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PubMed1973

Glyceride lipases in nerve endings of guinea-pig brain and their stimulation by noradrenaline, 5-hydroxytryptamine and adrenaline.

1. Combined guinea-pig cortex and cerebellum was shown to contain triglyceride lipase, diglyceride lipase and monoglyceride lipase, which were assayed by the release of [1-(14)C]palmitate from [1-(14)C]palmitoylglycerol esters. Triglyceride lipase and diglyceride lipase were found in all particulate fractions. 2. With osmotically ruptured synaptosomes the rates of release of palmitate from glyceryl tripalmitate and glyceryl dipalmitate were 7-25mumol/h per g of protein and 0.18-0.69mmol/h per g of protein respectively. The logarithm of the rate of hydrolysis of glyceryl monopalmitate increased linearly with the logarithm of protein concentration. The pH optima of triglyceride lipase and diglyceride lipase were between 7 and 8. The pH optimum for monoglyceride lipase was approx. 8. 3. Triglyceride lipase and diglyceride lipase of osmotically ruptured synaptosomes were stimulated by noradrenaline, 5-hydroxytryptamine and adrenaline. Triglyceride lipase of isolated synaptic membranes was stimulated by 0.01-1mm-noradrenaline. Aging of membranes at 0 degrees C decreased activity, which could still be stimulated by noradrenaline. Diglyceride lipase of isolated membranes was stimulated by 1mum-1mm-noradrenaline. The activity of triglyceride lipase in isolated synaptic vesicles was diminished by 1mm-5-hydroxytryptamine.

Animals↗
PubMed2006

Different specificity of two types of Pseudomonas lipases for C20 fatty acids with a Delta5 unsaturated double bond and their application for selective concentration of fatty acids.

Two kinds of lipases, AK-lipase and HU-lipase, produced by two different Pseudomonas fluorescens strains, AK102 and HU380, respectively, were evaluated as to fatty acid hydrolysis specificity using six types of oil containing higher amounts of C20 fatty acids such as arachidonic acid (5,8,11,14-eicosatetraenoic acid, AA, or 20:4omega6), dihomo-gamma-linolenic acid (8,11,14-eicosatrienoic acid, DGLA, or 20:3omega6), 5,8,11,14,17-eicosapentaenoic acid (EPA or 20:5omega3), mead acid (5,8,11-eicosatrienoic acid, MA, or 20:3omega9), 8,11-eicosadienoic acid (20:2omega9) and 8,11,14,17-eicosatetraenoic acid (20:4omega3). Although HU-lipase did not show any specificity for C20 fatty acids with respect to the presence or absence of a Delta5 unsaturated bond, it exhibited comparatively low reactivity for 4,7,10,13,16,19-docosahexaenoic acid (DHA or 22:6omega3). In contrast, AK-lipase was less reactive for C20 fatty acids with a Delta5 unsaturated bond. However, the specificity of hydrolysis of AK-lipase gradually decreased as the reaction proceeded. Utilizing this fatty acid specificity, we concentrated either EPA or DHA from fish oils containing both EPA and DHA by means of lipase-catalyzed hydrolysis and urea adduction. Hydrolysis and urea adduction of refined cod oil including 12.2% EPA and 6.9% DHA with HU-lipase provided free fatty acids with 43.1% EPA and 7% DHA, respectively. The resulting yield of concentrated total fatty acids comprised 2.6% of the fatty acids from the cod oil. Thus, EPA was particularly concentrated in the fatty acids derived from refined cod oil on partial hydrolysis with HU-lipase followed by urea adduction. On the other hand, hydrolysis of cuttlefish oil with AK-lipase followed by urea adduction increase slightly the EPA composition from 14.2% to 16.8%, and markedly enhanced the composition of DHA from 16.3% to 44.6% in the hydrolyzed fatty acids. The yield of purified total fatty acids by urea concentrate was 9.4% of the fatty acids from the cuttlefish oil. Thus, DHA was particularly concentrated in the fatty acids derived from on partial hydrolysis with AK-lipase followed by urea adduction. We concluded that EPA and DHA concentrates can be easily and inexpensively obtained using HU-lipase and AK-lipase, respectively. Furthermore, it might be possible to separate and concentrate C20 polyunsaturated fatty acids (PUFAs) with or without a Delta5 double bond from PUFAs rich oils including both fatty acids.

