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PubMed2006

Spectrophotometric assay for online measurement of the activity of lipase immobilised on micro-magnetic particles.

A spectrophotometric assay has been adapted to directly measure the activity of enzymes immobilised on insoluble magnetic particles. Three different types of lipases (Candida antarctica lipase A and B and Thermocatenulatus lanuginosus lipase) were immobilised on two types of magnetic beads. The activity of the resulting immobilised lipase preparations was measured directly in the reaction solution by using a modified p-nitrophenol ester assay using a spectrophotometer. Removal of the solid particles was not necessary prior to spectrophotometric measurement, thus allowing reliable kinetic measurements to be made rapidly. The method was effective for a wide range of magnetic bead concentrations (0.01-0.2 mg ml(-1)). In all cases the assay could determine the bead-related specific enzyme activity. The assay was validated by comparing with a pH-stat method using p-nitrophenol palmitate as the substrate with an excellent correlation between the two methods. The utility of the spectrophotometric assay was demonstrated by applying it to identify the best combination of lipase type, activation chemistry and magnetic particle. Epoxy activation of poly vinyl alcohol-coated magnetic particles prior to immobilisation of commercial C. antarctica lipase A gave the best preparation.

Coated Materials, Biocompatible↗
PubMed1980

Characterization of triacylglycerol lipase activity in human amniotic fluid.

A radiochemical assay was used to measure the triacylglycerol lipase activity found in normal human amniotic fluid at term. Enzyme activity was characterized in a partially purified extract of amniotic fluid and was found to be optimal at pH 8.0 +/- 0.2 in the presence of 5mM sodium taurocholate, with the use of emulsified tri-[3H]oleoyl glycerol as the substrate. The assay described made it possible to determine the lipase activity in as little as 25 microliters of a 12,000 x g supernatent of whole amniotic fluid as the source of enzyme. The lipase appeared to be distinct from another triacylglycerol lipase measurable in fetal membranes. In turn, it was shown that the amniotic fluid enzyme exhibited several catalytic properties which resembled those of pancreatic lipase. i.e., its substrate specificity, the bimodal effect of bile salt, and the influence of authentic pancreatic colipase on the catalytic process. The results suggest that the assay of triacylglycerol lipase activity may be clinically useful in the detection of enzyme abnormalities in human amniotic fluid.

Amniotic Fluid↗
PubMed1986

Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart.

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.

Animals↗
PubMed1992

Purification, molecular cloning, and expression of lipase from Pseudomonas aeruginosa.

An extracellular lipase secreted by Pseudomonas aeruginosa TE3285 was purified. A genomic library of this strain was constructed in lambda EMBL3, and a DNA fragment 2.7 kb long containing the lipase gene, lipA, was isolated with an oligonucleotide probe synthesized on the basis of the partial amino acid sequence of a purified preparation of the enzyme. Nucleotide sequence analysis showed an open reading frame of 933 bases, and the deduced amino acid sequence agreed well with the molecular mass and partial amino acid sequences of mature lipase. The results of alignment of the amino acid sequences of five lipases from Pseudomonas species considered together with the published crystal structure studied with human pancreatic lipase showed that Ser82, His251, and Asp209 were catalytic residues and that a surface loop from residues 172 to 204 was responsible for the substrate specificity. About 50 bases downstream of lipA, there was another gene, lipB. The sequence of lipB was highly homologous to that of putative modulators of the production of active lipases in other Pseudomonas species. Expression plasmids encoding lipA followed by the complete or incomplete lipB gene downstream of the lac promoter of pUC18 were constructed. lipA was expressed in Escherichia coli 1100 only in the presence of the complete lipB gene.

Amino Acid Sequence↗
PubMed1993

Lipid droplet accumulation and lipoprotein lipase activity in the rat salivary gland during the perinatal period.

