The phospholipase A1 of Trypanosoma brucei does not release myristate from the variant surface glycoprotein

[3H]Myristoyl-labeled variant surface glycoprotein (VSG) has been isolated from Trypanosoma brucei by reverse phase high performance liquid chromatography and used as substrate for the conversion by trypanosomal enzymes of membrane-form VSG to soluble VSG. Conversion is detected by the release of myristoyl-containing lipids. The major lipolytic enzyme of T. brucei, phospholipase A1, is effective for the hydrolysis of myristoyl esters of p-nitrophenol, in a colorimetric assay. However, the phospholipase is unable to cleave the myristoyl ester linkage of VSG. The phospholipase can be separated from the myristoyl-releasing activity of trypanosome homogenate by centrifugation, affinity chromatography, and anion-exchange chromatography. Elution profiles on anion-exchange high performance liquid chromatography also indicate that the phospholipase is inactive against VSG. A small amount of myristoyl-releasing activity associated with the purified phospholipase is probably due to contamination with a phosphodiesterase which releases myristoyl-containing diglyceride from VSG.     

Comparative studies of lipase and phospholipase A2 acting on substrate monolayers.

The kinetic aspects of lipolysis by pancreatic lipase and phospholipase A2 from different sources have been compared using monomolecular films of short chain lipids as the substrates. Phosphatidylcholine monolayers, in contrast to phosphatidylethanolamine and phosphatidylglycerol monolayers, were resistant to hydrolysis by pancreatic lipase. The induction time, measured during pre-steady state conditions, increased abruptly for a given value of the surface pressure. This appears to be due to a degree of lipid packing above which the enzyme no longer can penetrate the lipid film. The existence of an optimum in the velocity versus surface pressure profile is the result of at least two counterbalancing factors. As the surface pressure increases, the amount of enzyme present in the interface decreases, whereas the minimal specific activity of the enzyme increases. From this study with monolayers we can conclude that activity of lipolytic enzymes used as tools for probing biological membranes will be greatly influenced by the physiochemical nature of the membrane-water interface. Thus, studies such as this one which can measure the penetrating ability of various lipolytic enzymes can be useful in deriving a better understanding of biological membrane structure.     

Effects of lipolytic enzymes on the photochemical activities of spinach chloroplasts.

1. Spinach class II chloroplasts were treated with purified potato lipolytic acyl-hydrolase and venom phospholipase A2, and their lipid degradations and the effects on the photochemical activities were followed. 2. Potato lipolytic enzyme hydrolyzed monogalactosyldiacylglycerol at a faster rate than phospholipids such as phosphatidylglycerol and phosphatidylcholine. The treatment caused a rapid decrease of Photosystem I activity, and a less change of Photosystem II activity. 3. Venom phospholipase A2 which preferentially hydrolyzed phosphatidylglycerol, caused a rapid decrease of Photosystem II activity and only a slight decrease of photosystem I activity. 4. Potato enzyme and phospholipase A2 degraded the membrane lipids of glutaraldehyde-fixed chloroplasts at a rather slightly higher rate than those of non-treated chloroplasts. 5. The results suggested a possible correlation between monogalactosyldiacylglycerol degradation and decay of Photosystem I activity and between phosphatidylglycerol degradation and decay of Photosystem II activity. A possible mechanism is discussed.     

Bacterial phospholipase A: structure and function of an integral membrane phospholipase

Within the large family of lipolytic enzymes, phospholipases constitute a very diverse subgroup with physiological functions such as digestion and signal transduction. Most phospholipases may associate with membranes at the lipid-water interface. However, in many Gram-negative bacteria, a phospholipase is present which is located integrally in the bacterial outer membrane. This phospholipase (outer membrane phospholipase A or OMPLA) is involved in transport across the bacterial outer membrane and has been implicated in bacterial virulence. OMPLA is calcium dependent and its activity is strictly regulated by reversible dimerisation. Recently the crystal structure of this integral membrane enzyme has been elucidated. In this review, we summarise the implications of these structural data for the understanding of the function and regulation of OMPLA, and discuss a mechanism for phospholipase dependent colicin release in Escherichia coli.     

