. They gave linear responses with enzyme concentrations ranging from 1 x 10(-3) to 30 units x ml-1. We then applied them to a study of the optimal pH conditions for human gastric lipase which were found to range around 5, as previously observed. In the presence of variable concentrations of sodium taurodeoxycholate, these two methods also showed that human gastric lipase is active in the 8-13 dyn cm-1 range of interfacial tension. It is concluded that these two methods, based upon variations with time of the oil-water interfacial tension, constitute reliable, sensitive and convenient means of investigating lipase kinetics.

Animals↗
PubMed1992

Stereoselectivity of lipases: esterification reactions of octadecylglycerol.

Stereoselectivity of several triacylglycerol lipases (EC 3.1.1.3) has been investigated in the enzymatic esterification of rac-1-O-octadecylglycerol with oleic acid in the presence of organic solvents, such as hexane. X-1(3)-O-Octadecylmonooleoylglycerols were the only products formed with most lipases; considerable proportions of X-1(3)-O-octadecyldioleoylglycerols were also formed with the lipase from Candida cylindracea. The mixtures of unesterified enantiomeric substrates, i.e., X-1(3)-O-octadecylglycerols were converted to their 3,5-dinitrophenylurethane derivatives and subsequently resolved into sn-1 and sn-3 enantiomers by HPLC on a chiral stationary phase (Sumichiral OA 2100). The data on enantiomeric excess (ee) and enantiomeric ratio (E) in the unesterified substrate revealed for the lipases from porcine pancreas, Rhizopus sp., Pseudomonas sp., Candida cylindracea, Chromobacterium viscosum and Penicillium cyclopium a distinct preference for 1-O-octadecyl-sn-glycerol over its enantiomer indicating stereoselectivity for the sn-3 position. For the lipase from Rhizomucor miehei a slight stereoselectivity for the sn-1 position was observed. Solvents, such as diethyl ether and dichloromethane, strongly inhibited the esterification reaction, but the enzymatic activity could be restored upon removal of such solvents by washing with hexane indicating reversible inhibition.

Esterification↗
PubMed1985

Limited proteolysis of porcine pancreatic lipase. Lability of the Phe 335-Ala 336 bond towards chymotrypsin.

Mild chymotrypsin digestion of native lipase (449 amino acids) preferentially cleaved the Phe 335-Ala 336 bond. On SDS-gel electrophoresis, 3 major bands were observed: band 1 (52 kDa) representing native lipase, bands 2 and 3 (40 and 12 kDa) representing the two lipase fragments A and B. Fragment A does not retain lipase activity but maintains its ability to adsorb to interfaces. Fragment B was identified with the lipase C-terminal region (336-449). It does not exhibit any activity towards tributyrylglycerol emulsions and any ability to adsorb to interfaces.

Alanine↗
PubMed1990

Sequence identity between human pancreatic cholesterol esterase and bile salt-stimulated milk lipase.

Three overlapping cDNA clones covering the entire primary sequence of the bile salt stimulated lipase in human milk were isolated from a human breast lambda gt10 cDNA library by screening with the rat pancreatic cholesterol esterase cDNA. Nucleotide sequencing of the cDNA showed that the human milk lipase mRNA encodes a 748-residue protein, including a 23-residue signal peptide. The human milk lipase cDNA is highly homologous to rat pancreatic cholesterol esterase, suggesting that the milk lipase may be identical to the cholesterol esterase in human pancreas. This conclusion was confirmed by isolation and sequencing of the cDNA for human pancreatic cholesterol esterase. Analysis of the sequence for the human cholesterol esterase/milk lipase revealed similarities to other serine esterases in three distinct regions of the protein. These domains may represent the active site triads of these proteins.

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↗
PubMed1986

Characterization and solubilization of membrane bound diacylglycerol lipases from bovine brain.

Bovine brain contains two diacylglycerol lipases. One is localized in purified microsomes and the other is found in the plasma membrane fraction. The microsomal enzyme is markedly stimulated by the non-ionic detergent, Triton X-100, and Ca2+, whereas the plasma membrane diacylglycerol lipase is strongly inhibited by Triton X-100 and Ca2+ has no effect on its enzymic activity. Both enzymes were solubilized using 0.25% Triton X-100. The solubilized enzymes followed Michaelis-Menten kinetics. The apparent Km values for microsomal and plasma membrane enzymes are 30.5 and 12.0 microM respectively. Both lipases are strongly inhibited by RHC 80267, with Ki values for microsomal and plasma membrane diacylglycerol lipases of 70 and 43 microM, respectively. The retention of microsomal diacylglycerol lipase on a concanavalin A-Sepharose column and its elution by methyl alpha-D-mannoside indicates the glycoprotein nature of this enzyme.

