Neutral Lipase Research Articles

Stimulation of hormone-sensitive lipase activity by contractions in rat skeletal muscle.

Because the enzymic regulation of muscle triglyceride breakdown is poorly understood we studied whether neutral lipase in skeletal muscle is activated by contractions. Incubated soleus muscles from 70 g rats were electrically stimulated for 60 min. Neutral lipase activity against triacylglycerol increased after 1 and 5 min of contractions [0.36 +/- 0.02 (basal) versus 0.49 +/- 0.05 (1 min) and 0.54 +/- 0.05 (5 min) m-unit.mg of protein(-1), means +/- S.E.M., P < 0.05]. After 10 min the neutral lipase activity (0.40 +/- 0.05 m-unit.mg of protein(-1)) had decreased to basal values (P > 0.05). The contraction-mediated increase in lipase activity was increased by approximately 110% when muscle was stimulated in the presence of okadaic acid. Conversely, treatment of muscle homogenate with alkaline phosphatase completely reversed the contraction-mediated lipase activation. Lipase activity did not change during contractions when analysed in the presence of anti-hormone-sensitive-lipase (HSL) antibody [0.17 +/- 0.02 (basal) versus 0.21 +/- 0.02 (5 min) m-unit.mg of protein(-1), P > 0.05]. Furthermore, immunoprecipitation with affinity-purified anti-HSL antibody reduced muscle-HSL protein concentration by 81+/-4% and caused similar reductions in lipase activity against triacylglycerol and in the contraction-induced increase in this activity. Neither prior sympathectomy [0.33+/- 0.02 (basal) versus 0.53 +/- 0.06 (5 min) m-unit.mg of protein(-1), P < 0.05] nor propranolol impaired the lipase response to contractions. Glycogen phosphorylase activity in the absence of AMP increased after 1 min [27.3 +/- 3.1 versus 8.9 +/- 1.8% (activity without AMP/total activity with AMP), P < 0.05] and returned to basal levels after 5 min. In conclusion, skeletal-muscle-immunoreactive HSL is transiently stimulated by contractions and the mechanism probably involves phosphorylation. The time course of HSL activation is similar to that of glycogen phosphorylase. Apparently, the two enzymes are regulated in parallel by contraction-induced as well as hormonal mechanisms, allowing simultaneous recruitment of all major extra- and intra-muscular energy stores.

Hormone-sensitive lipase functions as an oligomer.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase whose activity is regulated by reversible phosphorylation and which is thought to be the rate-limiting enzyme for the mobilization of FFA from adipose tissue. In the current studies the subunit structure of HSL has been explored using sucrose gradient centrifugation and in vivo and in vitro protein-protein interactions. Evidence is provided to demonstrate that HSL exists as a functional dimer composed of homologous subunits. Dimeric HSL displayed approximately 40-fold greater activity against cholesteryl ester substrate when compared with monomeric HSL without any differences in affinity for the substrate. Truncations of HSL identified the importance of the N-terminal 300 amino acids, as well as other regions, in participating in the oligomerization of HSL. These studies support the notion that the N-terminal region of HSL represents a docking domain for protein-protein interactions and provide an additional mechanism for the posttranslational control of HSL activity in the cell via oligomerization.

Lipolysis in dry-cured ham maturation

Thirty light Parma hams were tested for muscle lipolytic activity (acid and neutral lipase activity) and free fatty acid (FFA) amounts in M. semimembranosus and biceps femoris, during progressive phases (0, 3, 6, 10 months) of dry-cured ham manufacturing. No correlation was found between the activities of acid and neutral lipases in fresh M. semimembranosus, while during processing the activities were positively related ( p<0.1), probably due to effects of muscle composition changes on lipolytic activities. In each processing step tested, acid lipase activities were higher in the M. semimembranosus than in the M. biceps femoris, and FFA amounts varied accordingly, the only exception being for the very dehydrated 10-month old M. semimembranosus, which yielded lower FFA than in the corresponding M. biceps femoris. FFAs in the end product correlated positively with acid and neutral lipase activities of green ham, suggesting that FFA production could be influenced by both raw meat properties and muscle composition during processing.

