Lipase-like Activity Research Articles

An artificial self-assembling peptide with carboxylesterase activity and substrate specificity restricted to short-chain acid p-nitrophenyl esters.

Natural enzymes possess remarkable catalytic activity and high substrate specificity. Many efforts have been dedicated to construct artificial enzymes with high catalytic activity. However, how to mimic the exquisite substrate specificity of a natural enzyme remains challenging because of the complexity of the enzyme structure. Here, we report artificial carboxylesterases that are specific for short chain fatty acids and were constructed via peptide self-assembly. These artificial systems have esterase-like activity rather than lipase-like activity towards p-nitrophenyl esters. The designer peptides self-assembled into nanofibers with strong β-sheet character. The extending histidine units and the hydrophobic edge of the fibrillar structure collectively form the active center of the artificial esterase. These artificial esterases show substrate specificity for short-chain acids esters. Moreover, 1-isopropoxy-4-nitrobenzene could function as a competitive inhibitor of hydrolysis of p-nitrophenyl acetate for an artificial esterase.

Characterization of digestive enzymes during early ontogeny of white Snook (Centropomus viridis).

The white snook (Centropomus viridis) is a species with great aquaculture potential, therefore, the present study aims two objectives; 1) to characterize digestive enzymatic activity during larval development and; 2) characterize the diversity and functionality of digestive enzymes in an early juvenile stage of C. viridis. On first feeding, larvae were 2.75 ± 0.09 mm total length and grew to 24.35 ± 4.35 mm after 42 days. The specific activity of acid proteases was detected with low activity from day 1 (Days after hatching, DAH) to day 21, and increased at 23 DAH, reaching maximum activity at 40 DAH. Meanwhile, digestive enzymatic activity of alkaline proteases was observed from hatching to day 40. From hatching until 40 DAH, the presence of alkaline proteases, trypsin, chymotrypsin, leucine aminopeptidase, α-amylase and lipase were detected with different activity patterns. Total and specific activity of digestive enzymes on juveniles showed higher activity of acid than alkaline proteases, while α-amylase had a higher activity than lipases. The maximum activity of acid proteases was observed at 40 °C and at pH 2.0, while digestive alkaline proteases showed maximum activity at 60 °C. α-amylases and lipases showed their maximum activities at 50 °C. The maximum alkaline protease and α-amylase activities were found at pH 7 to 9. The enzymatic activity of juvenile's acid proteases was highly inhibited by Pepstatin A (96%), however the activity of alkaline proteases incubated with PHEN, TLCK, SBTI, PMFS, and OVO showed reductions of 51.29, 30.16, 27.98, 27.26, and 24.68% respectively. Lipase activity was totally inhibited with Ebelactone B, and highly inhibited by Ebelactone A, Orlistat, 1% SDS, and PMFS (96.12, 77.16, 64.22, and 51.29%, respectively). Seven bands (102.6, 65.7, 58.9, 40.6, 27.4, 21.5, and 15.8 kDa) resolved by protein electrophoresis were detected with alkaline protease activity, identified as serine proteases and metalloproteases, and three bands (57, 43.9, and 32.6 kDa) with lipase-like activity were also identified. These results show that C. viridis ontogeny entails an indirect development of its digestive system with a functional stomach on day 23, and characterized by increases in pepsin activity. Indeed, white snook ontogeny shows a similar developmental trajectory to other carnivorous marine fish, showing a diversity of alkaline proteases that work in a wide range of pH and temperatures. Thus, the identification of these enzymes provides advantages to formulate white snook aqua-feeds.

