Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1,2-diacylglycero-3-phosphocholine

rac-1-[1-14C]Lauroyl-2-oleylglycero-3-phospho[methyl-3H]choline and rac-1-lauroyl-2-[1-14C]oleoylglycero-3-phospho[methyl-3H]choline along with rac-1-palmitoyl-2-oleylglycero-3-phosphocholine and sn-1-palmitoyl-2-oleylglycero-3-phosphocholine were synthesized and subjected to hydrolysis with phospholipase C (EC 3.1.4.3) from Clostridium perfringens and phospholipase D (EC 3.1.4.4) from cabbage. Kinetics of hydrolysis of the radioactive substrates were determined by measuring the 3H radioactivity retained in the aqueous phase due to free choline and phosphocholine and the 3H and 14C radioactivity recovered in the organic phase due to the released diacylglycerols and phosphatidic acids and the residual phosphatidylcholines. The rate of hydrolysis of the unlabelled substrates by phospholipase C was determined by thin-layer chromatography and gas-liquid chromatography of the methanolysis products. The relative initial rates of hydrolysis of sn-1,2,- and sn-2,3-enantiomers were 100-200:1 for phospholipase C and 40-50:1 for phospholipase D using rac-1-lauroyl-2-oleoylglycero-3-phosphocholine as the substrate. The substitution of the 2-acyl group by an alkyl group resulted in a loss of stereospecificity, which was partial for phospholipase C (relative rates equal to 8-13:1) and total for phospholipase D. There was a parallel dramatic decrease (500-1000-fold) in the initial rate of hydrolysis with phospholipase C but the activity of phospholipase D was only moderately reduced (18-fold). These findings are consistent with the earlier observed loss of the stereospecificity of lipoprotein lipase following introduction of a 2-alkyl group into triacylycerols, and point to a general unsuitability of 2-alkyl-linked acylglycerols as substrates for the assay of the stereospecificity of lipases, as well as for the isolation of enantiomeric 2-alkylacylglycerols by means of stereospecific lipases.     

Asymmetric short-chain phosphatidylcholines: defining chain binding constraints in phospholipases

Several short-chain asymmetric lecithins with a total of 14 carbons in the acyl chains (ranging from 1-lauroyl-2-acetylphosphatidylcholine to 1-hexanoyl-2-octanoylphosphatidylcholine) have been synthesized and characterized. The specific activities of phospholipase A2 from cobra venom, phospholipase A2 from porcine pancreas, and phospholipase C from Bacillus cereus toward these lecithins as micelles have been determined. The results of these kinetic studies allow the definition of hydrophobic binding requirements in the active sites of these water-soluble phospholipases. For phospholipase C, with the exception of monomyristoylphosphatidylcholine, each of the asymmetric short-chain lecithins exhibits high activity, comparable to the 14-carbon symmetric short-chain species, diheptanoylphosphatidylcholine. Therefore, for phospholipase C, in addition to the acyl linkages, only a certain degree of hydrophobicity in the fatty acyl chains is requisite for substrate binding and appreciable hydrolysis; there is no chain specificity. The activity of phospholipase A2 from cobra venom toward the same asymmetric lecithins is quite different. As the sn-2 chain lengthens, activity is increased to a maximum for diheptanoyl-PC. Further increase in the number of carbons in the sn-2 chain has no effect on hydrolysis rates. For this enzyme, seven carbons in the sn-2 chain are necessary for optimal activity. In contrast, porcine pancreatic phospholipase A2 activity shows very little dependence on sn-2 chain length.     

Substrate specificity of phospholipase B from guinea pig intestine. A glycerol ester lipase with broad specificity.

The substrate specificity of a calcium-independent, 97-kDa phospholipase B purified from guinea pig intestine was further investigated using various natural and synthetic lipids. The enzyme was equally active toward enantiomeric phosphatidylcholines under conditions allowing a strict phospholipase A activity. The lysophospholipase activity declined with the following substrates: 1-acyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-propanediol-3-phosphocholine greater than 1-palmitoyl-glycol-2-phosphocholine, suggesting some influence of the polar residue vicinal to the cleavage site. The enzyme also acted on various neutral lipids including triacylglycerol, diacylglycerol, and monoacylglycerol, whereas cholesteryl oleate remained refractory to enzymatic hydrolysis. The lipase hydrolyzed sequentially the sn-2 and sn-1 acyl ester bonds of diacylglycerol, although some direct cleavage of the external acyl ester bond could also occur, as shown with diacylglycerol analogues bearing a nonhydrolyzable alkyl ether or amide bond in the sn-1 or sn-2 position. The three main activities of the enzyme (phospholipase A2, lysophospholipase, and diacylglycerol lipase) were resistant to 4-bromophenacyl bromide, but they were inhibited by N-ethylmaleimide, 5,5'-dithiobis-(2-nitrobenzoic acid), and diisopropyl fluorophosphate, suggesting the possible involvement of both cysteine and serine residues in a single active site. It is concluded that guinea pig intestinal phospholipase B, which was also detected in rat and rabbit, is actually a glycerol ester lipase with broad substrate specificity and some unique enzymatic properties.     