Millennium fat-cell lipolysis reveals unsuspected novel tracks.

Adipose tissue lipolysis, i.e., the catabolic process leading to the breakdown of triglycerides into fatty acids and glycerol, is often considered as a simple and well-understood metabolic pathway. However, progress on the hormonal regulation and molecular mechanism of fat-cell lipolysis is opening new avenues and points to a number of unanswered questions. Recent studies on the lipolytic beta- and antilipolytic alpha2-adrenergic control of lipolysis has allowed a better understanding of the relative contribution of the two types of receptors and provide strong evidence for the in vivo implication of alpha2-adrenoceptors in the physiological control of subcutaneous adipose-tissue lipolysis. A novel lipolytic system has been characterized in human fat cells. Natriuretic peptides stimulate lipolysis through a cGMP-dependent pathway. The molecular details of the lipolytic reaction are not fully understood. Translocation of hormone-sensitive lipase, the rate-limiting enzyme of lipolysis, to the lipid droplet seems to be an important step during lipolytic activation. Reorganization of the lipid droplet coating by perilipins may also facilitate the access of the enzyme. Unexpectedly, hormone-sensitive lipase-deficient mice are not obese and show residual adipose-tissue lipolysis, which suggests the existence of another triglyceride lipase. Whether the expression of this uncharacterized neutral lipase is compensatory for the lack of hormone-sensitive lipase is an important question yet to be resolved. In humans, alterations of hormone-sensitive lipase expression are associated with changes in lipolysis in various physiological and pathological states. Genetic studies show that beta2-adrenoceptor and hormone-sensitive lipase genes may participate in the polygenic background of obesity.

Recent advances in lipid nutrition in fish larvae.

Due to the importance of dietary lipid utilization for larval rearing success, increasing attention has been paid during the last years to different aspects of larval lipid nutrition such as digestion, absorption, transport and metabolism, which are frequently studied by different research groups. The present study reviews the published information on these aspects, including some recent results obtained in our laboratory, that contribute to a better understanding of larval lipid nutrition. Neutral lipase activity was found in the digesta of larval gilthead seabream as early as first feeding, followed by a significant increase which reached up 8 times the initial levels at day 15 and was clearly influenced by the fatty acid composition of dietary lipids. Accordingly, the capacity for lipid absorption by the intestinal epithelium has been also observed at the onset of exogenous feeding, although the specific location in the different digestive tract segments differ with species. Whereas the capacity to absorb lipid increases with development in live prey-fed larvae, this improvemment is delayed in larvae fed formulated diet. Increasing dietary phosphatidyl cholines levels enhanced lipid absorption regardless of whether it is of soybean or marine origin, but the latter improved hepatic lipid utilization. Enzymatic, histological and biochemical evidences suggest that marine fish larvae are able to effectively digest and absorb n-3 HUFA-rich triacylglycerols, but feeding with phosphoacylglycerols, particularly if they are rich in n-3 HUFA, would enhance phosphoacylglycerols digestion and specially lipid transport alowing a better n-3 HUFA incorporation into larval membrane lipids and promoting fish growth. Although the essentiality of n-3 HUFA for larval marine fish has been studied extensively, only recently has the importance of dietary arachidonic acid in the larvae of few species been recognised. Evidences for competitive interactions among these essential fatty acids suggest that besides a minimum dietary requirement for each essential fatty acid, their relative ratios must also be considered.