Chemical Fractionation↗
PubMed1986

Iodine-125-labeled lipoprotein lipase as a tool to detect and study spontaneous lipolysis in bovine milk.

The distribution of lipoprotein lipase among cream, casein, and milk serum can be evaluated by addition of a trace amount of 125I-labeled lipoprotein lipase to milk. Radioactive lipase was distributed in parallel to endogenous lipase under several conditions. In some milk samples, binding of lipase to cream increased when the milk was cooled. Correlation was good between bound labeled lipase and degree of cold-induced lipolysis in corresponding milk samples. Binding of lipase to cream or to casein was not saturable by addition of two-to threefold more lipase than is normally present in milk. In milk with a relatively high fraction of lipase bound to cream, a correspondingly lower fraction was associated with casein, whereas the fraction of lipase in milk serum was similar in all milk samples. Cold-induced binding of lipoprotein lipase to cream was not fully reversed when the milk was warmed again. Heparin released lipase from casein and increased the amount of lipase bound to cream after cooling.

Animals↗
PubMed1982

Hepatic triacylglycerol lipase activities after induction of diabetes and administration of insulin or glucagon.

Triacylglycerol lipase activities of homogenates and subcellular fractions of rat liver were measured under optimal conditions at pH 7.5 using emulsified tri[1-14C]oleoylglycerol as substrate. Twenty-four hr after administration of streptozotocin, hepatic alkaline lipase activity was 39% of normal, and this lower level of activity was observed at 72 hr and 7 days, after streptozotocin injection. After 24 hr of starvation, lipase activity also was significantly lower (35%) than normal. Insulin (35 U regular/kg body weight) had no acute (90 min) effect on the hepatic lipase activity of either normal or diabetic rats. Chronic insulin administration (4 subcutaneous injections of 10 U protamine zinc insulin/kg at 16-hr intervals) to normal rats provoked a 40% increase in hepatic lipase activity. Diabetic rats given the same insulin treatment showed lipase activity that was significantly higher (155%) than normal. Lipase activity fell to 65% of normal when insulin was withheld (32 hr) from diabetic rats given chronic insulin therapy. Intracardial injection of glucagon (1 mg/kg) into normal rats had no acute (30 min) effect on hepatic alkaline lipase activity. Hepatic alkaline lipase activity varied independently from the concentrations of either glucose or triacylglycerol in the plasma. However, there was an apparent negative correlation between this lipase activity and the concentration of fatty acids in the plasma; lipase activity was highest when fatty acid concentrations were lowest, and lowest when fatty acid concentrations were elevated. From these data we conclude: 1) changes in hepatic alkaline lipase activity ware provoked by chronic, but not acute, alteration of the hormonal and metabolic status of the rat, and 2) changes in hepatic alkaline lipase activity may be mediated through changes in the levels of circulating fatty acids presented to the liver, but the effect is not an immediate one.

Animals↗
PubMed1995

Comparative biochemical and molecular analysis of the Staphylococcus hyicus, Staphylococcus aureus and a hybrid lipase. Indication for a C-terminal phospholipase domain.