Animals↗
PubMed1977

Localization of lipase-like immunoreactivity in porcine adipose, aortic and myocardial tissue.

Lipase has been purified from pig adipose tissue and antibodies have been produced in rabbit. By indirect immunofluorescence and immunoenzyme techniques lipase-like immunoreactivity was demonstrated in the intima of pig aorta, in the endothelial cells of the myocardium and in plasma membranes of adipocytes and skeletal muscle cells. Lipase activity in extracts of some of these tissues was inhibited by the addition of anti-lipase antibodies. At least part of the immunoreactivity in the examined tissues is due to active lipases.

Adipose Tissue↗
PubMed1988

Regulation of lipoprotein lipase activity in the sand rat: effect of nutritional state and cAMP modulation.

The sand rat (Psammomys obesus) is a desert rodent in which obesity and diabetes mellitus appeared only subsequent to feeding laboratory animal chow. To study the role of lipoprotein lipase in the development and maintenance of obesity in the sand rat, enzyme activity in various organs and in plasma of sand rats or albino rats was determined following a 20-hour fast, or 16 hours after injection of cholera toxin. Despite comparable change in body weight, an altered pattern of enzyme distribution and regulation was observed in the sand rat. Neither fasting nor cholera toxin had an effect on heart and daiphragm muscle lipoprotein lipase activity of the sand rat, but caused a 1.5- to 2-fold increase in the treated albino rats. By using an isolated perfused heart system, we were able to measure enzyme activity present in the heparin-releasable fraction that represents the functional pool of the enzyme. In both species, the heparin-releasable fraction of the heart increased twofold following fasting, though initial values were lower in sand rat. In both species, fasting and cholera toxin administration resulted in an increase in plasma and liver lipoprotein lipase activity. Adipose tissue lipoprotein lipase activity of sand rat, unlike the albino rats, was similar in the various fat regions and was not lowered by food deprivation or cholera toxin administration. After both treatments, sand rat plasma insulin levels exceeded fivefold those of albino rats. Adipose tissue lipoprotein lipase activity of fed and fasted normal and diabetic sand rats correlated negatively with plasma insulin and glucose levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗
PubMed1989

Familial lipoprotein lipase-activity deficiency: study of total body fatness and subcutaneous fat tissue distribution.

Total body fatness and subcutaneous fat tissue distribution were evaluated in 19 hyperchylomicronemic patients. Eleven were males, aged 10 to 57 years, and eight were females, aged 13 to 46 years. Familial lipoprotein-lipase-activity deficiency was diagnosed by the absence of lipoprotein-lipase activity in the plasma withdrawn ten and 20 minutes after intravenous injection of ten units of heparin per kilogram of body weight. The 19 patients had skin-fold measurements for evaluation of subcutaneous fat distribution. Fifteen also underwent body density measurements by underwater weighing. Percent body fat was calculated from body density. These anthropometric data were plotted against the regression curves of 1638 normal controls of both sexes (aged 10 to 54 years) for fat tissue weight, percent body fat, subcutaneous fat/total fat mass ratio and trunk/extremity skin-fold ratio. Impairments in the process of building fat tissue reserves could not be shown in the 19 hyperchylomicronemic patients, in spite of the absence of lipoprotein-lipase activity in their postheparin plasma. It is hypothesized that normal fat tissue mass in these patients could be due partly to de novo synthesis of fatty acids by adipocytes, hydrolysis of plasma triglycerides by hepatic lipase, and/or contribution of a specific fat-tissue lipase to the catabolism of plasma triglyceride-rich lipoproteins.

Adipose Tissue↗
PubMed1991

Effect of physical training on lipids, lipoproteins, apolipoproteins, lipases, and endogenous sex hormones in men with premature myocardial infarction.