Surface dilution kinetics using substrate analog enantiomers as diluents: Enzymatic lipolysis by bee venom phospholipase A2

A novel assay employing d-enantiomers of phospholipids as diluents for characterizing surface kinetics of lipid hydrolysis by phospholipases is introduced. The rationales of the method are (i) d-enantiomers resist hydrolysis because of the stereoselectivity of the enzymes toward l-enantiomers and (ii) mixtures of l+d-lipids at various l/d ratios but constant l+d-lipid concentrations yield a surface dilution series of variable l-lipid concentration with constant medium properties. Kinetic characterization of bee venom phospholipase A₂ activity at bile salt + phospholipid aggregate-water interfaces was performed using the mixed l+d-lipid surface dilution assay, and interface kinetic parameters were obtained. The assay applies to biomembrane models as well. Activity was measured by pH-stat methods. Aggregation numbers and interface hydration/microviscosity measured by time-resolved fluorescence quenching and electron spin resonance, respectively, confirmed that interface properties were indeed invariant in a surface dilution series, supporting rationale (ii), and were used to calculate substrate concentrations. Activity data show excellent agreement with a kinetic model derived with d-enantiomers as diluents and also that d-phospholipids bind to the enzyme but resist hydrolysis; underscoring rationale (i). The assay is significant for enabling determination of interface-specific kinetic parameters for the first time and thereby characterization of interface specificity of lipolytic enzymes.     

Cytosolic phospholipase A2: biochemical properties and physiological roles

Phosphatidylcholine (PC) is a major constituent of biological membranes and a component of serum lipoproteins and pulmonary surfactants. The PC and other glycerophospholipid compositions of membranes change dynamically through stimulus-dependent and independent pathways, principally by the action of two different types of enzymes; phospholipase A2 [EC 3.1.1.4] and acyl-CoA:lysophospholipid acyltransferase [EC 2.3.1.23]. Phospholipase A2 is a key enzyme that catalyzes deacylation of the sn-2 position of glycerophospholipids. This enzyme is critical in the remodeling of membrane lipids and formation of two subclasses of lipid mediators, fatty acid derivatives and lysophospholipids. Among many different subtypes of phospholipase A2 enzymes, we found that cytosolic phospholipase A2alpha (cPLA2alpha) is important in various pathological and physiological responses. Here, we summarize the phenotypes resulting from genetic ablation of cPLA2alpha, and the properties of newly discovered enzymes in the cPLA2 family. Comprehensive analysis of lipid mediators using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is useful for understanding the roles of individual mediators in physiological and pathological processes.     

Adipose triglyceride lipase activity is inhibited by long-chain acyl-coenzyme A

Adipose triglyceride lipase (ATGL) is required for efficient mobilization of triglyceride (TG) stores in adipose tissue and non-adipose tissues. Therefore, ATGL strongly determines the availability of fatty acids for metabolic reactions. ATGL activity is regulated by a complex network of lipolytic and anti-lipolytic hormones. These signals control enzyme expression and the interaction of ATGL with the regulatory proteins CGI-58 and G0S2. Up to date, it was unknown whether ATGL activity is also controlled by lipid intermediates generated during lipolysis. Here we show that ATGL activity is inhibited by long-chain acyl-CoAs in a non-competitive manner, similar as previously shown for hormone-sensitive lipase (HSL), the rate-limiting enzyme for diglyceride breakdown in adipose tissue. ATGL activity is only marginally inhibited by medium-chain acyl-CoAs, diglycerides, monoglycerides, and free fatty acids. Immunoprecipitation assays revealed that acyl-CoAs do not disrupt the protein–protein interaction of ATGL and its co-activator CGI-58. Furthermore, inhibition of ATGL is independent of the presence of CGI-58 and occurs directly at the N-terminal patatin-like phospholipase domain of the enzyme. In conclusion, our results suggest that inhibition of the major lipolytic enzymes ATGL and HSL by long-chain acyl-CoAs could represent an effective feedback mechanism controlling lipolysis and protecting cells from lipotoxic concentrations of fatty acids and fatty acid-derived lipid metabolites.     

