How gastric lipase, an interfacial enzyme with a Ser-His-Asp catalytic triad, acts optimally at acidic pH

Gastric lipase is active under acidic conditions and shows optimum activity on insoluble triglycerides at pH 4. The present results show that gastric lipase also acts in solution on vinyl butyrate, with an optimum activity above pH 7, which suggests that gastric lipase is able to hydrolyze ester bonds via the classical mechanism of serine hydrolases. These results support previous structural studies in which the catalytic triad of gastric lipase was reported to show no specific features. The optimum activity of gastric lipase shifted toward lower pH values, however, when the vinyl butyrate concentration was greater than the solubility limit. Experiments performed with long-chain triglycerides showed that gastric lipase binds optimally to the oil-water interface at low pH values. To study the effects of the pH on the adsorption step independently from substrate hydrolysis, gastric lipase adsorption on solid hydrophobic surfaces was monitored by total internal reflection fluorescence (TIRF), as well as using a quartz crystal microbalance. Both techniques showed a pH-dependent reversible gastric lipase adsorption process, which was optimum at pH 5 (Kd = 6.5 nM). Lipase adsorption and desorption constants (ka = 147,860 M(-1) s(-1) and kd = 139 x 10(-4) s(-1) at pH 6) were estimated from TIRF experiments. These results indicate that the optimum activity of gastric lipase at acidic pH is only "apparent" and results from the fact that lipase adsorption at lipid-water interfaces is the pH-dependent limiting step in the overall process of insoluble substrate hydrolysis. This specific kinetic feature of interfacial enzymology should be taken into account when studying any soluble enzyme acting on an insoluble substrate.     

A highly stable Yarrowia lipolytica lipase formulation for the treatment of pancreatic exocrine insufficiency

Yarrowia lipolytica lipase has been assumed to be a good candidate for the treatment of fat malabsorption in patients with pancreatic insufficiency. Nevertheless, no systematic studies on its stability under physiological conditions pertaining to the human GI (gastrointestinal) tract have been published. Stability of various Y. lipolytica lipase powder formulations at various physiological pH values as well as the effect of digestive proteases and bile salts on enzyme activity were investigated. Results were compared with those obtained from another competing fungal lipase sourced from Candida rugosa. Among the studied formulations, Y. lipolytica lipase stabilized with gum arabic and skimmed milk powder was the most promising powder formulation. Under acidic conditions (pH 3-5), this formulation showed higher stability than those observed with the other Y. lipolytica lipase formulations and C. rugosa lipase. In addition, in the presence of gum arabic and skimmed milk powder as additives, Y. lipolytica lipase exhibited markedly higher resistance to pepsin, trypsin and chymotrypsin actions. Resistance to proteolytic degradation by digestive proteases was also by far higher than that observed with C. rugosa lipase. Similar behaviour was, however, observed when these two fungal lipases were incubated with increased concentrations of bile salts. Residual lipase activity of both fungal lipases showed a slight decrease in NaTDC (sodium taurodeoxycholate) concentration above 4 mM. Consequently, Y. lipolytica lipase formulated with gum arabic and milk powder seemed to have great potential for use as a therapeutic tool for patients with pancreatic insufficiency.     

Use of naturally fluorescent triacylglycerols from Parinari glaberrimum to detect low lipase activities from Arabidopsis thaliana seedlings

The aim of this study was to design a convenient, specific, sensitive, and continuous lipase activity assay using natural long-chain triacylglycerols (TAGs). Oil was extracted from Parinari glaberrimum seed kernels and the purified TAGs were used as a substrate for detecting low levels of lipase activities. The purified TAGs are naturally fluorescent because more than half of the fatty acids from Parinari oil are known to contain 9,11,13, 15-octadecatetraenoic acid (parinaric acid) in its esterified form. The presence of detergents (sodium taurodeoxycholate, CHAPS, Sulfobetaine SB12, Tween 20, Brij 35, Dobanol, n-dodecylglucoside) above their critical micellar concentration dramatically increases the fluorescence of the parinaric acid released by various lipases. This increase in the fluorescence intensity is linear with time and proportional to the amount of lipase added. This new method, performed under non-oxidative conditions, was applied successfully to detecting low lipase levels in crude protein extracts from plant seeds and could be scaled down to microtiterplate measurements. Quantities as low as 0.1 ng of pure pancreatic lipase could be detected under standard conditions (pH 8). Lipase activity can also be assayed in acidic media (pH 5) using human gastric lipase. This simple and continuous assay is compatible with a high sample throughput and might be applied to detecting true lipase activities in various biological samples.     

