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.     

Human plasma platelet-activating factor acetylhydrolase. Oxidatively fragmented phospholipids as substrates

Human plasma platelet-activating factor (PAF) acetylhydrolase hydrolyzes the sn-2 acetyl residue of PAF, but not phospholipids with long chain sn-2 residues. It is associated with low density lipoprotein (LDL) particles, and is the LDL-associated phospholipase A2 activity that specifically degrades oxidatively damaged phospholipids (Stremler, K. E., Stafforini, D. M., Prescott, S. M., Zimmerman, G. A., and McIntyre, T. M. (1989) J. Biol. Chem. 264, 5331-5334). To identify potential substrates, we synthesized phosphatidylcholines with sn-2 residues from two to nine carbon atoms long, and found the V/k ratio decreased as the sn-2 residue was lengthened: the C5 homolog was 50%, the C6 20%, while the C9 homolog was only 2% as efficient as PAF. However, the presence of an omega-oxo function radically affected hydrolysis: the half-life of the sn-2 9-aldehydic homolog was identical to that of PAF. We oxidized [2-arachidonoyl]phosphatidylcholine and isolated a number of more polar phosphatidylcholines. We treated these with phospholipase C, derivatized the resulting diglycerides for gas chromatographic/mass spectroscopic analysis, and found a number of diglycerides where the m/z ratio was consistent with a series of short to medium length sn-2 residues. We treated the polar phosphatidylcholines with acetylhydrolase and derivatized the products for analysis by gas chromatography/mass spectroscopy. The liberated residues were more polar than straight chain standards and had m/z ratios from 129 to 296, consistent with short to medium chain residues. Therefore, oxidation fragments the sn-2 residue of phospholipids, and the acetylhydrolase specifically degrades such oxidatively fragmented phospholipids.     

Metabolism of extracellular phospholipids in Tetrahymena pyriformis

We studied the metabolism of phospholipids exogenously added to cultures of the protozoan, Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated via endocytosis of phospholipid vesicles, as judged from the following facts: Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. Phospholipid vesicles were incorporated into a phagosome-like structure in Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with 14C and 3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with Tetrahymena cells, the glycerol moiety and fatty acyl chain at the sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in Tetrahymena (J. Biochem. 99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells via endocytosis were hydrolyzed through the action of lysosomal phospholipases A1 and C, and also that one of the products, sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol via the microsomal "monoacylglycerol pathway."     

Synthetic phospholipids as substrates for phospholipase C from Bacillus cereus.

The substrate requirement of phospholipids for hydrolysis with phospholipase C from Bacillus cereus was studied with synthetic lipids well-defined in structure and configuration. For optimal activity, the glycerol molecule must contain three substituents: phosphocholine in sn-3-, an ester bond in sn-2- and an ether- or ester bond in sn-1-position. The length of the ester or ether chains is of minor importance. Any deviation from these structural requirements results in a large decrease in the hydrolysis rate. These essential structural and configurational elements for optimal activity for the B. cereus enzyme are perfectly combined in the platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. This molecule is one of the best substrates for hydrolysis with the bacterial phospholipase C.     

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.     

Energy-minimized structures and packing states of a homologous series of mixed-chain phosphatidylcholines: a molecular mechanics study on the diglyceride moieties.

Phosphatidylcholines or C(X):C(Y)PC, quantitatively the most abundant lipids in animal cell membranes, are structurally composed of two parts: a headgroup and a diglyceride. The diglyceride moiety consists of the glycerol backbone and two acyl chains. It is the wide diversity of the acyl chains, or the large variations in X and Y in C(X):C(Y)PC, that makes the family of phosphatidylcholines an extremely complex mixture of different molecular species. Since most of the physical properties of phospholipids with the same headgroup depend strongly on the structures of the lipid acyl chains, the energy-minimized structure and steric energy of each diglyceride moiety of a series of 14 molecular species of phosphatidylcholines with molecular weights identical to that of dimyristoylphosphatidylcholine without the headgroup are determined in this communication by molecular mechanics (MM) calculations. Results of two types of trans-bilayer dimer for each of the 14 molecular species of phosphatidylcholines are also presented; specifically, the dimeric structures are constructed initially based on the partially interdigitated and mixed interdigitated packing motifs followed subsequently by the energy-minimized refinement with MM calculations. Finally, tetramers with various structures to model the lateral lipid-lipid interactions in a lipid bilayer are considered. Results of laborious MM calculations show that saturated diacyl C(X):C(Y)PC with delta C/CL values greater than 0.41 prefer topologically to assemble into tetramers of the mixed interdigitated motif, and those with delta C/CL values less than 0.41 prefer to assemble into tetramers with a repertoire of the partially interdigitated motif. Here, delta C/CL, a lipid asymmetry parameter, is defined as the normalized acyl chain length difference between the sn-1 and sn-2 acyl chains for a C(X):C(Y)PC molecule; an increase in delta C/CL value is an indication of increasing asymmetry between the two lipid acyl chains. These computational results are in complete accord with the calorimetric data presented previously from this laboratory (H-n. Lin, Z-q. Wang, and C. Huang. 1991. Biochim. Biophys. Acta. 1067:17-28).     