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.     

The lipolytic activities of the isolated cell envelope fractions of baker''s yeast

1. The existence of phospholipase and lipase activities in the isolated cell envelopes of baker's yeast was demonstrated. 2. The content of phospholipase was found to be markedly higher than that of lipase. 3. After partial enzymic digestion of the isolated cell envelopes, the bulk of the lipolytic activities was recovered in the sedimentable preparations, which consisted of the fragments of the plasma membrane. 4. During repeated washings, the lipase was completely released from the cell envelopes, as were also the bulk of the lipid components and most of the Mg(2+)-dependent adenosine triphosphatase, an enzyme connected with the plasma membrane. The phospholipase was more firmly bound to the preparation but not so firmly as the external saccharase. 5. These results indicate that the lipolytic enzymes found in the cell envelopes are mostly located in the plasma membrane.     

Biomodulator-mediated susceptibility of endogenous lipid droplets from rat adipocytes to hormone-sensitive lipase

The amount of fatty acid release by a fat cell homogenate without pretreatment with epinephrine was found to be slightly more than that released from fat cells by epinephrine, suggesting that fat cells contain high lipolytic activity even in the absence of lipolytic agents. Fat cells contain high hormone-sensitive lipase activity (1383 mumole free fatty acids/g/hr) in the absence of epinephrine, and addition of epinephrine to the cells did not increase the activity, significantly. Like epinephrine, DBcAMP and/or theophylline also elicited marked release of glycerol from fat cells without activating the hormone-sensitive lipase activity. However, although fat cells contain a large amount of hormone-sensitive lipase, lipolysis was negligible in the absence of these lipolytic agents. These results suggest that lipolytic agents such as epinephrine, DBcAMP, and theophylline induce lipolysis in fat cells through some mechanism other than activation of hormone-sensitive lipase and that in the absence of lipolytic agents, some system in fat cells inhibits lipolysis of endogenous lipid droplets by hormone-sensitive lipase. The lipid droplets in fat cells consist mainly of triglyceride with phospholipids, cholesterol, carbohydrate, and protein as minor constituents. The phospholipid fraction was found to consist of 75% phosphatidylcholine and 25% phosphatidylethanolamine. Of the minor constituents of endogenous lipid droplets, only phosphatidylcholine strongly inhibited hormone-sensitive lipase activity in a [3H]triolein emulsion. These results suggest that phosphatidylcholine in endogenous lipid droplets may be responsible for inhibition of hormone-sensitive lipase. Then, a cell-free system was established in which epinephrine, DBcAMP, and theophylline stimulated lipolysis of endogenous lipid droplets from fat cells by lipase solution. In this system, these lipolytic agents did not induce lipolysis in the absence of added lipase. Lipolysis in the mixture of the endogenous lipid droplets and lipase solution was accelerated by phospholipase C with concomitant loss of epinephrine-induced lipolysis. After pretreatment of the endogenous lipid droplets with phospholipase C, these lipolytic agents no longer induced lipolysis. Pretreatment of the endogenous lipid droplets with phospholipase C reduced their phospholipid content with the formation of phosphorylcholine, but did not affect their triglyceride and cholesterol contents. Treatment of the endogenous lipid droplets with phospholipase D did not affect lipolysis in the cell-free system. These results suggest that phosphatidylcholine in the endogenous lipid droplets may inhibit their lipolysis by hormone-sensitive lipase in fat cells and also be involved in the mechanisms of the stimulatory effects of epinephrine, DBcAMP, and theophylline on lipolysis.     