The lipase gene, geh, from Staphylococcus aureus NCTC8530 was cloned in Staphylococcus carnosus. DNA sequencing revealed an open reading frame (ORF) of 2046 nucleotides encoding a 682-amino-acid protein with a molecular mass of 76900 Da. Determination of the transcriptional start site revealed a 203-nucleotide mRNA leader. Expression of geh in the protease-negative S. carnosus (pT181copSA22) resulted in overexpression of a 83-kDa lipase found in the culture supernatant. N-terminal protein sequencing and sequence comparison with three other staphylococcal lipases suggest that this lipase is organised as a pre-pro-enzyme. The substrate specificity of this lipase is different from the Staphylococcus hyicus lipase. The S. hyicus lipase expressed both a high Ca(2+)-dependent phospholipase and lipase activity while the S. aureus lipase lacked this phospholipase activity and its activity with tributyrylglycerol or p-nitrophenyl octanoate is hardly stimulated by Ca2+ ions. A hybrid protein was constructed in which the C-terminal 146 residues of the S. hyicus lipase were substituted by 145 residues of the C-terminal of the S. aureus lipase, which contains the proposed active-site amino acids Asp602 and His641. The hybrid enzyme was still active and revealed an intermediary enzymic activity. The most striking effect was that it had lost the S. hyicus-specific phospholipase activity and that, in contrast to the two parental enzymes, its activity with p-nitrophenyl octanoate became highly sensitive to the presence of Ca2+. These observations suggest that the C-terminal domain of the S. hyicus lipase strongly contributes to the binding pocket of the polar headgroup of phospholipids. The Ca(2+)-binding site seems to be located in the N-terminal fragment of the S. hyicus lipase. The fact that two closely related enzymes differ in the need for Ca2+ underscores the notion that it plays a structural rather than a catalytic role.

Amino Acid Sequence↗
PubMed1993

Apolipoprotein E modulates low density lipoprotein retention by lipoprotein lipase anchored to the subendothelial matrix.

Lipoprotein lipase (lipase), a key enzyme in lipoprotein triglyceride metabolism, has been shown to markedly increase low density lipoprotein (LDL) retention by subendothelial matrix. In the present study we assessed the role that lipoprotein and matrix components play in retention of LDL by lipase anchored to the subendothelial matrix. Lipase addition to subendothelial matrix increased LDL retention by 66-fold. Scatchard analysis of LDL binding to lipase-containing matrix yielded an association constant of 12 nM. Exogenous addition of the matrix components, heparan sulfate and dermatan sulfate (i.e. chondroitin sulfate B), reduced LDL retention by greater than 90%. These glycosaminoglycans (GAGs) also reduced lipolytic activity associated with the matrix, suggesting that lipase was released from its binding sites on the matrix. In contrast, other matrix components (collagen, fibronectin, vitronectin, and chondroitin sulfate A) neither affected LDL release nor matrix lipolytic activity. Thus, heparan sulfate and dermatan sulfate function to anchor lipase to the subendothelial cell matrix. The effects of apolipoprotein E (apoE) and apoA-I were also examined. Preincubation of the subendothelial matrix with apoE, followed by washing, did not affect subsequent lipase binding to the matrix nor its ability to retain LDL. However, the direct addition of apoE alone or in combination with phospholipid liposomes decreased lipase-mediated LDL retention in a concentration-dependent fashion. Addition of apoA-I had no effect. Thus, in these studies apoE functions to displace LDL bound to lipase, but not lipase anchored to the matrix. To further examine the physiologic implications of this process, we assessed the ability of human apoE-rich and apoE-poor high density lipoproteins (HDL) to displace LDL from matrix-anchored lipase. ApoE-rich HDL reduced LDL retention dramatically (86% at 2.5 micrograms/ml). In contrast, apoE-poor HDL, at the highest concentration evaluated (400 micrograms/ml), decreased LDL retention by only 32%. Overall, these data suggest apoE and specifically apoE-containing HDL reduce the lipase-mediated retention of LDL by subendothelial matrix. This observation, in part could explain the protective effects of apoE and apoE-containing HDL against atherosclerosis.

Animals↗
PubMed1980

Lipase Induction in Mucor hiemalis.