Conformation of phosphatidylcholine in neat and cholesterol-containing liquid-crystalline bilayers. Application of a novel method.

The conformation of phosphatidylcholine in liquid-crystalline bilayers was studied with a novel, high-resolution method employing phosphatidylcholine species containing pyrenyl moieties in both acyl chains of variable length. Analysis of the intramolecular pyrene-pyrene collision data obtained for 30 such species in terms of a simple geometrical model showed that the sn-1 acyl chain penetrates, on the average, 0.84 +/- 0.11 methylene units (0.8 A) deeper into the bilayer than the sn-2 chain at 22 degrees C. A similar value was obtained at 37 degrees C. Since the penetration difference of the sn-1 and sn-2 acyl chains is inherently coupled to the conformation of the glycerol moiety, these data mean that the glycerol moiety of phosphatidylcholine is, on the average, only moderately tilted with respect to the bilayer plane in the liquid-crystalline state. This contrasts the perpendicular orientation observed previously for phosphatidylcholine crystals [Pearson, R. H., & Pascher, I. (1979) Nature 281, 499-501]. Importantly, addition of 50 mol % cholesterol, which is known to reduce dramatically the interactions between phosphatidylcholine molecules in bilayers, had only a small effect on the penetration difference of the acyl chains, strongly suggesting that the conformation of phosphatidylcholine in the liquid-crystalline state is determined largely by intramolecular, rather than intermolecular, 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.     

NMR studies of interactions between inhibitors and porcine pancreatic phospholipase A2.

Two-dimensional NMR studies were performed on the complexes of porcine pancreatic phospholipase A2, bound to a micellar lipid-water interface of fully deuterated dodecylphosphocholine, with competitive inhibitors derived from the following general structure: [formula: see text] X and Y are alkyl chains with various 'reporter groups'. The interactions between the inhibitor and the enzyme were localized by comparison of 2-D nuclear Overhauser effect spectra using protonated and selectively deuterated inhibitors, and inhibitors with groups having easily identifiable chemical shifts. These experiments led us to the following conclusions for the phospholipase A2/inhibitor/micelle complex: i) the His48 C2 ring proton is in close proximity to both the amide proton and the methylene protons at the sn-1 position of the glycerol skeleton of the inhibitor, ii) the acyl chain of the inhibitor at the sn-2 position makes hydrophobic contacts near Phe5, Ile9, Phe22 and Phe106; iii) no interactions between the acyl chain at the sn-1 position and the protein could be identified. Comparison of our results on the enzyme/inhibitor/micelle ternary complex with the crystal structure of the enzyme-inhibitor complex shows that the mode of inhibitor binding is similar. However, in several cases we found indications that the hydrophobic chains of the inhibitors can have multiple conformations.     

Membrane-bound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase.