Postprandial serum triacylglycerols and oxidative stress in mice after consumption of fish oil, soy oil or olive oil: Possible role for paraoxonase-1 triacylglycerol lipase-like activity

Objective Postprandial triacylglycerols and oxidative stress responses are influenced by the type of fat consumed. We investigated the effect of individual unsaturated fatty acids or oils (fish, soy, or olive) on postprandial triglyceridemia response in association with serum resistance to oxidation and paraoxonase-1 (PON1) activity. Methods Balb/C mice were supplemented with phosphate buffered saline (control), docosahexaenoic acid (ω-3), linoleic acid (ω-6), or oleic acid (ω-9; 500 μg/300 μL of phosphate buffered saline) and with fish, soy, or olive oil (300 μL); blood samples were collected 2 h after feeding. Results Serum triacylglycerol and oxidative stress responses increased after intake of all unsaturated fatty acids and oil supplements. However, ingestion of fish oil or its major fatty acid, docosahexaenoic acid, induced the most remarkable increase in postprandial serum triacylglycerols and in the susceptibility of serum to in vitro oxidation. Serum PON1 activity was decreased by 24% after fish oil ingestion. The increase in postprandial serum susceptibility to oxidation was lower after soy oil supplementation to PON1-transgenic mice in comparison with Balb/C mice, showing that PON1 attenuates the postprandial serum oxidative response. In parallel, in PON1-transgenic mice, a decreased postprandial triacylglycerol response was noted, suggesting PON1 involvement in triacylglycerol metabolism. PON1 exhibited a triacylglycerol lipase-like activity on chylomicrons. Conclusion PON1 attenuates the postprandial oxidative stress response, and this could have resulted from PON1 lipase-like activity on chylomicron triacylglycerols.

Serum Opacity Factor, a Streptococcal Virulence Factor That Binds to Apolipoproteins A-I and A-II and Disrupts High Density Lipoprotein Structure

Serum opacity factor (SOF) is a virulence determinant of group A streptococci that opacifies mammalian sera. We analyzed the specificity and mechanism of the opacity reaction using a recombinant form of the amino-terminal opacification domain of SOF, rSOF. Our data indicate that rSOF is neither a protease nor a lipase, but rather it is the binding of rSOF to high density lipoprotein (HDL) that triggers the opacity reaction. rSOF did not opacify plasma from apoA-I(-/-) mice or purified low or very low density lipoproteins but readily opacified HDL. rSOF binding to HDL was characterized by two high affinity binding sites; it bound to apoA-I (K(d) = 6 nm) and apoA-II (K(d) = 30 nm), and both apoA-I and apoA-II blocked the binding of rSOF to HDL. Electron microscopic examination and biochemical analyses of HDL treated with rSOF revealed the formation of lipid droplets devoid of apolipoproteins. Thus, SOF interacts with HDL in human blood by binding to apoA-I and apoA-II and causing the release of HDL lipid cargo, which coalesces to form lipid droplets, resulting in opacification. The disruption of HDL may attenuate its anti-inflammatory functions and contribute to the pathogenesis of group A streptococcal infections.

Lipase and antibacterial activities of a recombinant protein from the accessory glands of femalePhlebotomus papatasi(Diptera: Psychodidae)

The reproductive accessory glands of the adult female have a functional versatility in insects, contributing to reproduction in various ways. The major protein secreted by the accessory glands of female Phlebotomus papatasi (Diptera, Psychodidae) has already been characterised and named PhpaLIP (for P. papatasi lipase) because, in terms of its amino-acid sequence, it is very similar to a wide range of vertebrate lipases. The gene coding for PhpaLIP has now been cloned into a pQE30 vector and expressed in Escherichia coli. When the recombinant PhpaLIP was tested in vitro, it was found to have not only lipase-like activity (when p-nitrophenyl caprylate was used as the substrate) but also specific antibacterial activity against some Gram-positive and Gram-negative bacteria. The possible physiological roles of PhpaLIP in P. papatasi are discussed, in the light of these results.

Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila.

Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

Enzymatic degradation of cellulose acetate plastic by Novel degrading bacterium Bacillus sp. S2055