Screening of preduodenal lipases in several mammals

The tissular localization of preduodenal lipases was studied from the tongue to the pyloric portion of the stomach in 11 mammals. Lipolytic activities were clearly differentiated from those of pancreas. All lipase activities show an acidic pH optimum, except the gastric enzyme from hog. For every mammal tested, preduodenal lipase activity was associated mainly with only a single tissue located either in tongue, or in the pharyngeal area, or in the stomach. Resistance to acidic pH medium allows the classification of lipase activities into three groups. These results are related to the dietary habits and zoologic classification of the different animal species.     

Production of a novel extracellular acidic lipase from Pseudomonas gessardii using slaughterhouse waste as a substrate

An isolate exhibiting high extracellular lipolytic activity was identified as Pseudomonas gessardii by 16S rDNA gene sequence analysis. The slaughterhouse waste, goat tallow, was used as a lipid substrate for the production of acidic lipase by P. gessardii. The maximum lipase activity of 156 U/ml was observed at an acidic pH of 3.5 and at 0.31 g substrate concentration. The purification steps resulted in the isolation of acidic lipase with a specific activity of 1,473 U/mg and a molecular weight of 94 kDa. One interesting feature of this purified lipase is its stability at highly acidic pH ranging from 2.0 to 5.5 with a high molecular weight. The amino acid composition was determined using HPLC. This acidic lipase has potential applications in the medicinal field as a substitute for pancreatic lipases for enzyme therapy, oleochemical and in biotechnological industries.     

Green tea extract (AR25) inhibits lipolysis of triglycerides in gastric and duodenal medium in vitro

In this study, we aimed to evaluate in vitro the inhibitory activity of a green tea extract (AR25 standardized at 25% catechins) on gastric and pancreatic lipase activities. We first used tributyrin as a substrate to evaluate the capability of AR25 to induce digestive lipase inhibition. Gastric lipase was totally inhibited by 40 mg AR25/g tributyrin whereas pancreatic lipase inhibition was maximum (78.8 +/- 0.7%) with 80 mg AR25/g tributyrin. We then used triolein, a long-chain triglyceride, to check whether AR25 could alter lipase activities on a physiologic substrate. AR25 60 mg/g triolein induced a dramatic inhibition of gastric lipase (96.8 +/- 0.4%) whereas pancreatic lipase activity was partially reduced (66.50 +/- 0.92%). Finally, the concerted action of gastric and pancreatic lipases was studied with an excess of enzymes to mimic the physiologic conditions observed in vivo. Incubation of AR25 with an excess of digestive lipases resulted in a drastic decrease in gastric lipolysis but the inhibitory effect on pancreatic lipase was less marked. On the whole, as compared to the control, lipolysis of triolein under the successive action of the two digestive lipases was reduced by 37 +/- 0.6% in the presence of AR25. Because a lipid/water interface is necessary for lipolysis to occur, lipid emulsification and emulsion droplet size were measured in gastric and duodenal media in the presence of AR25. In gastric and duodenal conditions, AR25 inhibited the lipid emulsification process. From these data we conclude that (1) in vitro, fat digestion is significantly inhibited by 60 mg AR25/g triolein, and (2) gastric as well as pancreatic lipase inhibition could be related to altered lipid emulsification in gastric or duodenal media. The green tea extract AR25 exhibiting marked inhibition of digestive lipases in vitro is likely to reduce fat digestion in humans.     