Coordinate assembly of lipids and enzyme proteins into epidermal lamellar bodies

Formation of the epidermal permeability barrier requires delivery of lamellar body (LB) contents to the stratum corneum interstices. LB are enriched in a mixture of polar lipids and a family of hydrolytic enzymes, required for the extracellular processing of the secreted polar lipids into the more hydrophobic products which mediate barrier function. Prior non-quantitative studies show that acute barrier disruption leads to immediate secretion of the contents of performed LB from the outermost layer of granular cells, followed by the synthesis and accelerated secretion of newly-formed (= nascent) organelles over 0.5–4 h. We asked here whether lipids and hydrolytic enzymes are packaged into nascent organelles separately, or in a parallel, linked process. We first quantified the rate of appearance of lipids (by the content of internal lamellae within LB) and enzyme content (by cytochemistry of neutral lipase and acid sphingomyelinase); both are concentrated in LB, and in nascent organelles. Immediately after barrier disruption, the density of LB in the cytosol of the outermost granular cell decreased by > 50% reduction at 30 min, returning to near-normal densities by 4 h. Nascent organelles budded off a trans-Golgi-like reticulum, in the outermost granular cells as early as 30 min. In quantitative studies, LB progressively accumulated lipid and enzyme contents in parallel. However, when lipid/lamellae generation was inhibited with lipid synthesis inhibitors, enzymes did not accumulate in organelles. Likewise, when exogenous physiologic lipids were delivered to sites of LB generation in the face of brefeldin A blockade of organellogenesis, or when lipids were delivered in conjunction with treatment with lipid synthesis inhibitors, enzymes accumulated only in those organelles that displayed lipid content. These studies demonstrate: (a) quantitative changes in the density of LB in the outermost granular cell at various time points after acute barrier disruption; (b) the origin of nascent organelles in a trans-Golgi-like reticulum; (c) co-ordinate packaging of lipid and enzyme contents into nascent organelles; (d) that lipid deposition in nascent organelles is required for enzyme accumulation; and (e) that enzymes can be delivered to nascent organelles, even if the source of lipid is of exogenous rather than endogenous origin.

Partitioning and resolution of mixture of two lipases from Bacillus stearothermophilus SB-1 in aqueous two-phase system

The partitioning and resolution of two lipases (acidic and neutral) was achieved in an aqueous two-phase system from crude culture supernatant of Bacillus stearothermophilus SB-1. The effects of changes in PEG molecular weight, varying PEG/phosphate system, pH and sodium chloride concentration were examined on the partition coefficient, K in PEG/phosphate system. The best system was PEG 6000 (70% w/v)/phosphate (40% w/v) and NaCl (3% w/v) at pH 7.0 where the total lipase partitioned to the top PEG phase. The two lipases were then selectively eluted by addition of fresh salt phase in a pH dependent manner with acidic lipase moving to the bottom phase at pH 4.0 with 5.27-fold purification while neutral lipase shifted to the bottom phase at pH 6.0 with 15.25-fold purity. The two lipases were easily recovered from the salt phase by immobilisation on accurel. Thus the mixture of two lipases was resolved and purified into acidic and neutral within 2 h with complete recycling of both the phase components. It has also been found that addition of PEG 6000 leads to an increase in the enzyme activity of the lipase mixture.

Expression of hormone-sensitive lipase and its regulation by adrenaline in skeletal muscle

The enzymic regulation of triacylglycerol breakdown in skeletal muscle is poorly understood. Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying demonstrated the presence of immunoreactive hormone-sensitive lipase (HSL), with the concentrations in soleus and diaphragm being more than four times the concentrations in extensor digitorum longus and epitrochlearis muscles. Neutral lipase activity determined under conditions optimal for HSL varied directly with immunoreactivity. Expressed relative to triacylglycerol content, neutral lipase activity in soleus muscle was about 10 times that in epididymal adipose tissue. In incubated soleus muscle, both neutral lipase activity against triacylglycerol (but not against a diacylglycerol analogue) and glycogen phosphorylase activity increased in response to adrenaline (epinephrine). The lipase activation was completely inhibited by anti-HSL antibody and by propranolol. The effect of adrenaline could be mimicked by incubation of crude supernatant from control muscle with the catalytic subunit of cAMP-dependent protein kinase, while no effect of the kinase subunit was seen with supernatant from adrenaline-treated muscle. The results indicate that HSL is present in skeletal muscle and is stimulated by adrenaline via beta-adrenergic activation of cAMP-dependent protein kinase. The concentration of HSL is higher in oxidative than in glycolytic muscle, and the enzyme is activated in parallel with glycogen phosphorylase.