The influence on lipase induction in Mucor hiemalis of different types of triglycerides containing mainly oleic acid (olive oil), erucic acid (mustard oil), or saturated fatty acids of 8 to 16 carbons (coconut oil) was studied. The fungus was grown in shake flasks in a fermentation medium containing peptone, minerals, and glucose or one of the oils as the carbon source. Maximum lipase was produced when the initial pH of the fermentation medium was kept at 4.0. Addition of Ca to the medium did not increase lipase production. The optimum pH for activity of both the mycelial and extracellular lipases was found to be 7.0. The fungus produced a significant amount of lipase in the presence of glucose, but the lipase activity increased markedly when olive oil was added to the medium at the beginning of the fermentation. Addition of olive oil at a later stage did not induce as much enzyme. Studies with washed mycelia showed that a greater amount of lipase was released when olive oil was present than when glucose was present. Among the various types of triglycerides used as the carbon source, olive oil was found to be most effective in inducing the lipase. Olive oil and mustard oil fatty acids inhibited the lipase more than those of coconut oil. The lipase induced by a particular type of triglyceride did not seem to be specific for the same triglyceride, nor was it inhibited specifically by it. Irrespective of the triglyceride used in the fermentation medium, the lipase produced was most active against coconut oil triglyceride, and this specificity, as shown by lipase activities in an n-heptane system, was not found to be due to a better emulsification of this oil. The lipase of M. hiemalis can be considered to be both constitutive and inducible.

Journal Article↗
PubMed1989

The rabbit as an animal model of hepatic lipase deficiency.

A natural deficiency of hepatic lipase in rabbits has been exploited to gain insights into the physiological role of this enzyme in the metabolism of plasma lipoproteins. A comparison of human and rabbit lipoproteins revealed obvious species differences in both low-density lipoproteins (LDL) and high-density lipoproteins (HDL), with the rabbit lipoproteins being relatively enlarged, enriched in triacylglycerol and depleted of cholesteryl ester. To test whether these differences related to the low level of hepatic lipase in rabbits, whole plasma or the total lipoprotein fraction from rabbits was either kept at 4 degrees C or incubated at 37 degrees C for 7 h in (i) the absence of lipase, (ii) the presence of hepatic lipase and (iii) the presence of lipoprotein lipase. Following incubation, the lipoproteins were recovered and subjected to gel permeation chromatography to determine the distribution of lipoprotein components across the entire lipoprotein spectrum. An aliquot of the lipoproteins was subjected also to gradient gel electrophoresis to determine the particle size distribution of the LDL and HDL. Both hepatic lipase and lipoprotein lipase hydrolysed lipoprotein triacylglycerol and to a much lesser extent, also phospholipid. There were, however, obvious differences between the enzymes in terms of substrate specificity. In incubations containing hepatic lipase, there was a preferential hydrolysis of HDL triacylglycerol and a lesser hydrolysis of VLDL triacylglycerol. By contrast, lipoprotein lipase acted primarily on VLDL triacylglycerol. When more enzyme was added, both lipases also acted on LDL triacylglycerol, but in no experiment did lipoprotein lipase hydrolyse the triacylglycerol in HDL. Coincident with the hepatic lipase-induced hydrolysis of LDL and HDL triacylglycerol, there were marked reductions in the particle size of both lipoprotein fractions, which were now comparable to those of human LDL and HDL3, respectively.

Chromatography, Gel↗
PubMed1980

Modification of Rhizopus delemar lipase by its binding with phospholipids.