The submandibular and sublingual glands of foetal and newborn rats aged 21 days in utero to 7 days after birth were examined morphologically and biochemically. Lipid droplets tended to be localized in secretory cells, especially in their basal cytoplasm. The degree of droplet accumulation varied with the age of the rat. No droplets were observed before and immediately after birth. The number of accumulated droplets peaked 24-48 h after birth, then gradually decreased and reached normal levels by 5 days. In the salivary glands of fasted newborn rats, no lipid droplets were observed throughout the experiment. The amount of triacylglycerol reached its maximum level 1 day after birth; it then decreased gradually until 5 days and after that did not change. The amount of cholesterol did not change during postnatal development. Lipase activity attained its maximum level in the salivary glands immediately after birth and then decreased rapidly. It was higher in the glands of fasted than fed 1-day-old rats. Antiserum against lipoprotein lipase inhibited the salivary gland lipase activity in a dose-dependent manner, with 5 microliters of antiserum producing 60-70% inhibition. Non-immune serum had little effect. It was concluded that (1) accumulated lipid in the secretory cell cytoplasm of the salivary glands originates from ingested milk; (2) the principal component of accumulated lipid droplets is triacylglycerol; (3) 60-70% of the total lipase activity represents lipoprotein lipase; (4) an increase of lipoprotein lipase activity is recognizable before the accumulation of triacylglycerol.

Animals↗
PubMed1975

Release of lipoprotein lipase from fat cells in vitro.

Release of lipoprotein lipase from rat fat cells incubated at 20 degrees in medium with albumin, but without glucose proceeded at a constant rate for 30 min. The initial rate of release was increased when serum was present in the medium. Maximal stimulation (100-300%) was produced with 3.8% serum. The maximal increment in release caused by serum was always greater than that produced by heparin and when both were added release was greater than it was with either one alone. The active component(s) of serum, nondialyzable and stable for 30 min at 56 degrees C, was present in sera from humans and rats in the fed or fasted state. Glucose plus insulin (but neither alone) enhanced the rate of lipase release in the presence of serum but not in its absence. The half-life of the lipase in basal medium of 20 degrees C was 90 min. Heparin decreased this to about 50 min and serum markedly prolonged it whether or not heparin was present. Lipoprotein lipase activity in cells and fractions thereof was assayed in extracts of acetone powders. After centrifugation of fat cell homogenates at 600 times g for 15 min, only 50-60% of the activity was recovered in the supernatant. After centrifugation at 100 000 times g for 60 min, the supernatant contained about 10% of the total activity and the sediment 40%. In some experiments, most of the rest was recovered in the floating fat fraction. Total lipoprotein lipase activity of cells plus medium increased steadily during incubation of fat cells for 1h at 30 degrees C. The major increment occurred in the cells and activity in the medium was always less than 15% of the total. Our observations are consistent with the view that activation may be an important determinant of fat cell lipoprotein lipase activity as well as an integral part of the release process.

Adipose Tissue↗
PubMed1976

Lipoprotein lipase in rat lung. Effect of dexamethasone.

The effect of hormone administration on the activity of lipoprotein lipase in the lung was studied in the rat. The following hormones were administered: dexamethasone, L-thyroxine, estradiol-17beta and progesterone. In addition, lung lipoprotein lipase activity was studied in diabetic and lactating rats. Lipoprotein lipase activity was measured in dried, defatted preparations of rat lung using double labeled ([14C]palmitate, [3H]glycerol) chylomicron triacylglycerol as substrate. Dexamethasone administration caused a rise of 70% in the level of activity of lipoprotein lipase in acetone powders of lung and a 100% increase in the amount of enzyme released during heparin infusion into isolated, perfused lungs. Enzyme activity was higher in lungs of females than of male rats; however; the level of activity was unaffected by estrogen or progesterone administration to either male or ovariectomized rats. Diabetes, hyperthyroidism or lactation did not change lipoprotein lipase activity in the lung. The constant presence of lipoprotein lipase activity in the lung suggests that this organ is able to maintain a steady supply of triacylglycerol-fatty acids under a variety of physiological and pathological conditions. Stimulation of enzyme activity by dexamethasone could lead to increased uptake of triacylglycerol-fatty acids by the lung and may thus be a contributing factor to corticosteroid-induced enhanced surfactant synthesis.

Animals↗
PubMed1976

Hydrolysis of chylomicron phosphatidylcholine in vitro by lipoprotein lipase, phospholipase A2 and phospholipase C.