Design, synthesis, and characterization of bis-phosphatidylcholine, a mechanistic probe of phosphatidylcholine transfer protein catalytic activity

The design, synthesis, and characterization of 1-(17,18-dithiatetratriacontandioyl)-bis(2-hexadecanoyl-sn-glycero -3- phosphocholine) is described. Bis-phosphatidylcholine is a dimeric phospholipid comprised of two glycerophosphocholine groups linked together by a disulfide bond at the distal ends of the sn-1 fatty acyl chains. Electron microscopy and [14C]glucose trapping studies indicate that hydrated dispersions of bis-phosphatidylcholine form closed, spherical structures which have diameters in the range of 125-500 nm. Sensitivity to phospholipase hydrolysis suggests that this bipolar lipid is organized in a membrane such that the two polar head groups of the molecular are oriented at the same surface of the membrane. Using conditions in which bovine liver phosphatidylcholine transfer protein transfers both unsaturated and saturated diacyl phosphatidylcholines between fluid phosphatidylcholine vesicles, no transfer of the bipolar phospholipid is observed. The lack of activity toward bis-phosphatidylcholine suggests that this molecule may be a useful tool for elucidating the role of membrane phosphatidylcholine in the catalytic mechanism of the phosphatidylcholine transfer protein.     

Bacillus cereus phospholipase C: carboxylic acid ester specificity and stereoselectivity

Thiophosphate analogs of phosphatidylcholine have been synthesized with varying structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C (Bacillus cereus) to estimate the minimal structural requirements associated with the non-polar portion of the substrate phospholipid. The analogs were of three types containing zero, one or two carboxylic acid ester functionalities. The analogs with one or two ester groups acted as substrates for phospholipase C, while those without an ester functionality were not hydrolyzed. The rac-phosphatidylcholine analog with two ester functionalities gave biphasic time-course results, and was subsequently resolved into enantiomers by selective hydrolysis with a sterospecific phospholipase A2 (Crotalus atrox). The enantiomer with R absolute configuration was rapidly hydrolyzed by the phospholipase C while the enantiomer with the S configuration was slowly hydrolyzed after a long induction period. The results suggest that the B. cereus phospholipase C is specific for an ester functionality and is stereoselective for the R absolute configuration at glycerol C-2.     

Probing the role of C-1 ester group in Naja naja phospholipase A2-phospholipid interactions using butanetriol-containing phosphatidylcholine analogues.

To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A2-glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A2. Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A2-phospholipid interactions.     

Evidence for the involvement of tyrosine-69 in the control of stereospecificity of porcine pancreatic phospholipase A2

We have studied the role of Tyr-69 of porcine pancreatic phospholipase A2 in catalysis and substrate binding, using site-directed mutagenesis. A mutant was constructed containing Phe at position 69. Kinetic characterization revealed that the Phe-69 mutant has retained enzymatic activity on monomeric and micellar substrates, and that the mutation has only minor effects on kcat and Km. This shows that Tyr-69 plays no role in the true catalytic events during substrate hydrolysis. In contrast, the mutation has a profound influence on the stereospecificity of the enzyme. Whereas the wild-type phospholipase A2 is only able to catalyse the degradation of sn-3 phospholipids, the Phe-69 mutant hydrolyses both the sn-3 isomers and, at a low (1-2%) rate, the sn-1 isomers. Despite the fact that the stereospecificity of the mutant phospholipase has been altered, Phe-69 phospholipase still requires Ca2+ ions as a cofactor and also retains its specificity for the sn-2 ester bond. Our data suggest that in porcine pancreatic phospholipase A2 the hydroxyl group of Tyr-69 serves to fix and orient the phosphate group of phospholipid monomers by hydrogen bonding. Because no such interaction can occur between the Phe-69 side-chain and the phosphate moiety of the substrate monomer, the mutant enzyme loses part of its stereospecificity but not its positional specificity.     