Rhizopus delemar (ATCC 34612) lipase was modified by phospholipid (PL)-treatment so as to enhance its activity on lipoprotein. In order to detect change in lipase conformation in the modified state, a preliminary experiment was performed to remove PL from the PL-treated lipase solution which included PL nonessential to enhancement of lipoprotein lipase (LPL) activity. It was found that treatment with a mixture of isopropyl ether: n-butanol (3 : 1) was suitable for this purpose because of the stability of the enzyme. Changes in isoelectric point and alpha-helical content of lipase caused by PL-treatment were studied by means of isoelectric focusing and circular dichroism spectrum. The isoelectric point of lipase was found to shift to the acidic side on its binding with phosphatidylcholine (PC) or cardiolipin (CL). The circular dichroism spectra of the original lipase and PL-treated lipases indicated that the alpha-helical content of lipase decreased on its binding with PL. In CL-bound lipase, which was more greatly enhanced as to LPL activity than was PC-bound lipase, alpha-helical content was decreased to a larger extent than that of PC-treated lipase.

Cardiolipins↗
PubMed1975

Effect of fasting on two postheparin plasma triglyceride lipases and triglyceride removal in obese subjects.

A new method was used for selective measurement of lipoprotein lipase and hepatic lipase in human postheparin plasma. Hepatic lipase was assayed in 1.0 M NaCl withour addition of serum, and the activity of lipoprotein lipase was determined in 0.1 M NaCl after immunoprecipitation of hepatic lipase with specific antiserum. The activity of both these enzymes and the total lipolytic activity were measured in plasma samples taken during a 4-h infusion of heparin. Each of the activities was related to basal serum triglyceride concentration and to the fractional removal constant (K) of Intralipid in 13 obese subjects before and after prolonged fasting. During a normal isocaloric diet the lipolytic activities showed a biphasic response to heparin infusion in all subjects. A peak activity was reached within 30 minutes ("early response") and thereafter the lipase activities decreased to a constant level maintained during the rest of the heparin infusion ("late response"). The early response of lipoprotein lipase showed a significant inverse correlation with the basal serum triglyceride level (r = -0.85) and a significant positive correlation with the fractional removal rate of Intralipid (r = 0.84). The late response of lipoprotein lipase was not related to either of these parameters. The early response of hepatic lipase was not correlated with basal triglyceride concentration or Intralipid removal, whereas the late response of this enzyme showed a significant negative correlation with the removal rate of Intralipid (r = -0.82). After fasting for several days the acute response of all lipolytic activities to heparin was markedly decreased or totally abolished, but the magnitude of the late response was similar to that seen in the fed state. The fractional removal rate of Intralipid was slightly increased by starvation. All correlations between postheparin plasma lipases and serum triglyceride concentration and removal disappeared in fasting subjects. It is concluded that the rapidly releasable lipoprotein lipase probably reflects the activity of the tissue enzyme(s) which is responsible for the primary removal of very low density lipoprotein (VLDL) triglycerides and chylomicrons. It is probable that this component of the postheparin plasma lipolytic activity is derived from the endothelial lipoprotein lipase pool. This enzyme plays a key role in the efflux of plasma triglycerides under normal conditions, and it is thus one determinant of plasma triglyceride level. Prolonged fasting obviously changes the triglyceride removal sites and mechanism but does not impair the removal efficiency.

Adult↗
PubMed2000

Serum lipase levels as a diagnostic marker in cystic fibrosis patients with normal or borderline sweat tests.