Human plasma platelet activating factor acetylhydrolase (pPAF-AH) is a phospholipase A(2) that specifically hydrolyzes the sn-2 ester of platelet activating factor (PAF) and of phospholipids with oxidatively truncated sn-2 fatty acyl chains. pPAF-AH is bound to lipoproteins in vivo, and it binds essentially irreversibly to anionic and zwitterionic phospholipid vesicles in vitro and hydrolyzes PAF and PAF analogues. Substrate hydrolysis also occurs in the absence of vesicles, with a maximum rate reached at the critical micelle concentration. A novel pre-steady-state kinetic analysis with enzyme tightly bound to vesicles and with a substrate that undergoes slow intervesicle exchange establishes that pPAF-AH accesses its substrate from the aqueous phase and thus is not an interfacial enzyme. Such a mechanism readily explains why this enzyme displays dramatic specificity for phospholipids with short sn-2 chains or with medium-length, oxidatively truncated sn-2 chains since a common feature of these lipids is their relatively high water solubility. It also explains why the enzymatic rate drops as the length of the sn-1 chain is increased. pPAF-AH shows broad specificity toward phospholipids with different polar headgroups. Additional results are that PAF undergoes intervesicle exchange on the subminute time scale and it does not undergo transbilayer movement over tens of minutes.     

Synthesis of sn-1 functionalized phospholipids as substrates for secretory phospholipase A2

Secretory phospholipase A2 (sPLA2) represents a family of small water-soluble enzymes that catalyze the hydrolysis of phospholipids in the sn-2 position liberating free fatty acids and lysophospholipids. Herein we report the synthesis of two new phospholipids (1 and 2) with bulky allyl-substituents attached to the sn-1 position of the glycerol backbone. The synthesis of phospholipids 1 and 2 is based upon the construction of a key aldehyde intermediate 3 which locks the stereochemistry in the sn-2 position of the final phospholipids. The aldehyde functionality serves as the site for insertion of the allyl-substituents by a zinc mediated allylation. Small unilamellar liposomes composed of phospholipids 1 and 2 were subjected to sPLA2 activity measurements. Our results show that only phospholipid 1 is hydrolyzed by the enzyme. Molecular dynamics simulations revealed that the lack of hydrolysis of phospholipid 2 is due to steric hindrance caused by the bulky side chain of the substrate allowing only limited access of water molecules to the active site.     

The acylation of lipophilic alcohols by lysosomal phospholipase A2

A novel lysosomal phospholipase A(2) (LPLA2) with specificity toward phosphatidylethanolamine and phosphatidylcholine was previously purified and cloned. LPLA2 transfers sn-1 or sn-2 acyl groups of phospholipids to the C1 hydroxyl of the short-chain ceramide N-acetylsphingosine (NAS) under acidic conditions. The common features of lipophilic alcohols serving as acceptor molecules in the transacylase reaction were examined. 1-O-Hexadecyl-2-acetyl-sn-glycerol (HAG) was acylated by LPLA2 similar to NAS. HAG competed with NAS and inhibited NAS acylation. The transacylation of 1-O-hexadecyl-glycerol (HG), 1-O-palmityl-2-O-methyl-sn-glycerol (PMG), and monoacylglycerols was also investigated. HG, PMG, 1- or 3-palmitoyl-sn-glycerol, and 2-palmitoylglycerol were converted to 1,3-alkylacylglycerol, 1,2-dialkyl-3-acylglycerol, 1,3-diacylglycerol, and 1,2- or 2,3-diacylglycerol, respectively. HG and monoacylglycerol inhibited the acylation of NAS by the enzyme with IC(50) values of 35 and 45 microM, respectively. Additionally, the enzyme acylated glycerol to produce 1- or 3-acyl-sn-glycerol but not 2-acylglycerol. Therefore, the preferred acceptor molecules for LPLA2 are primary alcohols with one long carbon chain and one small nonpolar residue linked to the C2 position of ethanol. The enzyme acylated other natural lipophilic alcohols, including anandamide and oleoylethanolamide. Thus, LPLA2 may function to remodel acyl groups and modulate the biological and pharmacological activities of some lipophilic alcohols.     

Characterization of the transacylase activity of rat liver 60-kDa lysophospholipase-transacylase. Acyl transfer from the sn-2 to the sn-1 position.