The effects of lipoprotein lipase, phospholipase A2 and phospholipase C on chylomicron phosphatidylcholine and triacylglycerol were studied with rat lymph chylomicrons containing phosphatidylcholine labeled with [14C]oleic acid. Lipoprotein lipase purified from bovine milk readily hydrolyzed chylomicron phosphatidylcholine to lysophosphatidylcholine and fatty acid, and triacylglycerol to monoacylglycerol, fatty acid and glycerol. The rates of hydrolysis of phosphatidylcholine and triacylglycerol increased with enzyme concentration, and both decreased when fatty-acid binding sites on albumin in the incubation medium were limited. The proportion and amount of phosphatidylcholine hydrolyzed was always less than that of triacylglycerol. Analyses of hydrolytic products showed that lipoprotein lipase cleaved the 1-acyl ester bond of phosphatidylcholine. The findings indicate that lipoprotein lipase can account for some of the phospholipase A1 activity found in postheparin plasma. Phospholipase A2 and phospholipase C hydrolyzed chylomicron phosphatidylcholine, greater than 92% in 10 min, but not triacylglycerol. The resultant phosphatidylcholine-deficient chylomicrons, which could be concentrated by ultra-centrifugation and resuspended in incubation medium, were readily depleted of triacylglycerol when incubated with lipoprotein lipase. The findings indicate that phosphatidylcholine can be removed from the surface film of chylomicrons without disrupting the particles or blocking the action of lipoprotein lipase on the core triacylglycerol.

Animals↗
PubMed1977

Activatable cholesterol esterase and triacylglycerol lipase activities of rat adrenal and their relationship.

Activatable cholesterol esterase and triacylglycerol lipase of rat adrenal were 58-69% recovered in the 100 000 X g supernatant fraction. Activatable triacylglycerol lipase activity was differentiated from the activity of acid lipase and lipoprotein lipase also found in this fraction. Cholesterol esterase was activated 39.7 +/- 13.6% (S.D.) and triacylglycerol lipase 11.9 +/- 2.9% in a reaction dependent on ATP, cyclic AMP, and protein kinase. The two activities were shown by differential inhibition by an organophosphate, and by partial separation on salting out, to be largely due to separate enzymes. The two enzymes bound tightly to substrate emulsions with quantitatively similar distribution between competing emulsions, suggesting concerted binding. Coinciding gel filtration patterns reinforced, The hypothesis of a lipase complex. Cholesterol esterase comprised a major component of higher apparent Km for substrate and molecular weight 3-10(5)-6-10(5) by gel filtration and a minor component of lower apparent Km and heterogeneous molecular weight above 1 million, which was found mostly in complex and lipid.

Adrenal Glands↗
PubMed1979

Alteration of the lipase activities of muscle, adipose tissue and liver by rapeseed oil feeding of rats.

Feeding rapeseed oil, rich in erucic acid, for 4 days results in a significant increase of the lipoprotein lipase activities of heart and adipose tissue. The lipase activity of liver, which in earlier studies has been shown to be releasable by heparin perfusion, also increases by the dietary regimen. The increased lipoprotein lipase activity of heart may contribute to lipid accumulation in this organ. The higher intracellular lipid store probably results in higher (hormone-sensitive) tissue lipase activity. The increase of lipoprotein lipase of adipose tissue, however, is not accompanied by an increase of hormone-stimulated tissue lipase activity in fat cells. This activity may even become lower, and might contribute to the decrease of the lipid store in heart after an initial rapid phase of fat accumulation during erucic acid feeding.

Adipose Tissue↗
PubMed1982

The temperature-dependent interfacial inactivation of porcine pancreatic lipase. Effect of colipase and bile salts.

This paper confirms and extends the previous observation that colipase and bile salts stabilize pancreatic lipase against inactivation at its water/substrate interface. It is shown that colipase and bile salts above their critical micellar concentration offer better protection than either of them alone. Colipase has no effect on the catalytic efficiency of lipase against an emulsified substrate in the absence or presence of bile salts. Its reported activation of pancreatic lipolysis at high temperatures in the absence of bile salts is, most likely, fully explained by its protective effect on lipase inactivation. Colipase at high concentrations relative to lipase inhibits the enzyme activity in a competitive fashion. The temperature-dependent surface inactivation of lipase has certain consequences for the methodology of lipase activity determination.

Animals↗
PubMed1982

Hydrolysis of triacylglycerol emulsions by lingual lipase. A microscopic study.

The effect of lingual lipase on four different triacylglycerol emulsions was observed by light microscopy at pH 5-6. The extent of hydrolysis on the microscope slide was determined with the aid of radioactive emulsions or by analyzing the products by gas-liquid chromatography. Artificial emulsions that had been stabilized with amphiphilic lipids gradually coalesced during the unstirred lipase reactions. Gum arabic-stabilized emulsions and human milk fat droplets did not stick to each other or coalesce during lingual lipase hydrolysis. No visible liquid-crystalline product phases, as are seen with pancreatic lipase (Patton, J.S. and Carey, M.C. (1979) Science 204, 145-148), were observed with lingual lipase. The products of lingual lipase activity, protonated fatty acid and diacylglycerol, appear to remain dissolved in the oil phase of the triacylglycerol particle.