Purification of extracellular lipases from Trichosporon fermentans WU-C12

Two types of extracellular lipases (I and II) from Trichosporon fermentans WU-C12 were purified by acetone precipitation and successive chromatographies on Butyl-Toyopearl 650 M, Toyopearl HW-55F and Q-Sepharose FF. The molecular weight of lipase I was 53 kDa by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and 160 kDa by gel filtration, while that of lipase II was 55 kDa by SDS-PAGE and 60 kDa by gel filtration. For the hydrolysis of olive oil, the optimum pH and temperature of both the lipases were 5.5 and 35°C, respectively. The lipases showed stable activities after incubation at 30°C for 24 h in a pH range from 4.0 to 8.0. The thermostability of lipase I for 30 min at a reaction pH of 5.5 was up to 40°C, while that of lipase II under the same conditions was up to 50°C. Both lipases could hydrolyze the 1-, 2-, and 3-positions of triolein, and cleave all three ester bonds, regardless of the position in the triglyceride.     

Characterization of lipases from Staphylococcus aureus and Staphylococcus epidermidis isolated from human facial sebaceous skin

Two staphylococcal lipases were obtained from Staphylococcus epidermidis S2 and Staphylococcus aureus S11 isolated from sebaceous areas on the skin of the human face. The molecular mass of both enzymes was estimated to be 45 kDa by SDS-PAGE. S2 lipase displayed its highest activity in the hydrolysis of olive oil at 32 degrees C and pH 8, whereas S11 lipase showed optimal activity at 31 degrees C and pH 8.5. The S2 lipase showed the property of cold-adaptation, with activation energy of 6.52 kcal/mol. In contrast, S11 lipase's activation energy, at 21 kcal/mol, was more characteristic of mesophilic lipases. S2 lipase was stable up to 45° C and within the pH range from 5 to 9, whereas S11 lipase was stable up to 50 degrees C and from pH 6 to 10. Both enzymes had high activity against tributyrin, waste soybean oil, and fish oil. Sequence analysis of the S2 lipase gene showed an open reading frame of 2,067 bp encoding a signal peptide (35 aa), a pro-peptide (267 aa), and a mature enzyme (386 aa); the S11 lipase gene, at 2,076 bp, also encoded a signal peptide (37 aa), pro-peptide (255 aa), and mature enzyme (399 aa). The two enzymes maintained amino acid sequence identity of 98-99% with other similar staphylococcal lipases. Their microbial origins and biochemical properties may make these staphylococcal lipases isolated from facial sebaceous skin suitable for use as catalysts in the cosmetic, medicinal, food, or detergent industries.     

Production and activity of extracellular lipase from Luteibacter sp.

Microbial lipases are widely used in industrial applications due to their versatility, and the characterization of new lipase-producing microorganisms could provide new sources of these enzymes, with different specificities and better activities. In this context, we have improved lipase production by Luteibacter sp. by using basal medium supplemented with 2 % olive oil, a pH of 6 and a growth temperature of 37 °C. The enzyme extraction process with the addition of 0.25 % Tween 80 increased lipase activity. Implementation of these modifications increased lipase activity by approximately 430 %. The lipase activities produced in the culture supernatant (LCS) and extracted with Tween 80 (LCST80) were characterized. Both extracts hydrolyzed ρ-nitrophenyl (ρNP) esters with different acyl chain lengths, with a preference for short acyl lengths, and had optimum activity at 45 °C. The LCS was stable at acidic and alkaline pH, but LCST80 was only stable at alkaline pH. Methanol, SDS, Triton X-100, EDTA, and EGTA did not affect lipase activity, while divalent cations (Ca²⁺, Zn²⁺, Mg²⁺) - with the exception of Co²⁺— increased lipase activity. Both extracts showed transesterification activity on ρNP ester substrates, and both were able to hydrolyze different natural lipids. The characterization of lipase produced by Luteibacter sp. introduces this recently described genus as a new source of lipases with great biotechnological potential.     