Bicelles in structure-function studies of membrane-associated proteins

Bicelles are a novel form of long-chain/short-chain phospholipid aggregates, which are useful for biophysical and biochemical studies of membrane-associated biomolecules. In this work, we review the development of bicelles and their uses in structural characterization (primarily via NMR, circular dichroism, and fluorescence) of membrane-associated peptides. We also show that bicellar phospholipids are substrates for lipolytic enzymes. For this latter work, we employed a 31P NMR enzymatic assay system to examine the kinetic behavior of cobra venom phospholipase A(2) toward a variety of bicellar substrates. This enzyme hydrolyzed all bicelle lipids at rates comparable to those found for the enzyme action on traditional micellar substrates, which are the best substrates for this enzyme. In addition, we found that this PLA(2) showed no significant preference for long-chain or short-chain phospholipids when they were presented as mixtures in bicelles.     

Phosphoglycerides and phospholipase C in membrane fractions of Escherichia coli B.

The phospholipid composition and the phospholipase C activity of envelope fractions of Escherichia coli B were determined with special consideration of fractions containing sites at which an attachment of inner and outer membranes had been observed in the electron microscope (Int.M). Phosphoglycerides labeled with [14C]palmitic acid and [3H]serine were extracted from membrane fractions and identified by two-dimensional thin-layer chromatography. The amount of phosphatidylethanolamine was highest in the outer membrane, whereas the amounts of phosphatidylglycerol and cardiolipin were highest in the inner membrane. The Int.M fractions were observed to have concentrations of phospholipids intermediate to those of the inner and outer membranes. This result supports the assumption that a concentration gradient of inner membrane-outer membrane lipids might exist at the membrane contact sites. The highest phospholipase C activity was detected in the inner membrane and Int.M fractions. The presence of phospholipase C and other lipolytic enzymes in the Int.M fractions suggests a possible involvement of adhesion sites in lipid metabolism, adding a further set of activities to the function of these domains.     

Electrostatic method of determination of phospholipase A activity

An electrostatic assay of phospholipase A activity has been developed. This assay is based on the monitoring of changes in the boundary potential of the planar lipid bilayer. The initial rate of the potential changes induced by phospholipase hydrolysis is proportional to the enzyme activity. The sensitivity of the method is up to 0.0002 Units/ml; the effect of impurities is ignorable. The method allows the use of natural phospholipase substrates and is rapid, i.e., the phospholipase activity assay takes approximately 5 minutes.     

Activities of some enzymes of phospholipid metabolism in cultured rat ventricular myocytes in normoxic and hypoxic conditions

Phospholipid catabolism is thought to be one of the critical events in membrane injury during heart ischemia. In this work, the enzymes involved in phospholipid metabolism were studied in purified cultured ventricular myocytes in normoxic and hypoxic conditions. Purified ventricular myocytes exhibited an alkaline phospholipase A activity which had sn-2 specificity and which was calcium dependent, and an acid phospholipase A activity with sn-1 specificity. These cells also exhibited lysophospholipase and acyl-CoA/lysophosphatidylcholine acyltransferase activities. Oxygen deprivation of the myocardial cells for 4 h resulted in a sharp reduction of both phospholipase A2 and A1 activities. The activities of the other lipolytic enzymes were unaffected by hypoxia. Although hypoxia resulted in a marked increase of lactate dehydrogenase leakage in the bathing fluid, no additional release of the lipolytic enzymes and mitochondrial enzyme was observed. However, we noted an important alkaline phospholipase A2 leakage during normoxia. It is suggested that ventricular myocytes, under hypoxia, tend to prevent phospholipid degradation by reducing their phospholipase A activities.     