Patients with normal or borderline sweat test present a diagnostic challenge. In spite of the availability of different methods such as genetic analysis and measurements of nasal potential difference, uncertainty in diagnosing cystic fibrosis (CF) in some patients still exists. Neonates with CF have high serum lipase levels, which decline over time in pancreatic-insufficient patients, whereas pancreatic-sufficient patients demonstrate high serum lipase levels beyond infancy. Because patients with borderline or normal sweat test are almost always pancreatic sufficient, this study was aimed to assess whether serum lipase levels may be of help in establishing the diagnosis of CF in these patients. Serum lipase levels were measured in 100 CF patients and in 17 healthy individuals. Patients were grouped according to their genotype. Group A patients (n = 70) carried two mutations previously found to be associated with a pathologic sweat test and pancreatic insufficiency (delta F508, W1282X, G542X, N1303K, S549R). Group B (n = 30) were compound heterozygote patients who carried one mutation known to cause mild disease with borderline or normal sweat tests and pancreatic sufficiency (3849+10kb C-->T, 5T). Group C included 17 healthy controls. Serum lipase levels ranged between 2 and 104.4 U/L (mean +/- SD 16.9 +/- 14.7), 6.1-200 U/L (mean +/- SD 53.9 +/- 47.9), and 8.5-27.8 U/L (mean +/- SD 16.9 +/- 5.1) in Groups A, B, and C, respectively, with some overlapping between groups. The distribution of lipase levels was significantly different in Group B vs Groups A and C (P < 0.01). High lipase levels were found in 63.3% (19/30) of Group B patients, but in only 4.3% (3/70) and 0% (0/17) of Group A and C, respectively. Lipase levels were found to be inversely related to sweat chloride concentrations (r = -0.19, P < 0.05). Patients with borderline or normal sweat tests had high lipase levels, whereas low lipase levels were associated with pathologic sweat tests. Our findings indicate that the serum lipase level is genetically determined and that it has a useful role in the diagnosis of CF. Thus, in patients with borderline sweat tests and high lipase levels, the diagnosis of CF should be considered.

Adult↗
PubMed1988

Interference of Staphylococcus aureus lipase with human granulocyte function.

The influence of purified Staphylococcus aureus lipase on granulocyte function and morphology was studied. The lipase itself was strongly chemotactic; in addition preincubation of granulocytes with low concentrations of lipase enhanced the directed movement, as assayed in the agarose system. Higher concentrations of lipase, in contrast, gave a progressive reduction of granulocyte chemotaxis; at 12 micrograms lipase per ml, cells were almost immobilized. Phagocytic killing of Staphylococcus aureus by granulocytes preincubated with lipase was reduced in a dose-dependent manner. At 12 micrograms lipase per ml almost no staphylococcal killing occurred. This was mainly accounted for by a reduction of bacterial uptake, but some decrease in intragranulocytic killing was also noted. These functional alterations, which can all be ascribed to an interference with membrane functions, were associated with marked changes of the granulocyte surface structure, which was denuded and lacked normal microvilli. The effects of lipase were partly retained after heat inactivation of lipase activity, indicating that the effects of staphylococcal lipase on granulocyte function are not due to enzymatic activity alone. These effects of lipase may be an important virulence factor and contribute to the preferential location of lipase-producing Staphylococcus aureus strains at deep sites of infection.

Chemotaxis, Leukocyte↗
PubMed2004

Bacterial lipases: an overview of production, purification and biochemical properties.

Lipases, triacylglycerol hydrolases, are an important group of biotechnologically relevant enzymes and they find immense applications in food, dairy, detergent and pharmaceutical industries. Lipases are by and large produced from microbes and specifically bacterial lipases play a vital role in commercial ventures. Some important lipase-producing bacterial genera include Bacillus, Pseudomonas and Burkholderia. Lipases are generally produced on lipidic carbon, such as oils, fatty acids, glycerol or tweens in the presence of an organic nitrogen source. Bacterial lipases are mostly extracellular and are produced by submerged fermentation. The enzyme is most commonly purified by hydrophobic interaction chromatography, in addition to some modern approaches such as reverse micellar and aqueous two-phase systems. Most lipases can act in a wide range of pH and temperature, though alkaline bacterial lipases are more common. Lipases are serine hydrolases and have high stability in organic solvents. Besides these, some lipases exhibit chemo-, regio- and enantioselectivity. The latest trend in lipase research is the development of novel and improved lipases through molecular approaches such as directed evolution and exploring natural communities by the metagenomic approach.

Bacillus↗
PubMed2001

Cloning and expression analysis of NhL1, a gene encoding an extracellular lipase from the fungal pea pathogen Nectria haematococca MP VI (Fusarium solani f. sp. pisi) that is expressed in planta.