A comparative study of the effect of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine and some homologous compounds on PKC alpha and PKC epsilon

The effects of the anti-neoplastic ether lipid ET-18-OCH3 and some structural homologues on the activity of protein kinase C alpha (PKC alpha) were studied and compared with the effects the same had on the activity of PKC epsilon. ET-18-OCH3 progressively inhibited the activity of PKC alpha as the concentration was increased up to 30 mol% of the total lipid, above which the effect was one of activation. The experiments carried out with the homologues showed that the methoxy group bound at the sn-2 position of the glycerol of ET-18-OCH3 is essential for both the initial inhibitory effect and the subsequent activation effect. On the other hand, variations in the type of bond linking substitutions in the sn-1 position, ether or ester, do not seem to play an important role in determining the activity of the enzyme. The effects were different on PKC epsilon since ET-18-OCH3 had a triphasic effect, activating the enzyme at low concentrations, inhibiting it at slightly higher concentrations and then activating it again at higher concentrations. In this case, when the homologues were used, it was observed that the presence of the methoxy group linked to the sn-2 position of glycerol and the type of bond linking substitutions to the sn-1 position were important for activating the enzyme, so that only homologues with ester bonds as LPC and PAPC were able to induce the initial activation step in a way similar to ET-18-OCH3. Substitution of the phosphocholine group of ET-18-OCH3 by phosphoserine led to a greater activation of PKC alpha, an effect that comes from the Ca(2+)-phospholipid binding site probably because of the specific interaction of this site with the phosphoserine group. The action of ET-18-OCH3 and its homologues, as demonstrated in this paper, may permit the selective inhibition or activation of PKC alpha and PKC epsilon by using the most suitable range of concentrations.     

Infrared characterization of conformational differences in the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine

Fourier transform infrared spectroscopy was used to characterize the lamellar phases of 1,3-dipalmitoyl-sn-glycero-2-phosphocholine (1,3-DPPC), a positional isomer of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (1,2-DPPC). The molecule exists in three distinct phases over the temperature interval 0-70 degrees C. In the low-temperature (LC) phase, the spectra are indicative of acyl chains packed in an orthorhombic subcell, while the carbonyl groups and phosphate ester at the head group show evidence of only partial hydration. The transition from the low-temperature (LC) phase to the intermediate-temperature (L beta) phase at 25 degrees C corresponds to a temperature-induced head-group hydration in which the hydration of the phosphate and carbonyl ester groups results in the reorganization of the hydrocarbon chain-packing subcell from orthorhombic to hexagonal. The transition from the intermediate (L beta) to the high-temperature (L alpha) phase at 37 degrees C is a gel-to-liquid-crystalline phase transition analogous to the 41.5 degrees C transition of 1,2-DPPC. The spectra of the acyl-chain carbonyl groups show evidence of significant differences in molecular conformation at the carbonyl esters in the LC phase. In the L beta and L alpha phases, the carbonyl band contour becomes much more symmetric. However, two components are clearly present in the spectra indicating that the sn-1 and sn-3 carbonyls experience slightly different environments. The observed differences are likely due to a preferred conformation of the phosphocholine group relative to the glycerol backbone. Indications from the infrared spectra of differences in the structure of the C = O groups provide a possible explanation for the selection of the sn-1 chain of 1,3-DPPC by phospholipase A2 on the basis of a preferred head group conformation.     

Purification and regiospecificity of multiple enzyme activities of phospholipase A(1) from bonito muscle

Phospholipase A(1) (PLA(1)), which catalyzes the hydrolysis of the sn-1 ester bond of diacyl phospholipids, was purified from 100,000 x g supernatant of bonito muscle to homogeneity by ammonium-sulfate precipitation and four consecutive column chromatographies (DEAE anion-exchange, ether-Toyopeal, hydroxylapatite and Toyopeal HW 50S columns). The final preparation showed a single band above the 67-kDa molecular marker on SDS-PAGE, and the molecular mass was determined to be 71.5 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using bovine serum albumin as a standard for calibration. The N-terminal 8 amino residues were determined to be Ala-Pro-Ala-Glu-Lys-Val-Lys-Try. Regiospecificity of multiple enzyme activities of the PLA(1) was examined using positionally defined synthetic phosphatidylcholine (PC) and lysophosphatidylcholines (LPC). An acyl ester bond at the sn-1 position of PC was exclusively hydrolyzed by phospholipase activity, and 1-acyl LPC was cleaved to fatty acid and glycerophosphocholine by lysophospholipase (LPL) activity. However, the positional isomer, 2-acyl LPC was a poor substrate for LPL activity. PC/transacylation activity was also observed when excess 2-acyl LPC was supplied in the reaction mixture, and fatty acid at the sn-1 position of donor PC was transferred to the sn-1 position of acceptor LPC. These results demonstrate that the multiple enzyme activities of PLA(1), this is lysophospholipase, transacylase as well as phospholipase, have a strict regiospecificity at the sn-1 position of substrates.     

