Purification and characterization of an a type phospholipase from potato and its effect on potato mitochondria

A potato (Solanum tuberosum) phospholipid acyl-hydrolase, which - in the pH range 7.5 to 8.5—is at least 10,000 times more effective with phospholipids than with galactolipids, has been purified and characterized. It is a soluble enzyme readily distinguished from a neutral lipid lipase and a third lipid acyl-hydrolase which, while acting on phospholipid, shows a decided preference for glyceryl monoolein. The phospholipase in question has a pH optimum of 8.5, is stimulated by Ca²⁺ at pH above 7.5 and inhibited by Ca²⁺ at lower pH, is not dependent on detergents although stimulated by Triton X-100 to a moderate extent, and remains very active at temperatures close to zero. The phospholipids of intact potato mitochondria are highly susceptible to degradation by potato phospholipase, and it is suggested that this enzyme is involved in the extensive lipid breakdown which occurs in fresh potato slices following cutting, and in the deterioration of mitochondria during their preparation and aging.     

The enzymic deacylation of phospholipids and galactolipids in plants. Purification and properties of a lipolytic acyl-hydrolase from potato tubers

An enzyme preparation that catalyses the deacylation of mono- and di-acyl phospholipids, galactosyl diglycerides, mono- and di-glycerides has been partially purified from potato tubers. The preparation also hydrolyses methyl and p-nitrophenyl esters and acts preferentially on esters of long-chain fatty acids. Triglycerides, wax esters and sterol esters are not hydrolysed. The same enzyme preparation catalyses acyl transfer reactions in the presence of alcohols and also catalyses the synthesis of wax esters from long-chain alcohols and free fatty acids. Gel filtration, DEAE-cellulose chromatography and free-flow electrophoresis failed to achieve any separation of the acyl-hydrolase activities towards different classes of acyl lipids (phosphatidylcholine, monogalactosyl diglyceride, mono-olein, methyl palmitate and p-nitrophenyl palmitate) or any separation of these activities from a major protein component. For each class of lipid the acyl-hydrolase activity was subject to substrate inhibition, was inhibited by relatively high concentrations of di-isopropyl phosphorofluoridate and the pH responses were changed by Triton X-100. The hydrolysis of phosphatidylcholine was stimulated 30-40-fold by Triton X-100. The specific activities of the potato enzyme with galactolipids were at least 70 times higher than those reported for a homogeneous galactolipase enzyme purified from runner bean leaves. The possibility that a single lipolytic acyl-hydrolase enzyme is responsible for the deacylation of several classes of acyl lipid is discussed.     

SUGAR-DEPENDENT1 encodes a patatin domain triacylglycerol lipase that initiates storage oil breakdown in germinating Arabidopsis seeds

Triacylglycerol hydrolysis (lipolysis) plays a pivotal role in the life cycle of many plants by providing the carbon skeletons and energy that drive postgerminative growth. Despite the physiological importance of this process, the molecular mechanism is unknown. Here, a genetic screen has been used to identify Arabidopsis thaliana mutants that exhibit a postgerminative growth arrest phenotype, which can be rescued by providing sugar. Seventeen sugar-dependent (sdp) mutants were isolated, and six represent new loci. Triacylglycerol hydrolase assays showed that sdp1, sdp2, and sdp3 seedlings are deficient specifically in the lipase activity that is associated with purified oil bodies. Map-based cloning of SDP1 revealed that it encodes a protein with a patatin-like acyl-hydrolase domain. SDP1 shares this domain with yeast triacylglycerol lipase 3 and human adipose triglyceride lipase. In vitro assays confirmed that recombinant SDP1 hydrolyzes triacylglycerols and diacylglycerols but not monoacylglycerols, phospholipids, galactolipids, or cholesterol esters. SDP1 is expressed predominantly in developing seeds, and a SDP1-green fluorescent protein fusion was shown to associate with the oil body surface in vivo. These data shed light on the mechanism of lipolysis in plants and establish that a central component is evolutionarily conserved among eukaryotes.     