Rat liver 60-kDa lysophospholipase-transacylase catalyzes not only the hydrolysis of 1-acyl-sn-glycero-3-phosphocholine, but also the transfer of its acyl chain to a second molecule of 1-acyl-sn-glycero-3-phosphocholine to form phosphatidylcholine (H. Sugimoto, S. Yamashita, J. Biol. Chem. 269 (1994) 6252-6258). Here we report the detailed characterization of the transacylase activity of the enzyme. The enzyme mediated three types of acyl transfer between donor and acceptor lipids, transferring acyl residues from: (1) the sn-1 to -1(3); (2) sn-1 to -2; and (3) sn-2 to -1 positions. In the sn-1 to -1(3) transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1(3) positions of glycerol and 2-acyl-sn-glycerol, producing 1(3)-acyl-sn-glycerol and 1,2-diacyl-sn-glycerol, respectively. In the sn-1 to -2 transfer, the sn-1 acyl residue of 1-acyl-sn-glycero-3-phosphocholine was transferred to not only the sn-2 positions of 1-acyl-sn-glycero-3-phosphocholine, but also 1-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. 1-Acyl-sn-glycero-3-phospho-myo-inositol and 1-acyl-sn-glycero-3-phosphoserine were much less effectively transacylated by the enzyme. In the sn-2 to -1 transfer, the sn-2 acyl residue of 2-acyl-sn-glycero-3-phosphocholine was transferred to the sn-1 position of 2-acyl-sn-glycero-3-phosphocholine and 2-acyl-sn-glycero-3-phosphoethanolamine, producing phosphatidylcholine and phosphatidylethanolamine, respectively. Consistently, the enzyme hydrolyzed the sn-2 acyl residue from 2-acyl-sn-glycero-3-phosphocholine. By the sn-2 to -1 transfer activity, arachidonic acid was transferred from the sn-2 position of donor lipids to the sn-1 position of acceptor lipids, thus producing 1-arachidonoyl phosphatidylcholine. When 2-arachidonoyl-sn-glycero-3-phosphocholine was used as the sole substrate, diarachidonoyl phosphatidylcholine was synthesized at a rate of 0.23 micromol/min/mg protein. Thus, 60-kDa lysophospholipase-transacylase may play a role in the synthesis of 1-arachidonoyl phosphatidylcholine needed for important cell functions, such as anandamide synthesis.     

Platelet-activating factor acetylhydrolases: broad substrate specificity and lipoprotein binding does not modulate the catalytic properties of the plasma enzyme

Platelet-activating factor acetylhydrolases (PAF-AHs) are a group of enzymes that hydrolyze the sn-2 acetyl ester of PAF (phospholipase A(2) activity) but not phospholipids with two long fatty acyl groups. Our previous studies showed that membrane-bound human plasma PAF-AH (pPAF-AH) accesses its substrate only from the aqueous phase, which raises the possibility that this enzyme can hydrolyze a variety of lipid esters that are partially soluble in the aqueous phase. Here we show that pPAF-AH has broad substrate specificity in that it hydrolyzes short-chain diacylglycerols, triacylglycerols, and acetylated alkanols, and displays phospholipase A(1) activity. On the basis of all of the substrate specificity results, it appears that the minimal structural requirement for a good pPAF-AH substrate is the portion of a glyceride derivative that includes an sn-2 ester and a reasonably hydrophobic chain in the position occupied by the sn-1 chain. In vivo, pPAF-AH is bound to high and low density lipoproteins, and we show that the apparent maximal velocity for this enzyme is not influenced by lipoprotein binding and that the enzyme hydrolyzes tributyroylglycerol as well as the recombinant pPAF-AH does. Broad substrate specificity is also observed for the structurally homologous PAF-AH which occurs intracellularly [PAF-AH(II)] as well as for the PAF-AH from the lower eukaryote Physarum polycephalum although pPAF-AH and PAF-AH(II) tolerate the removal of the sn-3 headgroup better than the PAF-AH from P. polycephalum does. In contrast, the intracellular PAF-AH found in mammalian brain [PAF-AH(Ib) alpha 1/alpha 1 and alpha 2/alpha 2 homodimers] is more selectively operative on compounds with a short acetyl chain although this enzyme also displays significant phospholipase A(1) activity.     