Phosphatidylcholine metabolism in endothelial cells: evidence for phospholipase A and a novel Ca2+-independent phospholipase C

The metabolism of phosphatidylcholine (PC) was investigated in sonicated suspensions of bovine pulmonary artery endothelial cells and in subcellular fractions using two PC substrates: 1-oleoyl-2-[3H]oleoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phospho[14C]choline. When these substrates were incubated with the whole cell sonicate at pH 7.5, all of the metabolized 3H label was recovered in [3H]oleic acid (95%) and [3H]diacylglycerol (5%). All of the 14C label was identified in [14C]lysoPC (92%) and [14C]phosphocholine (8%). These data indicated that PC was metabolized via phospholipase(s) A and phospholipase C. Substantial diacylglycerol lipase activity was identified in the cell sonicate. Production of similar proportions of diacylglycerol and phosphocholine and the low relative activity of phospholipase C compared to phospholipase A indicated that the phospholipase C-diacylglycerol lipase pathway contributed little to fatty acid release from the sn-2 position of PC. Neither phospholipase A nor phospholipase C required Ca2+. The pH profiles and subcellular fractionation experiments indicated the presence of multiple forms of phospholipase A, but phospholipase C activity displayed a single pH optimum at 7.5 and was located exclusively in the particulate fraction. The two enzyme activities demonstrated differential sensitivities to inhibition by p-bromophenacylbromide, phenylmethanesulfonyl fluoride and quinacrine. Each of these agents inhibited phospholipase A, whereas phospholipase C was inhibited only by p-bromophenacylbromide. The unique characteristics observed for phospholipase C activity towards PC indicated the existence of a novel enzyme that may play an important role in lipid metabolism in endothelial cells.     

Substrate specificity of Staphylococcus aureus (TEN5) lipases with isomeric oleoyl-sn-glycerol ethers as substrates

For the first time fully protected substrates with only one hydrolyzable ester bond have been used to analyze the substrate specificity of microbial lipases. In these substrates the ester is attached to the glycerol molecule in a precisely defined position. The use of three different substituents generates chirality and thus allows the analysis of positional specificities of individual lipases. Therefore, these new substrates have been used to study the enzymatic activities of two closely related lipases isolated from Staphylococcus aureus (TEN5) designated the 44 and 43 kDa lipase. The lipases, especially the 44 kDa molecule, show a high specificity for the hydrolysis of the ester in the sn-1 position (S-configuration), which is hydrolyzed by a factor of ten faster than that in the sn-3 position. In addition, the study demonstrates for the first time that the rate of hydrolysis of a fatty acid ester attached to the sn-2 position of glycerol by microbial lipases depends on the configuration of the substrate molecule.     

Cyclopentanoid analogs of phosphatidylcholine: susceptibility to phospholipase A2

Six isomers of dipalmitoylcyclopentanetriol phosphocholine (cyclopentano-lecithin) were tested as potential substrates for phospholipase A2. Since each of these analogs possesses a configuration that mimics a narrow range of conformations of a glycerophospholipid molecule, the analogs were used to assess the enzyme's conformational requirements. Studies showed that all of the analogs containing the phosphocholine at the C-1 (or C-3) position could be hydrolyzed, while only one of the three analogs that contains the polar head group at the C-2 position was susceptible. Kinetic studies, however, revealed that only the all-trans-(1,3/2-1P)-cyclopentano-lecithin gave initial rates of hydrolysis that were measurable by pH-stat. Acyl group specificity of the enzyme towards the all-trans isomer was determined with an analog was acyl groups were distinguishable. The synthesis of this mixed-acid-cyclopentano-PC is described herein. When this analog was enzymatically assayed, results unequivocally showed the enzyme to be specific for C-2 acyl hydrolysis. This specificity, and data showing that the all-trans analog is stereospecifically hydrolyzed, indicate that it is acted on in an analogous manner to dipalmitoylphosphatidylcholine. These studies indicate that although the configuration of the analog is not necessarily a prerequisite for hydrolysis, there does appear to be an optimal spatial orientation for enzymatic activity. The analogy between the susceptibilities of all-trans-(1,3/2-1P)-cyclopentano-lecithin and glycero-lecithin suggests that the conformation of the glycero-lecithin during phospholipase A2-mediated hydrolysis may be best simulated by the all-trans orientation of C-O bonds in the artificial substrate.     

Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana.

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full-length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild-type yeast showed that its phospholipid content was lower than that of the control (void plasmid-transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3-transformed yeast were incubated with di-[1-14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3-transformed yeast were incubated with either sn-1- or sn-2-[1-14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn-1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1-myristoyl, 2-oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6-6.5 and 60-65 degrees C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.     

Suicide-inhibitory bifunctionally linked substrates (SIBLINKS) as phospholipase A2 inhibitors. Mechanistic implications

Novel phospholipids that function as mechanism-based inhibitors for phospholipase A2 (PLA2) are described. PLA2-catalyzed hydrolysis of the sn-2 ester of these suicide-inhibitory bifunctionally linked substrates (SIBLINKS) followed by a cyclization reaction generates a cyclic anhydride at the active site of the enzyme which leads to inhibition. Structure/activity relationships for the SIBLINKS substituents in the sn-1 and sn-2 position are delineated. Time courses and efficiency of SIBLINKS inhibition are reported and compared for extracellular PLA2s obtained from Naja naja naja, porcine pancreas, bee venom, Crotalus atrox and Crotalus adamanteus. SIBLINKS-inhibited PLA2s cannot process either monomeric or micellar substrates consistent with inhibition at the catalytic site. Some SIBLINKS efficiently inactivate 1 mol of N. naja naja and C. adamanteus PLA2/6-10 mol of SIBLINKS hydrolyzed. Inhibition of N. naja naja PLA2 can be reversed by hydroxylamine, suggesting that a tyrosine residue is acylated.     

Conformationally constrained analogues of diacylglycerol (DAG). Effect on protein kinase C (PK-C) binding by the isosteric replacement of sn-1 and sn-2 esters in DAG-lactones

In order to determine the importance of the two ester pharmacophores in high affinity, conformationally constrained DAG-lactones (Lac-1-5) as PK-C ligands, we have independently replaced the sn-1 and sn-2 carbonyl esters in these compounds by ketone (2, 10, 11), amide (3, 25-28), and hydroxyl (12, 13) isosteres. Although the ketone analogue of the sn-1 ester (2) exhibited comparable activity to the parent Lac-1 when taking into account the difference in lipophilicities, the other isosteres were significantly poorer PK-C alpha ligands compared to the parent DAG-lactones. This study demonstrates that the ester functionality in DAG-lactone plays an important role in the ligand's capacity to form a strong hydrogen bond with Gly253 at the active site. The discrete K(i) analysis from the sn-1 and sn-2 isosteres further confirms that the DAG-lactones bind preferentially to the C1-domain in the sn-2 binding mode, as previously suggested.     

Conversion of Bacillus thermocatenulatus lipase into an efficient phospholipase with increased activity towards long-chain fatty acyl substrates by directed evolution and rational design.

The thermoalkalophilic lipase from Bacillus thermocatenulatus BTL2 exhibits a low phospholipase activity (lecithin/tributyrin ratio 0.03). A single round of random mutagenesis of the BTL2 gene followed by screening of 6000 transformants on egg-yolk plates identified three variants with 10-12-fold increased phospholipase activities, corresponding to lecithin/tributyrin ratios of 0.16-0.36. All variants were specific for the sn-1 acyl ester bond of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine. Mutations occurred predominantly in the N-terminal part of BTL2 with regions surrounding the predicted helix alpha(4) and lid as hotspots. Two mutations, L184P located in the predicted helix alpha(4) and H15P found in the highly conserved oxy-anion hole motif among hydrolases, were identified to account for increased phospholipase activity. Two of the three variants showed reduced activities towards medium- and long-chain fatty acyl methyl esters compared to the wild-type enzyme. Substitution of Leu353 with Ser, which is located adjacent to the active site histidine and is important for phospholipase activity in the Staphylococcus hyicus lipase, increased the absolute phospholipase activities of the variants, but not of BTL2, approximately 2-fold. The engineered best variant displayed a lecithin/tributyrin ratio of 0.52, corresponding to a 17-fold increase compared to the wild-type enzyme. Moreover, this variant exhibited a 1.5-4-fold higher activity towards long-chain fatty acyl methyl ester (C18:1, C18:2, C18 and C20) compared to BTL2. A second round of mutagenesis and screening on lecithin-plates yielded no new variants with further increased phospholipase/lipase activity ratios, but instead one variant with a 5-fold increased expression rate and two variants with a 3-fold reduced activity towards triolein were obtained.     