Cellulose acetate (CA)-degrading bacteria were isolated from samples obtained from environments at a population size of 6.7 × 10 1 to 1.0 × 10 8 halo-forming cfu/ml-water or g-solid, suggesting their ubiquitous presence. The classification of 35 isolated strains of CA-degrading bacteria into 15 genera indicates that CA-degrading activity is over a wide range of taxonomical groups. From these isolates, Bacillus sp. S2055 was found to be the most efficient CA-degrading bacterium, and its CA-degrading enzyme(s) was partially characterized. The weight loss of CA plastic film (degree of substitution (DS)=1.7) in the culture of S2055 was less than 12% after a 35-d culture while that in the crude enzyme solution extracted from the culture supernatant reached 62% after the same period. Lipase and cellulase activities were detected in the culture supernatant of strain S2055. The crude enzyme solution contained three major protein fractions that have different mean molecular weights (MWs). Fraction I with the highest MW exhibited both lipase and cellulase activities, while fraction II and III exhibited only lipase activity. Fraction I significantly deacetylated CA (DS 1.5) and fragmented CA plastic film into pieces while the other fractions failed to do so even when used in combination with commercially-available cellulases and lipases. The lipase activity of fraction I against various substrates differed considerably from those of known lipases. It was thus suggested that deacetylation of CA mediated by an enzyme with such a peculiar lipase-like activity is a requisite for the efficient biodegradation of CA plastics.

Functional molecular mass of rat hepatic lipase in liver, adrenal gland and ovary is different.

Lipoprotein lipase (LPL) is functionally active only as a dimer. It is also generally assumed that the highly homologous hepatic lipase functions as a dimer, but no clear evidence has been presented. A hepatic lipase-like activity, also indicated as L-type lipase, is present in adrenal and ovary tissues. This enzyme is thought to originate from the liver and to be identical to hepatic lipase. We determined the functional molecular mass of hepatic lipase in rat liver, adrenal gland and ovary by radiation inactivation, a method for determining the functional size of a protein without the need of prior purification. Samples were exposed to ionizing radiation at -135 degrees C. Hepatic lipase activity in liver homogenate showed a single exponential decay. The functional molecular mass was calculated to be 63 +/- 10 kDa. Hepatic lipase activity in adrenal homogenate was found to have a functional molecular mass of 117 +/- 16 kDa. The functional molecular masses of the lipases partially purified from rat liver perfusate, adrenal homogenate or ovarian homogenate showed the same pattern, a target mass for the liver enzyme of 56 +/- 6 kDa and a target mass of 117 +/- 14 kDa for the enzyme from adrenal gland or ovary. In Western blot analysis the mass of the structural units of hepatic lipase in liver was 57 kDa and in adrenal and ovary tissue 51 kDa. We conclude that the functional unit of hepatic lipase in the liver is a monomer. The enzyme in adrenal gland and ovary is different from the liver and the functional unit may be a dimer.

Hepatic Lipase in the Rat Ovary: Ovaries cannot synthesize hepatic lipase but accumulate it from the circulation

Hepatic lipase is proposed to have a role in steroidogenesis through its involvement in the metabolism of high density lipoproteins. We examined the activity, synthesis, distribution, and uptake of this enzyme and assessed the content of its mRNA in luteinized ovaries. We found that during peak steroidogenesis, ovaries of pregnant mare's serum gonadotropin-human chorionic gonadotropin-treated immature rats contained heparin-releasable hepatic lipase-like activity which was neutralized in a dose-dependent manner by purified antibodies to hepatic lipase isolated from post-heparin perfusates of rat livers. Quantitative immunoelectron microscopy revealed that ovarian hepatic lipase occurred along endothelial cells and was 3-fold more abundant in blood vessels of corpora lutea than those of stroma. However, hepatic lipase was not synthesized by the ovary since radiolabeled enzyme was not immunoisolated from the medium of dispersed luteinized granulosa cells incubated with [35S]methionine whereas it was present in the medium of control cells (hepatocytes). Similarly, hepatic lipase mRNA was detectable in liver but not ovaries or kidneys by Northern or slot blot analyses or by the polymerase chain reaction. Finally, 125I-labeled hepatic lipase injected into tail veins was quickly cleared from the systemic circulation, accumulating in liver, ovaries, kidneys, and spleen. Subsequent heparin injection caused rapid reappearance of radioactivity in the bloodstream and a marked decline of radiolabel in liver and ovaries but a modest decrease of that in kidneys and none in spleen. Exogenous 125I-bovine serum albumin also accumulated in all four organs but was not displaced from liver or ovaries by subsequent administration of heparin. Taken together, these data suggest that steroidogenically active ovaries possess but do not synthesize hepatic lipase. Instead, hepatic lipase originating elsewhere, presumably in the liver, is accumulated from the circulation at heparin-sensitive sites in ovarian blood vessels.