Non-lipolytic and lipolytic sequence-related carboxylesterases: A comparative study of the structure–function relationships of rabbit liver esterase 1 and bovine pancreatic bile-salt-activated lipase

To differentiate esterases from lipases at the structure–function level, we have compared the kinetic properties and structural features of sequence-related esterase 1 from rabbit liver (rLE) and bile-salt-activated lipase from bovine pancreas (bBAL). In contrast to rLE, bBAL hydrolyses water-insoluble medium and long chain esters as vinyl laurate, trioctanoin and olive oil. Conversely, rLE and bBAL are both active on water-soluble short chain esters as vinyl acetate, vinyl propionate, vinyl butyrate, tripropionin, tributyrin and p-nitrophenyl butyrate. However, the enzymes show distinctive kinetic behaviours. rLE displays maximal activity at low substrate concentration, below the critical micelle concentration, whereas bBAL acts preferencially on emulsified esters, at concentration exceeding the solubility limit. Comparison of the 3D structures of rLE and bBAL shows, in particular, that the peptide loop at positions 116–123 in bBAL is deleted in rLE. This peptide segment interacts with a bile salt molecule thus inducing a conformational transition which gives access to the active site. Inhibition studies and manual docking of a bulky ester molecule as vinyl laurate in the catalytic pocket of rLE and bBAL show that the inability of the esterase to hydrolyse large water-insoluble esters is not due to steric hindrance. It is hypothesized that esterases lack specific hydrophobic structures involved both in the stabilization of the lipase–lipid adsorption complex at interfaces and in the spontaneous transfer of a single substrate molecule from interface to the catalytic site.     

Papain catalyzed synthesis of glyceryl esters of N-protected amino acids and peptides for the use in trypsin catalyzed peptide synthesis

Papain catalyzed synthesis of glyceryl esters of BOC(Z)-protected amino acids and peptides was performed at 40-50 degrees C in a 50 molar excess of glycerol. Equilibrium was achieved in 6-7 h. The maximal yield of esters (50-70%) was obtained at 10% of water and pH 3.2-3.4. A lower water concentration resulted in a sharp decrease of the ester yield. The synthesized glyceryl esters of neutral amino acids are good substrates for trypsin and can be used for peptide synthesis catalyzed for trypsin. Glycerol esters are also good substrates for other enzymes possessing esterase activity. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 287-290, 1997.     

Glucuronoyl esterase--novel carbohydrate esterase produced by Schizophyllum commune

The cellulolytic system of the wood-rotting fungus Schizophyllum commune contains an esterase that hydrolyzes methyl ester of 4-O-methyl-d-glucuronic acid. The enzyme, called glucuronoyl esterase, was purified to electrophoretic homogeneity from a cellulose-spent culture fluid. Its substrate specificity was examined on a number of substrates of other carbohydrate esterases such as acetylxylan esterase, feruloyl esterase and pectin methylesterase. The glucuronoyl esterase attacks exclusively the esters of MeGlcA. The methyl ester of free or glycosidically linked MeGlcA was not hydrolysed by other carbohydrate esterases. The results suggest that we have discovered a new type of carbohydrate esterase that might be involved in disruption of ester linkages connecting hemicellulose and lignin in plant cell walls.     

Mutation of F417 but not of L418 or L420 in the lipid binding domain decreases the activity of triacylglycerol hydrolase

Human triacylglycerol hydrolase (hTGH) has been shown to play a role in hepatic lipid metabolism. Triacylglycerol hydrolase (TGH) hydrolyzes insoluble carboxylic esters at lipid/water interfaces, although the mechanism by which the enzyme adsorbs to lipid droplets is unclear. Three-dimensional modeling of hTGH predicts that catalytic residues are adjacent to an alpha-helix that may mediate TGH/lipid interaction. The helix contains a putative neutral lipid binding domain consisting of the octapeptide FLDLIADV (amino acid residues 417-424) with the consensus sequence FLXLXXXn (where n is a nonpolar residue and X is any amino acid except proline) identified in several other proteins that bind or metabolize neutral lipids. Deletion of this alpha-helix abolished the lipolytic activity of hTGH. Replacement of F417 with alanine reduced activity by 40% toward both insoluble and soluble esters, whereas replacement of L418 and L420 with alanine did not. Another potential mechanism of increasing TGH affinity for lipid is via reversible acylation. Molecular modeling predicts that C390 is available for covalent acylation. However, neither chemical modification of C390 nor mutation to alanine affected activity. Our findings indicate that F417 but not L418, L420, or C390 participates in substrate hydrolysis by hTGH.     