The filamentous fungus Nectria haematococca (anamorph Fusarium solani f. sp. pisi) resides in soil, and attacks pea seedlings in the area of the underground epicotyl and upper tap root, causing foot rot disease. We detected lipase activity during in vitro growth of N. haematococca. Subsequently, a lipase gene was cloned and functionally characterised by heterologous expression in Saccharomyces cerevisiae. The full-length cDNA of 1152 bp was cloned using a 3' RACE-PCR approach coupled with cDNA library screening. The genomic clone, comprising an ORF of 999 bp interrupted by two introns of 56 and 64 bp, was isolated from a newly constructed lambda phage library. Analysis of the deduced protein sequence revealed the presence of a typical signal peptide at the N-terminus, and of the three conserved amino acids forming the active site of lipases. The lipase of N. haematococca has a low degree of similarity to the lipases from Humicola lanuginosa (37.2%), Rhizomucor miehei (21.6%), Rhizopus delemar (23.1%), Rhizopus niveus (25.9%), and to mono- and diacylglycerol lipase from Penicillium camembertii (30.8%), and very high similarity (94.6%) to a lipase from Fusarium heterosporum. The lipase from N. haematococca shows maximal activity at 37 degrees C and pH 8.0. Based on Southern analysis, the lipase clone represents a single-copy gene in N. haematococca. Expression analysis was performed by RT-PCR. In vitro, the lipase gene shows a low basal expression, but is highly inducible by lipase substrates, and repressed by glucose. During plant infection, transcripts of this fungal lipase gene were detected 4, 8, and 10 days after infection.

Amino Acid Sequence↗
PubMed2002

Distinction between esterases and lipases: a kinetic study with vinyl esters and TAG.

The better to characterize enzymes hydrolyzing carboxyl ester bonds (carboxyl ester hydrolases), we have compared the kinetic behavior of various lipases and esterases against solutions and emulsions of vinyl esters and TAG. Short-chain vinyl esters are hydrolyzed at comparable rates by esterases and lipases and have higher limits of solubility in water than corresponding TAG. Therefore, they are suited to study the influence of the physical state of the substrate on carboxyl ester hydrolase activity within a large concentration range. Enzymes used in this study are TAG lipases from microorganisms, lipases from human and guinea pig pancreas, pig liver esterase, and acetylcholinesterase. This study also includes cutinase, a fungal enzyme that displays functional properties between esterases and lipases. Esterases display maximal activity against solutions of short-chain vinyl esters (vinyl acetate, vinyl propionate, and vinyl butyrate) and TAG (triacetin, tripropionin, and tributyrin). Half-maximal activity is reached at ester concentrations far below the solubility limit. The transition from solution to emulsion at substrate concentrations exceeding the solubility limit has no effect on esterase activity. Lipases are active on solutions of short-chain vinyl esters and TAG but, in contrast to esterases, they all display maximal activity against emulsified substrates and half-maximal activity is reached at substrate concentrations near the solubility limit of the esters. The kinetics of hydrolysis of soluble substrates by lipases are either hyperbolic or deviate from the Michaelis-Menten model and show no or weak interfacial activation. The presence of molecular aggregates in solutions of short-chain substrates, as evidenced by a spectral dye method, likely accounts for the activity of lipases against soluble esters. Unlike esterases, lipases hydrolyze emulsions of water-insoluble medium- and long-chain vinyl esters and TAG such as vinyl laurate, trioctanoin, and olive oil. In conclusion, comparisons of the kinetic behavior of carboxyl ester hydrolases against solutions and emulsions of vinyl esters and TAG allows the distinction between lipases and esterases. In this respect, it clearly appears that guinea pig pancreatic lipase and cutinase are unambiguously classified as lipases.

Acetylcholinesterase↗
PubMed1998

Studies of lipase-catalyzed esterification reactions of some acetylenic fatty acids.