Animals↗
PubMed1983

Heparin-independent release of lipoprotein lipase activity from perfused rat hearts.

Heparin-independent release of lipoprotein lipase activity from isolated perfused rat hearts was measured and related to the rapid turnover of the enzyme. Hearts consistently released lipoprotein lipase activity (2.1 +/- 0.2 U/g released per min) during 60 min of nonrecirculating perfusion without heparin. This rate of release did not significantly differ from that measured in heparin-perfused hearts after the first 10 min of perfusion (2.2 +/- 0.2 U/g released per min). The fractional release rate of lipoprotein lipase activity during nonheparin perfusion was 1.3% per min, which was higher than that calculated for alkaline phosphatase (0.002%) and creatine kinase (0.03%) activities. The lipase activity released was activated 4-fold by serum and inhibited 94 and 88% by 0.5 M NaCl and 3 mg/ml protamine sulfate, respectively. Lipoprotein lipase activity in the 1-min heparin-releasable (extracellular) and residual (intracellular) compartment remained stable during the last 40 min of nonheparin perfusion. During this period total heart, intracellular and extracellular enzyme t1/2 were calculated to be 52, 42 and 10 min, respectively. The results are consistent with the postulate that continuous release of lipoprotein lipase into the vascular compartment may be an important determinant of its rapid turnover in the heart, and possibly other tissues.

Animals↗
PubMed1984

Lipoprotein lipase mediated uptake of non-degradable ether analogues of phosphatidylcholine and cholesteryl ester by cultured cells.

Lipoprotein lipase mediated transfer of cholesteryl ester and its ether analog, cholesteryl linoleyl ether, from unilamellar liposomes, prepared from a nonhydrolyzable ether analog of 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleyl ether-sn-glycero-3-phosphocholine (DOEPC), was studied in various cells in culture. It was found that lipoprotein lipase enhanced the uptake of cholesteryl linoleyl ether and of DOEPC. These findings provided a definitive proof that hydrolysis of liposomal PC is not needed for the lipoprotein lipase catalyzed transfer of cholesteryl linoleyl ether and cholesteryl ester to cells. The lipids transferred by lipoprotein lipase to cells were localized in three compartments, trypsin-releasable, resistant and metabolic; the latter was a chloroquine-sensitive pool as evidenced by inhibition of cholesteryl ester hydrolysis. Labeled PC and, to a lesser extent DOEPC, in the trypsin-releasable pool was able to return to the medium, while cholesteryl linoleyl ether and cholesteryl ester required cholesteryl ester transfer protein for release. The transfer of cholesteryl linoleyl ether and cholesteryl ester into a trypsin-resistant compartment did not require metabolic energy and occurred also in formaldehyde-fixed cells. Metabolic energy was needed for the translocation of cholesteryl linoleyl ether and cholesteryl ester into the lysosomal compartment, presumably by a process of endocytosis. The physiological relevance of the present findings is that as intravascular hydrolysis of triacylglycerol-rich lipoproteins is mediated by lipoprotein lipase attached to endothelial cells, the latter can provide a very extensive surface for removal and metabolism of phospholipids and cholesteryl ester by a mechanism mediated by lipoprotein lipase.

Animals↗
PubMed1984

Lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine. Effect of lipid organization and apolipoprotein C-II on enzyme activity.

The effect of phospholipid organization on the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine was examined with sonicated vesicles and Triton X-100 or lysomyristoylphosphatidylcholine solubilized lipid. Triton X-100-dimyristoylphosphatidylcholine substrates were prepared at various ratios of detergent to phospholipid so as to produce lipid structures varying from bilayers to micelles. Apolipoprotein C-II, the activator protein for lipoprotein lipase, enhanced the rate of the lipoprotein lipase-catalyzed hydrolysis of dimyristoylphosphatidylcholine for each substrate tested. Although the absolute rate of lipoprotein lipase catalysis was different for each, the factor (the ratio of lipoprotein lipase activity with apolipoprotein C-II to that without the activator protein) was nearly constant, with a value of approximately 16. We conclude that the enhancement of lipoprotein lipase activity by apolipoprotein C-II is independent of the physical form of the phospholipid substrate.

Animals↗
PubMed1984

Synthesis and secretion of triacylglycerol lipase by cultured rat hepatocytes.