Characterization of a naturally occurring new version of the cholesterol ester transfer protein (CETP) from small intestine

The cholesterol ester transfer protein (CETP) is found in plasma mediating the transfer of cholesterol esters and triacylglycerides between lipoproteins. The last 26 amino acids of its carboxy-end correspond to an amphipathic a-helix whose hydrophobic side has been directly involved in the transfer of lipids. Alterations in this region lead to the reduction or loss of lipid transfer activity. To date, the only variant of the CETP messenger that has been reported lacks exon 9, which translates into an inactive isoform regarding neutral lipid transfer. In this study, we describe a new version of the messenger RNA of rabbit CETP identified exclusively in the small intestine of wild type (WT) rabbits. This isoform includes several of the intron bases prior to exon 16. The presence of a stop codon within this sequence prevents translation of exon 16, substituting the original carboxy-end sequence and therefore generating a random structure that does not contain the region responsible for neutral lipid transfer. Antibodies generated against a peptide within the carboxy-end sequence of the new isoform show the presence of this new protein in human plasma.     

Genetically modified mouse models to study hepatic neutral lipid mobilization

Excessive accumulation of triacylglycerol is the common denominator of a wide range of clinical pathologies of liver diseases, termed non-alcoholic fatty liver disease. Such excessive triacylglycerol deposition in the liver is also referred to as hepatic steatosis. Although liver steatosis often resolves over time, it eventually progresses to steatohepatitis, liver fibrosis and cirrhosis, with associated complications, including liver failure, hepatocellular carcinoma and ultimately death of affected individuals. From the disease etiology it is obvious that a tight regulation between lipid uptake, triacylglycerol synthesis, hydrolysis, secretion and fatty acid oxidation is required to prevent triacylglycerol deposition in the liver. In addition to triacylglycerol, also a tight control of other neutral lipid ester classes, i.e. cholesteryl esters and retinyl esters, is crucial for the maintenance of a healthy liver. Excessive cholesteryl ester accumulation is a hallmark of cholesteryl ester storage disease or Wolman disease, which is associated with premature death. The loss of hepatic vitamin A stores (retinyl ester stores of hepatic stellate cells) is incidental to the onset of liver fibrosis. Importantly, this more advanced stage of liver disease usually does not resolve but progresses to life threatening stages, i.e. liver cirrhosis and cancer. Therefore, understanding the enzymes and pathways that mobilize hepatic neutral lipid esters is crucial for the development of strategies and therapies to ameliorate pathophysiological conditions associated with derangements of hepatic neutral lipid ester stores, including liver steatosis, steatohepatitis, and fibrosis. This review highlights the physiological roles of enzymes governing the mobilization of neutral lipid esters at different sites in liver cells, including cytosolic lipid droplets, endoplasmic reticulum, and lysosomes. This article is part of a Special Issue entitled Molecular Basis of Disease: Animal models in liver disease.     

The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae

Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.     

Subcellular fractionation, partial purification and characterization of neutral triacylglycerol lipase from pig liver.

The subcellular distributions of acidic (pH 4.5) and neutral (pH 7.5) longchain triacylglycerol lipases (glycerol ester hydrolase, EC 3.1.1.3) of pig liver have been determined. The distribution of the acidic lipase closely paralleled that of the lysosomal marker enzyme, cathepsin D. Approx. 60% of the neutral lipolytic activity resided in the soluble fraction;the distribution of this activity failed to parallel that of marker enzymes for mitochondria, lysosomes, microsomes, or plasma membranes. A method has been developed for purification of the neutral lipase from the soluble fraction by ultracentrifugation. An approximate 90-fold purification was achieved, with recovery of 16% of the initial activity. The partially purified neutral lipase exhibited a pH optimum between 7.25 and 7.5. It required 30 mM emulsified triolein for optimal activity and ceased to liberate fatty acids after 30 min of incubation. The enzymatic activity was destroyed by heating at 60 degrees C. Neutral lipase was inhibited by sodium deoxycholate, Triton X-100 and iodoacetamide. The activity was not inhibited by sodium taurocholate, EDTA, heparin and diethyl-p-nitrophenyl phosphate. Neutral lipase failed to exhibit activity in assay systems specific for lipoprotein lipase, monoolein hydrolase, tributyrinase, and methyl butyrate esterase and showed little or no capacity to hydrolyze chyle chylomicrons or plasma very low density lipoproteins. It is suggested that the function of neutral lipase may be to supply the liver with fatty acids liberated from endogenously synthesized or stored triacylglycerols.     