Interaction of rat hormone-sensitive lipase with adipocyte lipid-binding protein.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that functions as the rate-limiting enzyme for the mobilization of free fatty acids in adipose tissue. By using the yeast two-hybrid system to examine the potential interaction of HSL with other cellular proteins, evidence is provided to demonstrate a direct interaction of HSL with adipocyte lipid-binding protein (ALBP), a member of the family of intracellular lipid-binding proteins that binds fatty acids, retinoids, and other hydrophobic ligands. The interaction was demonstrated in vitro by the binding of ALBP to HSL translated in vitro, to HSL in extracts of HSL overexpressing Chinese hamster ovary (CHO) cells, and to HSL in extracts of rat adipose tissue. Finally, the presence of ALBP was documented in immune complexes from rat adipose tissue immunoprecipitated with anti-HSL antibodies. The HSL-ALBP interaction was mapped to an N-terminal 300-aa region of HSL that is distinct from the C-terminal catalytic domain. These results suggest that HSL-derived fatty acids are bound by ALBP to facilitate intracellular trafficking of hydrophobic lipids.

Two acidothermotolerant lipases from new variants of Bacillus spp.

Two acidothermotolerant lipases from new isolates of Bacillus stearothermophilus SB-1 and Bacillus licheniformis SB-3 are reported. In addition, a thermotolerant, neutral lipase from Bacillus atrophaeus SB-2 that hydrolyses castor oil is also reported. The lipase from B. stearothermophilus SB-1 retained 70% activity and that from B. licheniformis SB-3 retained 50% activity at pH 3.0 at 50 °C. In addition, at 100 °C B. stearothermophilus SB-1 lipase had a half life of 25 min at pH 3.0 and 15 min at pH 6.0. Lipase activity was markedly stimulated by glycerol in case of B. stearothermophilus SB-1 and by diethylether in cases of B. atrophaeus SB-2 and B. licheniformis SB-3. The lipases varied in their substrate specificity towards triacylglycerols. The rate of hydrolysis of neem oil with B. stearothermophilus SB-1 and B. atrophaeus SB-2 lipases was, respectively, nearly 4-fold and 2-fold more than with olive oil.

Mutational analysis of structural features of rat hormone-sensitive lipase.

Hormone-sensitive lipase (HSL) is a cytosolic neutral lipase that hydrolyzes intracellular stores of triacylglycerols and cholesteryl esters. HSL activity is regulated via phosphorylation-dephosphorylation, with cyclic AMP-dependent protein kinase increasing activity following phosphorylation of a single serine and Ca2+/calmodulin-dependent protein kinase II phosphorylating another serine at a basal site. The current studies used site-directed mutagenesis to show that Ser-563 of rat HSL is phosphorylated by cyclic AMP-dependent protein kinase and that Ser-565 is phosphorylated by Ca2+/calmodulin-dependent protein kinase II. Mutation of Ser-563-->Ala eliminated HSL hydrolytic activity against cholesteryl ester, triacylglycerol, and diacylglycerol substrates to the same extent as mutation of Ser-423-->Ala, the presumed catalytic site. Mutation of Ser-565-->Ala modestly decreased HSL activity. In contrast, mutation of Ser-563-->Asp preserved HSL hydrolytic activity and even increased activity 20% above the control wild-type enzyme. Molecular modeling of the catalytic pocket of HSL suggested the involvement of Val-710. Mutation of Val-710-->Ala resulted in an 85% loss of HSL hydrolytic activity. The results of these studies illustrate the importance of the presence of a hydroxyl group or negative charge at residue 563, either for proper conformation of rat HSL or for proper stabilization of substrate to allow maintenance of hydrolytic activity, as well as the importance of the involvement of additional amino acids in the catalytic pocket of the enzyme.