Characterization of a Novel Alkalophilic Lipase From <Emphasis Type="Italic">Aneurinibacillus thermoaerophilus</Emphasis>: Lid Heterogeneity and Assignment to Family I.5

Recent investigations of Aneurinibacillus thermoaerophilus strains have allowed identification of a unique solvent tolerant lipase, distinct from known lipases. This work reports the expression and purification of this lipase (LipAT) and the first characterization of its structure and temperature and pH-dependent behaviour. LipAT has a secondary structural content compatible with the canonical lipase α/β hydrolase fold, and is dimeric at neutral pH. The protein was folded from pH 5 to 10, and association into folded aggregates at pH 7 and 8 likely protected its secondary structures from thermal unfolding. The enzyme was active from 25 to 65 °C under neutral pH, but its maximal activity was detected at pH 10 and 45 °C. The ability of LipAT to recover from high temperature was investigated. Heating at 70 °C and pH 10 followed by cooling prevented the restoration of activity, while similar treatments performed at pH 8 (where folded aggregates may form) allowed recovery of 50% of the initial activity. In silico analyses revealed a high conservation (85% or more) for the main lipase signature sequences in LipAT despite an overall low residue identity (60% identity compared to family I.5 lipases). In contrast, the active site lid region in LipAT is very distinct showing only 25% amino acid sequence identity to other homologous lipases in this region. Comparison of lids among lipases from the I.5 family members and LipAT reveals that this region should be a primary target for elucidation, optimisation and prediction of structure–function relationships in lipases.     

How do lipases and esterases work: the electrostatic contribution

This work explores the role of one of the factors explaining lipase/esterase activity: the contribution of electrostatic interactions to lipase/esterase activity. The electrostatic potential distribution on the molecular surface of an enzyme as a function of pH determines, to a large extent, the enzyme's pH activity profile. Other important factors include the presence and distribution of polar and hydrophobic residues in the active cleft. We have mapped the electrostatic potential distribution as a function of pH on the molecular surface of nine lipases/esterases for which the 3D structure is experimentally known. A comparison of these potential maps at different pH values with the corresponding pH-activity profile, pH optimum or pH range where the activity displayed by the enzyme is maximum, has revealed a considerable correlation. A negative potential in the active site appears correlated with maximum activity towards triglycerides, which has prompted us to propose a model for product release ('The electrostatic catapult model') after cleavage of an ester bond. At the same time as the bottom of the active site cleft becomes negatively charged, other nearby regions also titrate and become negatively charged when pH becomes more alkaline, for some of the studied lipases. If such lipases also show phospholipase activity (such as guinea pig lipase-related proteins 2 chimera) we raise the hypothesis that such other titratable regions after becoming negatively charged might stabilise the positive charge present in the polar head of phospholipids, such as phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The distribution of polar, weak polar and non-polar residues on the molecular surface of each studied lipase, in particular the active site region, was compared for all the lipases studied. The combination of graphical visualisation of the electrostatic potential maps and the polarity maps combined with knowledge about the location of key residues on the protein surface allows us to envision atomic models for lipolytic activity.     

Influence of N- and/or C-terminal regions on activity, expression, characteristics and structure of lipase from Geobacillus sp. 95

GD-95 lipase from Geobacillus sp. strain 95 and its modified variants lacking N-terminal signal peptide and/or 10 or 20 C-terminal amino acids were successfully cloned, expressed and purified. To our knowledge, GD-95 lipase precursor (Pre-GD-95) is the first Geobacillus lipase possessing more than 80 % lipolytic activity at 5 °C. It has maximum activity at 55 °C and displays a broad pH activity range. GD-95 lipase was shown to hydrolyze p-NP dodecanoate, tricaprylin and canola oil better than other analyzed substrates. Structural and sequence alignments of bacterial lipases and GD-95 lipase revealed that the C-terminus forms an α helix, which is a conserved structure in lipases from Pseudomonas, Clostridium or Staphylococcus bacteria. This work demonstrates that 10 and 20 C-terminal amino acids of GD-95 lipase significantly affect stability and other physicochemical properties of this enzyme, which has never been reported before and can help create lipases with more specific properties for industrial application. GD-95 lipase and its modified variants GD-95-10 can be successfully applied to biofuel production, in leather and pulp industries, for the production of cosmetics or perfumes. These lipases are potential biocatalysts in processes, which require extreme conditions: low or high temperature, strongly acidic or alkaline environment and various organic solvents.     