Concerted action of human carboxyl ester lipase and pancreatic lipase during lipid digestion in vitro: importance of the physicochemical state of the substrate

The pancreatic enzyme carboxyl ester lipase (CEL) has been shown to hydrolyse a large number of different esters, including triacylglycerols, cholesteryl esters and retinyl esters with an absolute requirement for bile salts. Some of the lipids that are substrates for CEL can also be hydrolysed by pancreatic lipase. In order to investigate the relative roles of human CEL and pancreatic lipase, the two enzymes were incubated on a pH-stat with isotope-labelled lipid substrate mixtures in physicochemical forms resembling the state of the dietary lipids in human intestinal contents. In the first set of experiments, cholesteryl oleate (CO) and retinyl palmitate (RP) were solubilised in an emulsion of triolein (TO) stabilised by egg phosphatidylcholine and bile salts. Lipase (always added together with its cofactor, colipase) hydrolysed TO, with monoolein and oleic acid as end-products, whereas CEL alone could not hydrolyse TO in the presence of phosphatidylcholine (PC). Lipase alone did not hydrolyse CO or RP, but CEL did hydrolyse these esters if lipase was present. Release of [3H]glycerol from labelled TO increased only slightly if CEL was added compared to lipase alone, suggesting that monoolein hydrolysis was slow under these conditions. In the second set of experiments, CO and RP were dissolved in bile salt/monoolein/oleic acid dispersions with varying bile salt concentrations. CEL hydrolysed CO and RP more rapidly in a system with a high bile salt concentration containing mixed micelles than in a system with a low bile salt concentration, where the lipids were dispersed in the form of mixed micellar and non-micellar aggregates; both types of aggregate have been reported to exist in human intestinal contents. In conclusion, these data suggest that the main function of CEL under physiological conditions is to hydrolyse cholesteryl and retinyl esters, provided that the triacylglycerol oil phase is hydrolysed by pancreatic lipase, which probably causes a transfer of the substrate lipids of CEL from the oil emulsion phase to an aqueous bile salt/lipolytic product phase. Depending on the bile salt/lipolytic product ratio, the substrate will reside in either micellar or non-micellar lipid aggregates, of which the micellar state is preferred by CEL.     

A phosphonate monoester hydrolase from Burkholderia caryophilli PG2982 is useful as a conditional lethal gene in plants

A bacterial phosphonate monoester hydrolase was evaluated in plants as a conditional lethal gene useful for cell ablation and negative selection. Glyphosate is a potent herbicide whereas its phosphonate monoester derivative, glyceryl glyphosate, is approximately 50-fold less active. A phosphonate monoesterase gene ( pehA ) encoding an enzyme that hydrolyzes phosphonate esters including glyceryl glyphosate to glyphosate and glycerol was cloned from the glyphosate metabolizing bacterium, Burkholderia caryophilli PG2982. Constitutive expression of the pehA gene in Escherichia coli and Arabidopsis thaliana RLD had no observable phenotypic effects on growth and development. However, cells and plants expressing the pehA gene were killed when treated with glyceryl glyphosate. The phytotoxicity resulted from the hydrolysis of glyceryl glyphosate to glyphosate and subsequent inhibition of aromatic amino acid biosynthesis. As an example of tissue-specific cell ablation, floral sterility without vegetative toxicity was demonstrated by expressing the pehA gene using a tapetal-specific promoter and treating the mature plants with glyceryl glyphosate. A chromogenic phosponate ester substrate, 5-bromo-4-chloro-indolyl phenylphosphonate, was used to monitor in situ expression of the pehA gene. The general utility of the pehA gene as a heterologous conditional lethal gene in plants is discussed.     