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).     

Solubilization and assay of phospholipase A2 activity from rat jejunal brush-border membranes

The phospholipase activity of rat jejunal brush-border membranes was examined in the presence of several solubilizing agents, by measuring the hydrolysis of endogenous membrane phospholipids, as well as the hydrolysis of exogenous, radiolabelled substrates. Enzyme activity was highly stimulated by dispersion in 1% solutions of bile salts, or in a synthetic, bile-salt derivative, 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate (CHAPS). Under these conditions the endogenous membrane phospholipids were largely degraded to free fatty acids and water-soluble phosphate. In the presence of 1% CHAPS, hydrolysis of exogenous phosphatidylcholine was shown to be due to an initial phospholipase A2-type attack followed by a subsequent lysophospholipase-type attack. These activities co-purified with the brush-border membrane. Maximal phospholipase A2 hydrolysis occurred at an alkaline pH of 8-11, with bile-salt detergents present at greater than their critical micellar concentrations. Hydrolysis was completely divalent-ion independent. Phospholipase A2 activity was not stimulated by 50% diethyl ether or ethanol, or in the presence of 1% solutions of Triton X-100, Zwittergent 3-12, sodium dodecyl sulphate, or n-octylglucoside. Stimulation of phospholipase activity by detergents was not related to their effectiveness at solubilizing the membrane proteins. When assayed individually phosphatidylcholine and lysophosphatidylcholine were each hydrolyzed (at the sn-2 and sn-1 positions, respectively) at a rate of approximately 125 nmol/mg protein per min. When assayed together, the two substrates appeared to compete for the same active site over a wide range of concentrations. It was concluded that the brush-border membrane contains an integral membrane protein with phospholipase A2 and lysophospholipase activities, which is specifically stimulated by bile salts and bile salt-like detergents.     

The interaction of phospholipase A2 with phospholipid analogues and inhibitors

A series of structurally modified phospholipids have been used to delineate the structural features involved in the interaction between cobra venom (Naja naja naja) phospholipase A2 and its substrate. Special emphasis has been placed on sn-2 amide analogues of the phospholipids. These studies have led to a very potent, reversible phospholipase A2 inhibitor. A six-step synthesis of this compound, 1-palmitylthio-2-palmitoylamino-1,2-dideoxy-sn-glycero-3- phosphorylethanolamine (thioether amide-PE), was developed. Other analogues studied included 1-palmitylthio-2-palmitoylamino-1,2-dideox-sn- glycero-3-phosphorylcholine, 1-palmityl-2-palmitoylamino-2- deoxy-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-palmitoylamino-2-deoxy-sn-glycero-3- phosphorylcholine, 1-palmitylthio- 2([(tetradecyloxy)carbonyl]amino)-1,2-dideoxy-sn-glycero-3- phosphorylcholine, 1-palmitoyl- 2([(octadecylylamino)carbonyl]amino)-2-deoxy-sn-glycero-3- phosphorylcholine, and sphingomyelin. Inhibition studies used the well defined Triton X-100 mixed micelle system and the spectroscopic thio assay. The phospholipid analogues showed varying degrees of inhibition. The best inhibitor was the thioether amide-PE which had an IC50 of 0.45 microM. In contrast, sphingomyelin, a natural phospholipid that resembles the amide analogues, did not inhibit but rather activated phosphatidylcholine hydrolysis. This systematic study of phospholipase A2 inhibition led to the following conclusions about phospholipid-phospholipase A2 interactions: (i) sn-2 amide analogues bind tighter than natural phospholipids, presumably because the amide forms a hydrogen bond with the water molecule in the enzyme active site, stabilizing its binding. (ii) Inhibitor analogues containing the ethanolamine polar head group appear to be more potent inhibitors than those containing the choline group. This difference in potency may be due solely to the fact that the cobra venom phospholipase A2 is activated by choline-containing phospholipids. Thus, choline-containing non-hydrolyzable analogues both inhibit and activate this enzyme. Both of these effects must be taken into account when studying phosphatidylcholine inhibitors of the cobra venom enzyme. (iii) The potency of inhibition of these analogues is significantly enhanced by increasing the hydrophobicity of the sn-1 functional group.(ABSTRACT TRUNCATED AT 400 WORDS)