In 17 men, aged 27 to 54 years, with myocardial infarction 2 to 10 months before the current exercise study, we aimed to determine whether 3 months of exercise training, at a level designed to elevate high-density lipoprotein cholesterol (HDLC), would be associated with changes in endogenous sex steroid hormones and postheparin lipoprotein and hepatic lipases, and whether the changes in sex hormones, lipids, lipoproteins, apolipoproteins, and physical activity were interrelated. Supervised bicycle ergometry, 30 minutes, 3 days per week, eliciting 75% of maximum heart rate, produced a significant training effect, with a 26% increase in the duration of the exercise test at a standardized, submaximal workload (P less than or equal to .001), and a reduction in heart rate measured at a standardized submaximal workload, P = .08. After 3 months' training, mean HDLC increased 23% (30 to 37 mg/dL), P less than or equal to .001, mean apo A2 increased 19% (43 to 51 mg/dL), P less than or equal to .001, and the ratio of total cholesterol (TC) to HDLC decreased 26% (P less than or equal to .01), while estradiol (E2) levels decreased 45% (50.1 to 27.8 pg/mL), P less than or equal to .0001. After 1 and 2 months' exercise, TC (12% [P less than or equal to .001], 11% [P less than or equal to .01]), and low-density lipoprotein cholesterol (LDLC) (13% [P less than or equal to .01], 12% [P less than or equal to .01]) were reduced. Hepatic lipase decreased 16% (P less than or equal to .01) and 16% (P less than or equal to .05) after 1 and 3 months' exercise. There were no significant changes in apo A1, lipoprotein lipase, testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), or weight. By stepwise regression analysis, after 3 months' training, 66% (P = .0025) of the variance for the increase in HDLC from baseline to day 90 was accounted for independently by a decrease in triglyceride (F = 13.2, P = .003), by reduced heart rate on a fixed submaximal load (F = 12.7, P = .0035), and by a decrease in hepatic lipase (F = 5.5, P = .036). A modest, achievable exercise program can have significant cardiovascular benefit for men after myocardial infarction by ameliorating their hyperestrogenemia, reducing TC and LDLC, improving the TC to HDLC ratio, and elevating HDLC and apo A2. The increment in HDLC was related independently to improved capacity to sustain submaximal exercise and to exercise-induced reductions in triglyceride and postheparin hepatic lipase.

Adult↗
PubMed1990

Cloning of an interleukin-4 inducible gene from cytotoxic T lymphocytes and its identification as a lipase.

Interleukin-4 (IL-4) has been demonstrated to be an important lymphokine for the generation of cytotoxic T lymphocytes (CTLs). Here we describe an IL-4 inducible gene specifically expressed in CTLs. By sequence homology, this gene is likely to be the mouse homolog of pancreatic lipase. Oocyte translation of in vitro transcribed mRNA results in the expression of a protein with lipase activity, and Northern analysis of various tissues and a large panel of hematopoietic cell types demonstrates that this gene is expressed only in the pancreas and CTLs. Lysates of CTLs grown in IL-4, but not in IL-2, exhibit lipase activity. Furthermore, Northern analysis of CTLs grown in the presence of IL-4 for as little as 5 days demonstrates a marked induction of lipase mRNA, which correlates with enhanced cytolysis by these cells. These results suggest that this lipase may have an important role in CTL effector function.

Amino Acid Sequence↗
PubMed1990

Lipoprotein lipase mRNA expression in brown adipose tissue: translational and/or posttranslational events are involved in the modulation of enzyme activity.

Lipoprotein lipase mRNA abundance in rat brown adipose tissue increases during the first 24 h of cold exposure. Lipoprotein lipase mRNA levels do not change in brown fat throughout pregnancy and lactation, whereas enzyme activity is significantly lowered. After 5 h of acute cold or noradrenaline administration there is a 2-fold increase in lipoprotein lipase mRNA abundance, whereas lipoprotein lipase activity is stimulated to more than 6-fold the basal values. It is concluded that translational and/or posttranslational mechanisms are involved in the noradrenergic modulation of lipoprotein lipase activity in brown fat.

Acclimatization↗
PubMed1992

Cloning, nucleotide sequence and expression in Escherichia coli of a lipase gene from Bacillus subtilis 168.