Lipid composition and lipolytic enzyme activities in porcine skeletal muscles with different oxidative pattern

Three muscles with different oxidative pattern ( m. longissimus dorsi, Ld; m. biceps femoris, Bf; m. triceps brachii, Tb) have been analysed for lipid composition and lipolytic activities. The results showed a relatively low lipid (around 30 g kg −1) and cholesterol (around 0.5 g kg −1) content being higher in the oxidative and intermediate muscles Tb and Bf, respectively, than in Ld. Muscle Tb had higher amount of phospholipids and PUFA. Muscle Tb also had the higher lipolytic activity (acid and neutral lipase and acid phospholipase activities). There were no differences for acid and neutral esterase activities between the three muscles studied. The use of principal component (PC) analysis might be used to distinguish among the three studied muscles since each one showed a different pattern based on the lipid composition and lipolytic activities.

Structure of a microbial homologue of mammalian platelet-activating factor acetylhydrolases: Streptomyces exfoliatus lipase at 1.9 A resolution.

Neutral lipases are ubiquitous and diverse enzymes. The molecular architecture of the structurally characterized lipases is similar, often despite a lack of detectable homology at the sequence level. Some of the microbial lipases are evolutionarily related to physiologically important mammalian enzymes. For example, limited sequence similarities were recently noted for the Streptomyces exfoliatus lipase (SeL) and two mammalian platelet-activating factor acetylhydrolases (PAF-AHs). The determination of the crystal structure of SeL allowed us to explore the structure-function relationships in this novel family of homologous hydrolases. The crystal structure of SeL was determined by multiple isomorphous replacement and refined using data to 1.9 A resolution. The molecule exhibits the canonical tertiary fold of an alpha/beta hydrolase. The putative nucleophilic residue, Ser131, is located within a nucleophilic elbow and is hydrogen bonded to His209, which in turn interacts with Asp177. These three residues create a triad that closely resembles the catalytic triads found in the active sites of other neutral lipases. The mainchain amides of Met132 and Phe63 are perfectly positioned to create an oxyanion hole. Unexpectedly, there are no secondary structure elements that could render the active site inaccessible to solvent, like the lids that are commonly found in neutral lipases. The crystal structure of SeL reinforces the notion that it is a homologue of the mammalian PAF-AHs. We have used the catalytic triad in SeL to model the active site of the PAF-AHs. Our model is consistent with the site-directed mutagenesis studies of plasma PAF-AH, which implicate Ser273, His351 and Asp296 in the active site. Our study therefore provides direct support for the hypothesis that the plasma and isoform II PAF-AHs are triad-containing alpha/beta hydrolases.

The determination of lipase and phospholipase activities in gut contents of turbot (Scophthalmus maximus) by fluorescence-based assays

To study the potential of fluorescence based assays in the study of lipid digestion in fish, acyl esters of 4-methylumbelliferone and 1-acyl-2-[6 (7 nitro-1,3 benzoxadiazol-4-yl)amino]caproyl labeled phosphatidylcholine compounds (NBD-PC) were used as substrates for the assay of neutral lipase and phospholipase, respectively, in the gut contents of turbot. 4-Methylumbelliferyl hepatanoate (4-MUH) was hydrolysed at a higher rate than the butyrate or oleate esters whilst the hexanoic (C6) ester of NBD-PC was a more convenient substrate for the phospholipase assay than the dodecanoic (C12) ester. Neutral lipase activity was almost 10% higher when 50 mm potassium phosphate buffer pH 7.8 was used instead of 0.01 m citrate/sodium phosphate buffer pH 7.2. Both assays were very sensitive: neutral lipase and phospholipase activities were detectable at a minimum protein concentration in the digesta of 0.04 and 1.25 mg/ml, respectively. When the variations in lipolytic activities with gut segment and with size of fish were examined neutral lipase activity was always found to be higher in the hindgut and rectum segments than in the foregut. Although phospholipase activity was also found to be highest in the hindgut of the largest fish examined (av. wt. 182.3g), in fish of average weight 8g fish the activity was similar in all three segments. In the digesta from the whole gut of smaller fish (av. wt. 0.2, 0.6 and 1.43g) neutral lipase and phospholipase activities increased with increasing body mass when expressed as per ml of digesta. It is concluded that fluorescence-based assays are applicable to the study of lipid digestion in fish of different size.