Isolation and characterization of extracellular lipase preparations from wild-type (V-10) and mutant (M-1)Serratia marcescens strains

—The cultivation conditions of wild-type strain V-10 and mutant strain M-l (overproducer of endonuclease and chitinase) ofSerratia marcescens optimal for extracellular lipase biosynthesis were determined. The strain V-10 displayed the maximum lipase yield (840 AU/ml) after 10–12 h of cultivation; the strain M-l (330 AU/ml), after 25–30 h. The data showed that extracellular lipases from V-10 and M-1 can be precipitated in a weakly acidic medium (pH 5.0 and 4.5, respectively). This property was used to obtain partially purified lipase preparations. The effect of the ionic composition of the reaction mixture on the activities of these enzymatic preparations was studied. Both preparations displayed the highest activities in weakly alkaline media (pH 8.0); however, the wild-type strain lipase displayed higher thermal stability and stability at alkaline pH compared with M-1 lipase. Both lipases were activated by various anionic and nonionic surfactants and were inactive in the presence of cetyltrimethylammonium bromide.     

Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

Identification and Characterization of Lipolytic Enzymes from a Peat-Swamp Forest Soil Metagenome

In this work, a metagenomic library was generated from peat-swamp forest soil obtained from Narathiwat Province, Thailand. From a fosmid library of approximately 15,000 clones, six independent clones were found to possess lipolytic activity at acidic pH. Analysis of pyrosequencing data revealed six ORFs, which exhibited 34–71% protein similarity to known lipases/esterases. A fosmid clone, designated LP8, which demonstrated the highest level of lipolytic activity under acidic conditions and demonstrated extracellular activity, was subsequently subcloned and sequenced. The full-length lipase/esterase gene, estPS2, was identified. Its deduced amino acid was closely related to a lipolytic enzyme of an uncultured bacterium, and contained the highly conserved motif of a hormone-sensitive family IV lipase. The EstPS2 enzyme exhibited highest activity toward p-nitrophenyl butyrate (C₄) at 37 °C at pH 5, indicating that it was an esterase with activity and secretion characteristics suitable for commercial development.     

In silico mutation on a mutant lipase from Acinetobacter haemolyticus towards enhancing alkaline stability

Abstract

Alkaline-stable lipases are highly valuable biocatalysts that catalyze reactions under highly basic conditions. Herein, computational predictions of lipase from Acinetobacter haemolyticus and its mutant, Mut-LipKV1 was performed to identify functionally relevant mutations that enhance pH performance under increasing basicity. Mut-LipKV1 was constructed by in silico site directed mutagenesis of several outer loop acidic residues, aspartic acid (Asp) into basic ones, lysine (Lys) at positions 51, 122 and 247, followed by simulation under extreme pH conditions (pH 8.0–pH 12.0). The energy minimized Mut-LipKV1 model exhibited good quality as shown by PROCHECK, ERRAT and Verify3D data that corresponded to 79.2, 88.82 and 89.42% in comparison to 75.2, 86.15, and 95.19% in the wild-type. Electrostatic surface potentials and charge distributions of the Mut-LipKV1 model was more stable and better adapted to conditions of elevated pHs (pH 8.0???10.0). Mut-LipKV1 exhibited a mixture of neutral and positive surface charge distribution compared to the predominantly negative charge in the wild-type lipase at pH 8.0. Data of molecular dynamics simulations also supported the increased alkaline-stability of Mut-LipKV1, wherein the lipase was more stable at a higher pH 9.0 (RMSD = ～0.3?nm, RMSF = ～0.05–0.2?nm), over the optimal pH 8.0 of the wild-type lipase (RMSD = 0.3?nm, RMSF = 0.05–0.20?nm). Thus, the adaptive strategy of replacing surface aspartic acid to lysine in lipase was successful in yielding a more alkaline-stable Mut-LipKV1 under elevated basic conditions.