Rat hepatocytes isolated by collagenase perfusion were cultured for 48-72 h and examined for synthesis and secretion of hepatic triacylglycerol lipase activity. Low levels of enzyme activity found in the culture medium increased with time of incubation, and a 3-10-fold rise was encountered in the presence of optimal concentrations of heparin (5 U/ml). After interruption of enzyme synthesis by cycloheximide, plateauing of enzyme activity in the medium occurred, indicating that addition of heparin may not only stabilize but also enhance hepatic triacylglycerol lipase secretion. Synthesis and secretion of hepatic triacylglycerol lipase was not related to cell density, and enzyme secretion was encountered in subconfluent cultures. Release of enzyme activity into the medium was not sensitive to chlorpromazine, a lysosomal enzyme inhibitor, but was completely inhibited by treatment with tunicamycin, an inhibitor of glycosylation. As release of enzyme activity could be maintained for 12 h in the absence of serum, possible hormonal regulation was sought. Under the present experimental conditions, no modulation of hepatic triacylglycerol lipase was encountered by either gonadal or thyroid hormones. Addition of cyclic AMP to the culture medium resulted in a 30% decrease in enzyme activity. The dependence of hepatic triacylglycerol lipase secretion on the intactness of the Golgi apparatus and on vesicular transport was demonstrated by the treatment with monensin. The present results show that cultured rat hepatocytes provide a good model system by which the regulation of synthesis and secretion of hepatic triacylglycerol lipase can be studied.

1-Methyl-3-isobutylxanthine↗
PubMed1984

Substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas. I. Studies on neutral glycerides.

The substrate specificity of two cationic lipases with high phospholipase A1 activity purified from guinea pig pancreas has been tested towards various neutral glycerides. Triolein hydrolysis proceeded in the absence of di- and monoolein accumulation. Optimal conditions for di- and monoolein hydrolysis included an alkaline pH (9-10), a substrate concentration of 10 mM, and the presence of sodium deoxycholate (12 and 24 mM, respectively). Pancreatic colipase (bovine) had no effect on the activity of the two lipases. The comparison between the rates of hydrolysis of various substrates revealed the following order of decreasing enzyme activity: diolein greater than 1(3)-monoolein greater than tributyrin = triacetin greater than or equal to triolein = 2-monoolein. No hydrolysis of p-nitrophenylacetate and cholesteryloleate could be detected. Using 1-[3H]palmitoyl-2-[14C]linoleoyl-sn-glycerol, both enzymes displayed a strong preference for the 1-position, leading to the accumulation of 2-[14C]linoleoyl-sn-glycerol. Identical activities were found for the two lipases. It is concluded that the two cationic lipases from guinea pig pancreas represent a unique group of lipolytic enzymes different from other previously described enzymes, including classical pancreatic lipase, gastric and lingual enzymes, mold lipases and carboxylesterhydrolase.

Animals↗
PubMed1985

Secretion of human gastric lipase from dispersed gastric glands.

The presence of a triacylglycerol lipase in human gastric juice was described in previous studies. Its source and role in intragastric lipolysis was, however, uncertain. Our study presents definitive evidence for gastric origin of a lipase and its release by secretagogues. Both carbachol and cholecystokinin-8 stimulate release of this enzyme for dispersed human gastric glands. While the two secretagogues had similar efficacies, with nearly a 3-fold stimulation over basal rates, cholecystokinin-8 was about four orders of magnitude more potent in releasing lipolytic activity than carbachol (maximum stimulation at concentrations of 1 X 10(-9) and 1 X 10(-5) M, respectively). Lipolytic activity measured against triolein (18:1), tricaprylin (8:0) and tributyrin (4:0) emulsions was 1.18 +/- 0.12, 4.48 +/- 0.64, and 12.17 +/- 0.88 units (1 unit = 1 mumol free fatty acid released/min per mg protein), respectively. Characterization of the pH optimum for each substrate showed maximum lipolysis at 4.5 for tributyrin, and at 5.5 for tricaprylin and triolein. These results indicate that a lipase which hydrolyzes long-, medium- and short-chain triacylglycerols is secreted by human gastric mucosa. At pH 6.0, the pH of the duodenum, there is appreciable lipolytic activity in the presence of bile salts. This suggests that gastric lipase, in addition to initiating lipolysis in the stomach, might also aid in the digestion of lipids in the duodenum. It remains to be determined whether gastric lipase is distinct from lingual lipase, or is the same enzyme secreted by the lingual serous glands and the gastric mucosa.

Bile Acids and Salts↗