Hormone-sensitive lipase (HSL) expression and regulation in skeletal muscle.

Because the enzymatic regulation of muscle triglyceride metabolism is poorly understood we explored the character and activation of neutral lipase in muscle. Western blotting of isolated rat muscle fibers demonstrated expression of hormone-sensitive lipase (HSL). In incubated soleus muscle epinephrine increased neutral lipase activity by beta-adrenergic mechanisms involving cyclic AMP-dependent protein kinase (PKA). The increase was paralleled by an increase in glycogen phosphorylase activity and could be abolished by antiserum against HSL. Electrical stimulation caused a transient increase in activity of both neutral lipase and glycogen phosphorylase. The increase in lipase activity during contractions was not influenced by sympathectomy or propranolol. Training diminished the epinephrine induced lipase activation in muscle but enhanced the activation as well as the overall concentration of lipase in adipose tissue. In agreement with the in vitro findings, in adrenalectomized patients an increase in muscle neutral lipase activity was found at the end of prolonged exercise only if epinephrine was infused. In accordance with feedforward regulation of substrate mobilization in exercise, our studies have shown that HSL is present in skeletal muscle cells and is stimulated in parallel with glycogen phosphorylase by both epinephrine and contractions. HSL adapts differently to training in muscle compared with adipose tissue.

Neutral lipids and lipase activity for actinorhodin biosynthesis of Streptomyces coelicolor A3(2)

Neutral lipid accumulation during early growth phase of Streptomyces coelicolor A3(2) was essential for the actinorhodin production during later growth. The activities of lipase and isocitrate dehydrogenase were increasing and decreasing, respectively, suggesting that the degradation products of neutral lipids serve actinorhodin biosynthesis as precursors.

EFFECT OF MYOGLOBIN ON THE MUSCLE LIPASE SYSTEM

The effect of different concentrations of myoglobin on the enzyme systems involved in lipid degradation were determined. Lipases, including both acid and neutral lipases and acid phospholipase, were inhibited by myoglobin. The neutral lipase was more susceptible to the inhibitory effect of myoglobin (35% of its initial activity at 1 mg/mL of myoglobin), followed closely by acid phospholipase ; acid lipase activity was affected to a lesser extent (less than 20% inhibition). This inhibition was also observed on the esterase activities, being greater on the neutral esterase activity (38% of its initial activity at I mg/mL of myoglobin) than on the acid one. In view of these results, myoglobin could be considered as an endogenous inhibitor which would determine the lipolytic activity in different type of muscles and therefore, the rate of generation of the free fatty acids.

Site-directed mutants designed to test back-door hypotheses of acetylcholinesterase function

The location of the active site of the rapid enzyme, acetylcholinesterase, near the bottom of a deep and narrow gorge indicates that alternative routes may exist for traffic of substrate, products or solute into and out of the gorge. Molecular dynamics suggest the existence of a shutter-like back door near Trp 84, a key residue in the binding site for acetylcholine, in the Torpedo californica enzyme. The homology of the Ω loop, bearing Trp 84, with the lid which sequesters the substrate in neutral lipases displaying structural homology with acetylcholinesterase, suggests a flap-like back door. Both possibilities were examined by site-directed mutagenesis. The shutter-like back door was tested by generating a salt bridge which might impede opening of the shutter. The flap-like back door was tested by de novo insertion of a disulfide bridge which tethered the Ω loop to the body of the enzyme. Neither type of mutation produced significant changes in catalytic activity, thus failing to provide experimental support for either back door model. Molecular dynamics revealed, however, substantial mobility of the Ω loop in the immediate vicinity of Trp 84, even when the loop was tethered, supporting the possibility that access to the active site, involving limited movement of a segment of the loop, is indeed possible.