Characterization of the lipolytic activity of endothelial lipase

Endothelial lipase (EL) is a new member of the triglyceride lipase gene family previously reported to have phospholipase activity. Using radiolabeled lipid substrates, we characterized the lipolytic activity of this enzyme in comparison to lipoprotein lipase (LPL) and hepatic lipase (HL) using conditioned medium from cells infected with recombinant adenoviruses encoding each of the enzymes. In the absence of serum, EL had clearly detectable triglyceride lipase activity. Both the triglyceride lipase and phospholipase activities of EL were inhibited in a dose-dependent fashion by the addition of serum. The ratio of triglyceride lipase to phospholipase activity of EL was 0.65, compared with ratios of 24.1 for HL and 139.9 for LPL, placing EL at the opposite end of the lipolytic spectrum from LPL. Neither lipase activity of EL was influenced by the addition of apolipoprotein C-II (apoC-II), indicating that EL, like HL, does not require apoC-II for activation. Like LPL but not HL, both lipase activities of EL were inhibited by 1 M NaCl. The relative ability of EL, versus HL and LPL, to hydrolyze lipids in isolated lipoprotein fractions was also examined using generation of FFAs as an end point. As expected, based on the relative triglyceride lipase activities of the three enzymes, the triglyceride-rich lipoproteins, chylomicrons, VLDL, and IDL, were efficiently hydrolyzed by LPL and HL. EL hydrolyzed HDL more efficiently than the other lipoprotein fractions, and LDL was a poor substrate for all of the enzymes.     

Critical evaluation of p-nitrophenylphosphorylcholine (p-NPPC) as artificial substrate for the detection of phospholipase C*

Phospholipase C (PLC) activity secreted by bacteria as a virulence factor is commonly detected by use of the artificial substrate p-nitrophenylphosphorylcholine (p-NPPC). We examined several commercially available enzymes (phosphodiesterases, phosphomonoesterases, phospholipase A, lipase, protease) for their hydrolytic activity towards p-NPPC and compared these results with those of PLC tests using phospholipid substrates. Our data indicate that, in addition to PLC, several other enzymes which can affect phosphate esters are able to hydrolyze p-NPPC. We therefore suggest to use lipid substrates for correct characterization of bacterial PLCs, especially when whole bacteria or crude enzyme preparations are investigated.     

Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities

We established a novel technique for differential activity-based gel electrophoresis (DABGE) of lipolytic enzymes from two different biological samples. For this purpose, a set of three fluorescent suicide inhibitors was developed. These probes possess the same substrate analogous structures but carry different cyanine dyes (Cy2b, Cy3, and Cy5) as reporter fluorophores. For comparison of enzyme profiles, two samples are individually labeled with a different probe followed by mixing, gel electrophoresis, fluorescence imaging, and identification of the tagged proteins by MS/MS. Protocols for quantitative determination of active enzymes were developed on the basis of lipolytic proteomes that had been admixed with defined amounts of known lipases and esterases. A detailed analysis of the fluorescence intensities showed that the found enzyme ratios very closely reflected the relative amounts of the labeled enzymes that were used for spiking. The DABGE method was used to compare the lipolytic proteomes of brown and white adipose tissue showing specific enzyme patterns of both samples. This study represents the first application of this technology for comparative analysis of lipases and esterases. Further applications of this technique can be expected to provide entirely new information on lipid enzymology in health and disease with high precision.     

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7-9 and 8-10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50-75 degrees C, with optimal activity at 70 degrees C and were stable at 70 degrees C for at least 120 min. Both PLP and Est1 exhibited high V(max) toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C(4) and C(5)) and long chain (C(14) and C(16)) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C(10). We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.     

Novel functions of phospholipase A2s: Overview

The phospholipase A₂ (PLA₂) family comprises a group of lipolytic enzymes that typically hydrolyze the sn-2 position of (glycerol) phospholipids to give rise to fatty acids and lysophospholipids. The mammalian genome encodes more than 30 (even 50) PLA₂s or related enzymes, which are classified into several subfamilies on the basis of their structures and functions. The PLA₂ family has been implicated not only in signal transduction by producing lipid mediators, but also in membrane homeostasis, energy production, and barrier function. Disturbance of PLA₂-regulated lipid pathways often hampers tissue and cellular homeostasis and can be linked to various diseases. This special issue overviews the current state of understanding of the classification, enzymatic properties, and physiological functions of various enzymes belonging to the PLA₂ family. This article is part of a Special Issue entitled Novel functions of phospholipase A₂ Guest Editors: Makoto Murakami and Gerard Lambeau.