Morphological characterization of the cholesteryl ester cycle in cultured mouse macrophage foam cells

Mouse peritoneal macrophages can be induced to accumulate cholesteryl esters by incubating them in the presence of acetylated low density lipoprotein. The cholesteryl esters are sequestered in neutral lipid droplets that remain in the cell even when the acetylated low density lipoprotein is removed from the culture media. Previous biochemical studies have determined that the cholesterol component of cholesteryl ester droplets constantly turns over with a half time of 24 h by a cyclic process of de-esterification and re-esterification. We have used morphologic techniques to determine the spatial relationship of cholesteryl ester, free cholesterol, and lipase activity during normal turnover and when turnover is disrupted. Lipid droplets were surrounded by numerous 7.5-10.0-nm filaments; moreover, at focal sites on the margin of each droplet there were whorles of concentrically arranged membrane that penetrated the matrix. Histochemically detectable lipase activity was associated with these stacks of membrane. Using filipin as a light and electron microscopic probe for free cholesterol, we determined that a pool of free cholesterol was associated with each lipid droplet. Following incubation in the presence of the exogenous cholesterol acceptor, high density lipoprotein, the cholesteryl ester droplets disappeared and were replaced with lipid droplets of a different lipid composition. Inhibition of cholesterol esterification caused cholesteryl ester droplets to disappear and free cholesterol to accumulate in numerous myelin-like structures in the body of the cell.     

Distribution of acetylcholinesterase along the dorso-ventral axis of the hippocampal formation in the rabbit

Abstract— The cholesterol ester hydrolase of rat brain, localized almost exclusively in the myelin sheath, has been solubilized from the acidic high-molecular-weight protein fraction of purified myelin. Solubilization required both high ionic strength and an amphoteric detergent, Miranol H2M. Solubilized preparations with apparent purification factors of 300–500 fold over the starting homogenate still contained approx 25% lipid but were retarded on the Sephadex G-200 column. The enzyme was reversibly precipitated when the concentration of either Miranol H2M or KCI was lowered below certain critical levels. The soluble enzyme was characterized for the pH optimum, linearity against incubation time and enzyme protein, and apparent K_m. Activity was dependent on the presence of exogenously added lipid. Phosphatidylserine at optimum concentrations stimulated the hydrolytic activity 25-Fold. Effects of other lipids, bile salts, cations, heating and potential inhibitors were examined. β-Naphthyl oleate was a competitive inhibitor but both β-naphthyl acetate and cholesteryl butyrate were non-competitive inhibitors. These results suggested a heterogenous nature of the rat myelin cholesterol ester hydrolase, possibly with different specificities with respect to the chain length of the acyl group of substrates.     

Effect of light on lipid composition of Ricinus cell cultures

The changes of lipid composition were determined in callus cultures from Ricinus communis endosperm upon transfer from darkness into light. Culture in light induced chlorophyll synthesis and formation of differentiated chloroplasts. In light-grown cultures the major lipid classes were phospho- and glycolipids, dark-grown cultures were rich in triacylglycerol. The major fatty acids were linolenic acid and palmitic acid in both cultures. In the green cultures linolenic acid was predominantly esterified in glycolipids whereas in the dark-grown cultures this fatty acid was the major component of phospholipids. Ricinoleic acid was not found.Abbreviations PC phosphatidylcholine - PE phosphatidylethanolamine - MGD monogalactosyldigylceride - DGD digalactosyldiglyceride - SE steryl esters - NL neutral lipids     

ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6

Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.     

Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase.     

Isolation and characterization of an enzyme with esterase activity from Micropolyspora faeni.

The isolation and the characterization of one of the enzymes of Micropolyspora faeni that hydrolyzes the substrate N-benzoyl-DL-phenylalanine-beta-naphthyl ester and that seems to be of medical importance are described. This enzyme (enzyme 1) was isolated with an 86-fold purification by using the following seven steps: ammonium sulfate precipitation, gel filtration through Sephadex G-150, heat treatment, chromatography on diethylaminoethyl-cellulose, rechromatography on diethylaminoethyl-Sephadex, gel filtration through Sephadex G-200, and affinity chromatography. Enzyme 1 has a molecular weight of approximately 500,000 and maximum activity at pH 7.8 to 8.0 and at 20 degrees C. The enzyme is stable between pH 7.5 and 10.5 and at temperatures up to 60 degrees C. Its activity is not inhibited by ethylenediaminetetraacetic acid. It is, however, sensitive to diisopropyl phosphofluoride and phenylmethyl sulfonyl fluoride. These properties and the ability to hydrolyze the esters of phenylalanine, tyrosine, and tryptophan without endopeptidasic activity and no marked proteolytic activity suggest that the enzyme is an esterase.