The Turnover of Cytoplasmic Triacylglycerols in Human Fibroblasts Involves Two Separate Acyl Chain Length-dependent Degradation Pathways

Cultured fibroblasts from patients affected with the genetic metabolic disorder named neutral lipid storage disease (NLSD) exhibit a dramatic accumulation of cytoplasmic triacylglycerols (Radom, J., Salvayre, R., Nègre, A., Maret, A., and Douste-Blazy, L. (1987) Eur. J. Biochem. 164, 703-708). We compared here the metabolism of radiolabeled short-, medium- and long-chain fatty acids in these cells. Short/medium-chain fatty acids (C4-C10) were incorporated into polar lipids (60-80%) and triacylglycerols (20-40%) at a lower rate (5-10 times lower) than long-chain fatty acids. Pulse-chase experiments allowed to evaluate the degradation rate of cytoplasmic triacylglycerols in normal and NLSD fibroblasts and to discriminate between two catabolic pathways of cytoplasmic triacylglycerols. Short/medium-chain (C4-C10) triacylglycerols were degraded at a normal rate in NLSD fibroblasts, whereas long-chain (C12 and longer) triacylglycerols remained undegraded. These data are confirmed by mass analysis. The use of diethylparanitrophenyl phosphate (E600) and parachloromercuribenzoate (PCMB) inhibitors allows to discriminate between the two triacylglycerol degradation pathways. E600 inhibited selectively the in situ degradation of short/medium-chain triacylglycerols without inhibition of the degradation of long-chain triacylglycerols, whereas PCMB inhibited selectively the in situ hydrolysis of long-chain triacylglycerols without affecting the degradation of long-chain triacylglycerols. This was correlated with the in vitro properties of cellular triacylglycerol-hydrolyzing enzymes characterized by their substrate specificity and their susceptibility to inhibitors; the neutral lipase specific to long-chain triacylglycerols is inhibited by PCMB, but not by E600, in contrast to short/medium-chain lipase, which is inhibited by E600 but not by PCMB. The data of in vitro and in situ experiments suggest the existence in fibroblasts of two separate acyl chain length-dependent pathways involved in the degradation of cytoplasmic triacylglycerols, one mediated by a neutral long-chain lipase and another one mediated by a short/medium-chain lipase.

Plasma Platelet-activating Factor Acetylhydrolase Is a Secreted Phospholipase A2 with a Catalytic Triad

Platelet-activating factor (PAF) is a potent pro-inflammatory autacoid with diverse physiological and pathological actions. These actions are modulated by PAF acetylhydrolase, which hydrolyzes the sn-2 ester bond to yield the biologically inactive lyso-PAF. In contrast to most secreted phospholipase A2s, plasma PAF acetylhydrolase is calcium-dependent and contains a GXSXG motif that is characteristic of the neutral lipases and serine esterases. In this study we tested whether the serine in this motif is part of the active site of plasma PAF acetylhydrolase and, if so, what the other components of the active site are. Using site-directed mutagenesis, we demonstrated that Ser-273 (of the GXSXG motif), Asp-296, and His-351 are essential for catalysis. These residues were conserved in PAF acetylhydrolase sequences isolated from bovine, dog, mouse, and chicken. The linear orientation and spacing of these catalytic residues are consistent with the alpha/beta hydrolase conformation of other lipases and esterases. In support of this model, analysis of systematic truncations of PAF acetylhydrolase revealed that deletions beyond 54 amino acids from the NH2 terminus and 21 from the COOH terminus resulted in a loss of enzyme activity. These observations demonstrate that although plasma PAF acetylhydrolase is a phospholipase A2 it has structural properties characteristic of the neutral lipases and esterases.