Communicated by Ramaswamy H. Sarma     

Presumptive mechanisms of peptic ulceration by Helicobacter pylori VacA involving mucoprotease and CagA.

Helicobacter pylori vacuolating toxin (VacA) appears to be unusually stable, not only against extreme pH conditions or high temperatures, but also against common organic solvents or detergents. Under acidic conditions, its activity was markedly increased in the manner of temperature-independent, suggesting a spontaneous activation. A similar finding was also observed under alkaline conditions, however, it should have an appropriate temperature. From these observations, the mechanisms of VacA activation were suggested to be so redundant that either the case of acidic or basic amino acid residues could be involved in the VacA activation. Separately, we also found that the VacA production by H. pylori was pH-dependent: Its production was increased at a low pH region with a broad range (1.0-5.0), and at a high pH region with a narrow range (8.0-9.0). Astonishingly, a highly immunogenic CagA did not appear to be expressed under the acidic conditions. Its expression, however, was shown to be enhanced when the surrounding pH of this bacterium was raised. In contrast, mucoproteolytic activity in the H. pylori membrane was found to be increased at acidic conditions. Considering these observations, together with the stomach and duodenal pH of humans, two presumptive mechanisms of H. pylori VacA-associated ulceration may be deduced; namely, an acid- and an alkali-dependent type, involving mucoprotease and CagA, respectively.     

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.     

A novel thermostable lipase from Basidiomycete <Emphasis Type="Italic">Bjerkandera adusta </Emphasis>R59: characterisation and esterification studies

Microbial lipases are widely diversified in their enzymatic properties and substrate specificities, which make them very attractive for industrial application. Partially purified lipase from Bjerkandera adusta R59 was immobilized on controlled porous glass (CPG) and its properties were compared with those of the free enzyme. The free and immobilized lipases showed optimal activities at 45 and 50°C, respectively. Both enzyme forms were highly thermostable up to 60°C. The enzymes were stable at pH from 6.0 to 9.0 and their optimal pH for activity was 7.0. The free lipase was more thermostable in n-hexane than in aqueous environment. Both lipase preparations had good stabilities in non-polar solvents and were capable of hydrolysing a variety of synthetic and natural fats. Non-immobilized lipase activity was inhibited by disulphide bond reagents, serine and thiol inhibitors, while EDTA and eserine had no effect on enzyme activity. All anionic detergents tested in experiments inhibited lipase activity. The free lipase showed good stability in the presence of commercial detergents at laundry pH and temperatures. Applications of free and immobilized lipases for esterification were also presented.     

Monoglyceride and diglyceride lipases from human platelet microsomes

In the present study, we have characterized the properties of both diglyceride lipase (lipoprotein lipase, EC 3.1.1.24) and monoglyceride lipases (acylglycerol lipase, EC 3.1.1.23) in an attempt to assess the potential roles of these two enzymes in the release of arachidonate in activated human platelets. Diglyceride lipase exhibited maximal activity at pH 3.5, whereas monoglyceride lipase showed optimal activity at pH 7.0. Neither of the lipases were inhibited by EDTA or stimulated by Ca2+, Mg2+ or Mn2+. Both enzymes, however, were strongly inhibited by Hg2+ and Cu2+, indicating the involvement of sulfhydryl groups in catalytic activity. This suggestion was further supported by their sensitivity toward sulfhydryl inhibitors, with monoglyceride lipase being more susceptible to inhibition. Both lipases were found to be inhibited to a different degree by a variety of antiplatelet drugs blocking aggregation and arachidonate release. Kinetic studies indicated that dichotomous metabolism of diacylglycerol to monoacylglycerol and to phosphatidic acid could occur concurrently, since the apparent Km values for diglyceride lipase and for diglyceride kinase were comparable. Further studies showed that the specific activity of monoglyceride lipase was at least 100-fold higher than that of diglyceride lipase, indicating that the rate-limiting step in the release of arachidonate was the reaction catalyzed by diglyceride lipase.