Esterification of five positional isomers of acetylenic fatty acids [viz. 9:1(2a), 11:1(10a), 18:1(6a), 18:1(9a) and 22:1(13a)] of different chain lengths with n-butanol in n-hexane in the presence of eight different lipases [Lipozyme IM 20 (Rhizomucor miehei), Lipolase 100T (R. miehei), Novozyme 435 (Candida antarctica), PPL (porcine pancreatic lipase), CCL (C. cylindracea), PS-D (Pseudomonas cepacia), Lipase A-12 (Aspergillus niger) and Lipase AY-30 (C. rugosa)] was studied. 2-Nonynoic acid was not esterified except when catalyzed by the lipase from C. antarctica (Novozyme 435) to give 42% butyl ester after 48 h. The lipases from A. niger (Lipase A-12) and C. rugosa (Lipase AY-30) showed poor biocatalytic behavior in the esterification of the acetylenic fatty acids studied. 10-Undecynoic acid gave the highest conversion rate of esterification with each kind of lipase used. 6-Octadecynoic acid showed a marked degree of resistance to esterification carried out in the presence of C. cylindracea (CCL), P. cepacia (PS-D), or porcine pancreatic (PPL) lipase but not significantly in the presence of the lipases of R. miehei (Lipozyme IM 20), R. miehei (Lipolase 100T), or Novozyme 435. 9-Octadecynoic acid and 13-docosynoic acid were not discriminated and were readily esterified by the remaining six lipases, but when compared to oleic acid the acetylenic fatty acids were comparatively much slower in conversion to the esters.

Acetylene↗
PubMed1987

Mechanisms for turnover of lipoprotein lipase in guinea pig adipocytes.

Guinea-pig adipocytes released lipoprotein lipase activity to the medium without depletion of cell-associated lipoprotein lipase activity. Heparin caused immediate release of 20-25% of the lipase activity to the medium, and also enhanced the continued release. After addition of cycloheximide, cell-associated lipoprotein lipase activity decreased rapidly. Release of lipase activity to the medium continued unabated for about 30 min, but there was little release thereafter. The release accounted for only about 25% of the initial lipoprotein lipase activity in the absence and about 50% in the presence of heparin. In pulse-chase experiments with [35S]methionine, labeled lipoprotein lipase appeared in the medium within 40 min, and most of the release occurred during the first h of chase. In a 4-h chase the total (cells + medium) amount of labeled lipase decreased to 34%. Thus, degradation was a main fate of the lipase. Heparin markedly increased the amount of labeled lipase that was released to the medium and decreased the amount that was degraded. Heparin did not change the time-course for the release, and the amount of labeled lipase degraded was proportional to the amount not released to the medium, indicating that the effect of heparin was primarily on release, not on degradation as such. This study demonstrates that adipocytes synthesize lipoprotein lipase in excess of what is being released, and that the excess is rapidly degraded.

Adipose Tissue↗
PubMed1992

Substrate specificities of lipases A and B from Geotrichum candidum CMICC 335426.

The mould Geotrichum candidum produces extracellular lipases with different substrate specificities according to strains. We purified two lipases - termed lipase A and lipase B - from Geotrichum candidum CMICC 335426. The specificity of the two lipases was investigated using hydrolysis assays on emulsions of pure acylglycerols and a wide range of fatty acid esters. Lipase B was very highly specific for hydrolysis of esters of cis-delta 9-fatty acids. Lipase A did not show such strict specificity, because it hydrolysed a wider variety of fatty acid esters, in particular those of palmitic acid and isomers of oleic acid. We think that differences in specificity previously observed for crude lipases from various strains of G. candidum can be explained by the presence of different levels of specific (lipase B) and non-specific (lipase A) lipases. As lipases A and B are structurally related proteins, a minor variation in structure may be responsible for the differing specificities.

Chromatography, Liquid↗