Mouse preheparin plasma contains high levels of hepatic lipase with low affinity for heparin

It was recently noted that newborn mice have much higher lipase activity in plasma than rats or humans, and that most of the activity is due to an enzyme related to the hepatic (heparin-releasable) lipase. Here we report that this lipase is present in plasma of adult mice also. In contrast to the high activity of hepatic lipase, the activity of lipoprotein lipase in plasma was low and similar to that in rats. The source of the plasma lipase was probably the liver, since we could not demonstrate hepatic lipase-like activity in any other organ. When human hepatic lipase was injected into mice, it rapidly disappeared from plasma. Most of the injected lipase located in the liver, and could be released back into circulation by injection of heparin. These results indicate that there are binding sites for hepatic lipase in mouse liver, and suggest that mouse hepatic lipase has an affinity for these sites which is lower than usual. It is currently believed that the endothelial acceptors are heparan-sulfate or similar molecules. Mouse hepatic lipase eluted from heparin-Sepharose at lower salt concentration than rat or human hepatic lipase, demonstrating that it has a relatively low affinity for heparin-like polysaccharides.

Intralipid administration induces a lipoprotein lipase-like activity in the livers of starved adult rats.

The administration of Intralipid to starved adult rats induces the appearance of lipoprotein lipase (LPL)-like activity in the liver, whereas the so-called hepatic triacylglycerol lipase is unaffected. This LPL-like activity is eluted by 1.5 M-NaCl from heparin-Sepharose columns. This partially purified fraction is inhibited by 1.0 M-NaCl (91%) and by 1.0 mg of protamine sulphate/ml (79%), whereas it is stimulated 69-fold by the presence of 8.0 micrograms of apolipoprotein C-II/ml and inhibited by anti-LPL antibodies. We conclude that Intralipid administration induces the appearance of LPL activity in livers of starved adult rats. Its possible origin is discussed.

Demonstration of hepatic triglyceride lipase-like activity in ascites fluid of mice with sarcoma 180

The contents of apolipoproteins of plasma and ascites fluid of mice with Sarcoma 180 were measured. The apolipoprotein A-I contents of plasma decreased with development of the tumor. The apolipoprotein C-II and C-III contents of plasma reached a maximum on day 7 after tumor inoculation and then decreased. The apolipoprotein A-I content of the ascites fluid was lower than that of normal mouse plasma. In contrast, the apolipoprotein C-II and C-III contents of the ascites fluid were higher than those of normal mouse plasma. The ascites fluid of mice with Sarcoma 180 was found to contain at least two lipases. One had a pH optimum of 5.5–7.0 and was strongly inhibited by chlorpromazine. The other hand an alkaline pH optimum and was inhibited only slightly by chlorpromazine. When the ascites fluid was applied to a heparin-Sepharose column 40–45% of the applied triglyceride lipase activity was retained on the column, which was eluted with 0.75 M NaCl. This fraction was inhibited by heat-inactivated ( 56°C, 10 min) human serum, and was relatively resistant to 1 M NaCl. These results suggest that one of the lipolytic enzymes present in the ascites fluid of mice with Sarcoma 180 is hepatic triglyceride lipase.

Lipoprotein lipase-like activity in the liver of mice with Sarcoma 180.