The gene coding for an extracellular lipase of Bacillus subtilis 168 was cloned and found to be expressed in Escherichia coli. Enzyme activity measurements showed no fatty acid chain length preference. A set of Tn5 insertions which inactivate the gene were localized and used to initiate its sequencing. The nucleotide sequence was determined on two independent clones expressed in E. coli. In one of these clones, the sequence revealed a frameshift, due to the presence of an additional adenine in the N-terminal region, which caused the interruption of the open reading frame, probably allowing translation to initiate at a second ATG codon. The sequence of the wild-type lip gene from B. subtilis was confirmed on the chromosomal fragment amplified by polymerase chain reaction (PCR). When compared to other lipases sequenced to date, the enzyme described here lacks the conserved pentapeptide Gly-X-Ser-X-Gly supposed to be essential for catalysis. However, alignments of several microbial lipase sequences suggest that the pentapeptide Ala-X-Ser-X-Gly present in the lipase B. subtilis may function as the catalytic site. Homologies were found in the N-terminal protein region with lipases from different Pseudomonas species. The predicted M(r) and isoelectric point for the mature protein are 19,348 and 9.7 respectively.

Amino Acid Sequence↗
PubMed1983

Purification and some properties of the staphylococcal extracellular lipase.

Staphylococcal lipase has been purified by application of a multistep procedure involving ammonium sulfate precipitation, and hydrophobic interaction chromatography followed by gel filtration through Sepharose CL-4B. A purified enzyme was obtained which appeared to be homogeneous by molecular sieving, polyacrylamide gel electrophoresis and sucrose gradient centrifugation. The enzyme was then subjected to physicochemical analysis. It has been found that staphylococcal lipase appears in two molecular forms: light (45 kDa) and heavy (300 kDa). Amino acid analysis indicates that lipase contains 17 amino acids with a prevalence of hydrophobic amino acids. No sulfur-containing amino acid was found in the enzyme molecule. The lipase contains about 2% sugars and some amount of lipids. The lipase preparation is stable within pH 5.0 to 9.0 and exhibits maximal activity at pH 8.0. The optimal temperature for the enzymatic reaction was established at 55 degrees C.

Amino Acids↗
PubMed1995

Molecular characterization of a Galactomyces geotrichum lipase, another member of the cholinesterase/lipase family.

Geotrichum candidum secretes several lipase isoenzymes, differing in their selectively towards esters of long chain fatty acids with a cis-9 double bond. One group shows an absolute selectively towards these fatty acid esters, the other group has a more relaxed specificity and will also hydrolyze other long chain fatty acid esters. Galactomyces geotrichum secrets a lipase that has the same specificity as the latter group. The corresponding lipase gene was cloned from Galactomyces geotrichum. From an alignment of our enzymes' primary structure with those of different strains of Geotrichum candidum, remarkable conservation is evident and it is not yet possible to identify residues/structures responsible for differences in fatty acid specificity. Comparison of the GCL/GGL family with a variety of lipases from other sources, indicated that they are more related to mammalian than microbial lipases.

Amino Acid Sequence↗
PubMed1995

Purification and molecular characterization of lamb pregastric lipase.

Lamb pregastric lipase (LPGL) was purified from pharyngeal tissues. The purification procedure was based on an aqueous extract containing 0.7% Tween 80 which was chromatographed on DEAE-cellulose anion-exchanger and adsorbed on HA-Ultrogel followed by gel filtration on Ultrogel AcA-54. The final enzymatic preparation, where the overall activity recovery was 3%, showed a single protein band on SDS-PAGE with a molecular mass of 50 kDa. LPGL is a glycoprotein containing approx. 14% (w/w) of carbohydrate. Extensive deglycosylation using peptide N-glycosidase F yielded a protein with an apparent molecular mass of 43 kDa. An uncontrolled proteolysis of LPGL during the purification lead to a 45 kDa form which was previously observed in human lysosomal acid lipase (HLAL) and rabbit gastric lipase (RGL). The labile bond X54-Leu55 was identified. Isoelectric focusing of LPGL reveals a major band corresponding to an isoelectric point of 4.8. The pure enzyme displayed specific activities of 950 U mg-1, 300 U mg-1 and 30 U mg-1 at pH 6.0, using tributyroylglycerol, trioctanoylglycerol and trioleoylglycerol as substrates, respectively. Using Western blot analysis, a cross-immunoreactivity of LPGL was observed with purified anti-human gastric lipase polyclonal antibodies. Determination of the amino-acid sequence of 62 residues revealed a high degree of homology with other known preduodenal lipases.