The triglyceride lipase (TGL) activity of liver homogenates of mice with Sarcoma 180 was measured. The liver homogenate of normal or tumor-bearing mice was treated with 0.25% Triton X-100 and centrifuged at 100,000 g for 60 min, and the supernatant was applied to a heparin-Sepharose column. In normal mice, most of the TGL activities in the supernatant was eluted with 0.75 M NaCl from the column. In mice with Sarcoma 180, the TGL gave two peaks on heparin-Sepharose column chromatography, which were eluted with 0.75 M and 1.5 M NaCl, respectively. The activity in the first peak (0.75 M NaCl eluate) decreased; that in the second peak (1.5 M NaCl eluate) increased, and the ratio of the second peak to the first peak increased during tumor development. The livers of normal mice and mice on day 10 after tumor inoculation were perfused with heparin. The highest rate of the TGL release occurred within 1 min of heparin perfusion, and the bulk of heparin-releasable activity appeared within 2 min of perfusion in both normal and tumor-bearing mice. The TGL activity in liver perfusate of tumor-bearing mice, as well as that of liver homogenate, was resolved on a heparin-Sepharose column into two peaks, which were eluted with 0.75 M and 1.5 M NaCl, and most of the activity was eluted with 1.5 M NaCl. The nature of the TGL activity eluted from a heparin-Sepharose column was investigated. In both liver homogenates and liver perfusates, the first peak did not require serum for maximal activity and was relatively resistant to a high concentration of NaCl or protamine sulfate.(ABSTRACT TRUNCATED AT 250 WORDS)

Very low density lipoproteins promote triglyceride accumulation in macrophages.

Incubation of mouse peritoneal macrophages with very low density lipoproteins (VLDL) from normal rats or rhesus monkeys markedly increased the levels of intracellular triglycerides by 10- to 56-fold and was accompanied by the production of oil red O positive vacuoles. The stimulation of triglyceride accumulation in macrophages was time- and concentration-dependent and was specific for VLDL. Three possible mechanisms for the VLDL-stimulated triglyceride accumulation in macrophages were explored: receptor-mediated uptake, action of lipoprotein lipase, and phagocytosis. Macrophage uptake and degradation of 125I-monkey and rat VLDL demonstrated saturable and nonsaturable components. Uptake of 125I-VLDL could be inhibited by unlabeled normal VLDL, although hyperlipemic VLDL was more effective. HDL did not compete to a significant extent. Heparin released lipoprotein lipase-like activity from peritoneal macrophages. Addition of heparin with VLDL resulted in a greater, more rapid elevation in intracellular triglycerides, which was partially inhibited by albumin. Free fatty acid and Intralipid also produced triglyceride accumulation in macrophages. The data showed that all three of the mechanisms examined could contribute to the metabolism of VLDL by macrophages and cause the production of triglyceride-rich cells with a "foamy" appearance, although the evidence suggested that the action of lipoprotein lipase was probably the most important in this process.

Liver lipase-like activity in human and hamster adrenocortical tissue

Human adrenocortical tissue was found to contain a lipase activity that could be measured in vitro in the presence of 0.6 M NaCl at pH 8.5. The lipase was largely (80%) inhibitable by an antibody raised against heparin-releasable liver lipase. The activity of the lipase per gram tissue wet weight was threefold higher in hyperplastic than in normal adrenocortical tissue (165 μU vs 50 μU). No or very low lipase activity was found in adrenocortical tumors. When, employing the same assay system, lipase activity was measured in the adrenal gland of golden hamsters a very low activity, compared with rat adrenal gland, — was found (13 mU/g w.w. vs 225 mU/g w.w.). Since liver lipase may be involved in the uptake of cholesterol from serum high density lipoproteins by the liver, the presence of a similar lipase in adrenal glands suggests that this tissue can also take up cholesterol by a lipase-mediated mechanism in human and rat. In human adrenocortical carcinomas and in the normal hamster gland such a mechanism seems to be absent.

Glucagon and plasma lipoprotein lipase.

SummarySubcutaneous administration of 1.0 mg glucagon to 15 fasted normal people caused significant increase of plasma lipoprotein lipase-like activity in 1 hr. The activity is termed lipase-like since coconut oil emulsion rather than isolated chylomicrons was used as substrate. A linear relationship between lipolysis and time of incubation obtained indicating zero order kinetics. pH optimum of enzyme activity was 8.4. There was no correlation between depression of plasma triglycerides and increase -of plasma lipoprotein lipase-like activity following glucagon administration. This was thought to be due to other effects of glucagon administration which alter plasma lipid levels. Incubation of whole blood with glucagon caused an increase of plasma lipoprotein lipase-like activity and a depression of plasma triglycerides that were significantly correlated. The in vitro action of glucagon when incubated with whole blood suggests that blood cell(s) may be a source of glucagon-induced plasma lipoprotein lipase ...