Amidohydrolases↗
PubMed1987

Characterization of the intracellular processing and secretion of hepatic lipase in FU5AH rat hepatoma cells.

The processing and secretion of newly synthesized hepatic lipase was characterized in FU5AH rat hepatoma cells. Pulse-chase experiments revealed two immunoreactive species with apparent molecular weights of 55,400 and 57,600. The 55.4 kDa species was detectable only in cell extracts, whereas the 57.6 kDa species was present in both cell extracts and media. Following a 5 min pulse with L-[35S]methionine and a 10 min chase, these two species represented only 0.003% of the total labelled protein. Quantitation of the 55.4 kDa and 57.6 kDa species in a chase time course taken together with their respective sensitivity and resistance to digestion with endo-beta-N-acetylglucosaminidase H indicates that the 55.4 kDa species is a high mannose precursor to the mature 57.6 kDa enzyme which contains only complex N-linked oligosaccharides. From a time course of endo-beta-N-acetylglucosaminidase H digestion, it was determined that hepatic lipase contains a minimum of two N-linked oligosaccharides. Treatment of the 55.4 kDa species with endo-beta-N-acetylglucosaminidase H yields a protein with a kDa value similar to that observed after treatment of the mature secreted enzyme with endo-beta-N-acetylglucosaminidase F or trifluoromethanesulfonic acid. Therefore, processing of N-linked oligosaccharides is probably the only post-translational modification responsible for the observed change in the apparent molecular weight of hepatic lipase. The half-residence times of hepatic lipase in the endoplasmic reticulum-cis Golgi region and in the cell were estimated at 34 min and 57 min, respectively. Newly synthesized hepatic lipase in Fu5AH cells is secreted constitutively and is not stored in an intracellular pool. Finally, little of the newly synthesized enzyme is degraded during the course of a 1 h chase.

Animals↗
PubMed1988

Effect of sodium taurodeoxycholate, CaCl2 and albumin on the action of pancreatic lipase on droplets of trioleoylglycerol and the release of lipolytic products into aqueous media.

1. Effects of various substances on the activity of pancreatic lipase and on the release of lipolytic products into aqueous media were studied with droplets of trioleoylglycerol suspended from a membrane filter at the top of a flow-through chamber. The droplets were perifused for 7 min with a commercial preparation of pancreatic lipase in 0.15 M NaCl solution at pH 6.5 and then perifused for 60 min with lipase-free media, either 0.15 M NaCl at pH 6.5 or basal medium at pH 7.4 (70 mM sodium barbital) containing different additives. 2. About 6% of the trioleoylglycerol in droplets was hydrolyzed during the perifusion with lipase. Another 15% was hydrolyzed in 30 min, but none thereafter, when the droplets were perifused with 0.15 M NaCl alone. The rate of hydrolysis was doubled and prolonged when droplets were perifused with basal medium at pH 7.4. Lipolytic products formed at pH 7.4 were 62% oleic acid, 20% monooleoylglycerol and 18% dioleoylglycerol, yet only 4% of the lipolytic products were released into the perifusate. 3. Sodium taurodeoxycholate (TDC) (17 mM ) added to basal medium increased 18 x the amount of lipolytic products released into the perifusate but increased lipolysis only 13%. The molar ratio of oleic acid to monooleoylglycerol in the perifusate was 5.7 during the first 30 min and 4.0 during the last 30 min. 4. Ca2+ (3.3 mM) added to basal medium increased lipolysis 87% but did not affect the amount (4%) of lipolytic products released into the perifusing medium. 5. TDC and Ca2+ added to basal medium produced the largest increase in lipolysis, with 59% of trioleoylglycerol hydrolyzed in 15 min and 91% in 60 min. The amount of lipolytic products released into the perifusing medium, however, was not increased above that released into medium containing TDC alone. 6. Serum albumin (0.6 mM) and Ca2+ added to basal medium increased 14 x the amount of lipolytic products released into the perifusate without affecting the basal lipolytic rate. Albumin, however, suppressed by 40% the stimulatory effect of Ca2+ on pancreatic lipase activity.

Albumins↗