Remodeling of rat hepatocyte phospholipids by selective acyl turnover

Acyl turnover of rat hepatocyte phospholipids and triacylglycerols was assessed by incubating the cells in media containing 40% H2(18)O and measuring the time-dependent incorporation of 18O into ester carbonyls by gas chromatography-mass spectrometry of hydrogenated methyl esters. Incorporation of 18O into 22-carbon acyl groups was low in phosphatidylcholine, phosphatidylinositol, and phosphatidylserine, whereas in phosphatidylethanolamine, it was about the same as in the other acyl groups. Incorporation of 18O into individual molecular species of phosphatidylcholine and phosphatidylethanolamine was determined after phospholipase C hydrolysis, derivatization to dinitrobenzoates, and separation by high-performance liquid chromatography. In most molecular species, acyl groups at the sn-1 and sn-2 positions became 18O-labeled at drastically different rates, indicating remodeling through deacylation-reacylation. Molecular species expected to arise de novo from acylation of glycerophosphate exhibited similar rates of 18O incorporation at the sn-1 and sn-2 positions. The data suggest that hepatocyte phospholipids are continually synthesized, remodeled by deacylation-reacylation at specific turnover rates up to 10-15%/h, and degraded. This acyl turnover probably does not involve the majority of intracellular unesterified fatty acids whose 18O incorporation was found to be very low. In contrast, the oxygens of extracellular unesterified fatty acids were readily exchanged with the media. This exchange was enzyme-catalyzed, possibly by lipases released into the media from damaged cells. Incorporation of 18O into exogenously added fatty acids was also rapid and resulted in enhanced uptake of 18O-labeled fatty acids into cellular lipids, primarily triacylglycerols and phosphatidylcholine, without drastic change of the intracellular free fatty acid pool.     

Lipoprotein lipase-catalyzed hydrolysis of phospholipid monolayers: effect of fatty acyl composition on enzyme activity

The phospholipase A1 activity of lipoprotein lipase (LpL) was determined with monomolecular phospholipid films. Rates of phospholipid hydrolysis were dependent on apolipoprotein C-II (the activator protein for LpL) phospholipid fatty acyl composition, and lipid-packing density. In sphingomyelin: cholesterol (2:1, molar) monolayers containing 5 mol % disaturated phosphatidylcholines (PC) and at a surface pressure of 22 mNm-1, rates of LpL hydrolysis of diC14:0PC, diC16:0PC, and diC18:0PC were 74, 207, and 65 nmol h-1 mg LpL-1, respectively. At 22 mNm-1, phospholipids containing unsaturated fatty acyl chains were hydrolyzed at rates 5-10 times greater than saturated lipids. At higher lipid packing densities, the difference in hydrolysis rates between saturated and unsaturated lipids was less apparent. Comparison of molecular areas indicate no simple dependency between the rate of LpL catalysis and phospholipid fatty acyl chain length and saturation/unsaturation.     

Preferable stimulation of PON1 arylesterase activity by phosphatidylcholines with unsaturated acyl chains or oxidized acyl chains at sn-2 position

To examine the effect of phospholipids on PON1 activities, purified PON1 was exposed to phospholipids prior to the determination of arylesterase and paraoxonase activities. Phosphatidylcholines with saturated acyl chains (C10-C16) showed a stimulation of both activities, chain length-dependent, with a greater stimulation of arylesterase activity, suggesting the implication of lipid bilayer in the stimulatory action. Such a preferable stimulation of arylesterase activity was more remarkable with phosphatidylcholines with polyunsaturated acyl chains or oxidized chains at sn-2 position, implying that the packing degree of acyl chain may be also important for the preferable stimulation of arylesterase activity. Separately, 1-palmitoyl-lysoPC also stimulated arylesterase activity preferably, indicating that the micellar formation of lipids around PON1 also contributes to the stimulatory action. Additionally, phosphatidylglycerols slightly enhanced arylesterase activity, but not paraoxonase activity. In contrast, phosphatidylserine and phosphatidic acid (≥0.1 mM) inhibited both activities Further, such a preferable stimulation of arylesterase activity by phosphatidylcholines was also reproduced with VLDL-bound PON1, although to a less extent. These data indicate that phosphatidylcholines with polyunsaturated acyl chains or oxidized chain, or lysophosphatidylcholine cause a preferable stimulation of arylesterase activity, thereby contributing to the decrease in the ratio of paraoxonase activity to arylesterase activity.     

On the substrate specificity of rat liver phospholipase A1

The substrate specificity of purified phospholipase A1 was studied using mixed micelles of phospholipid and Triton X-100. The kinetic analysis employed determined Vmax, Ks (a dissociation constant for the phospholipase A1-mixed micelle complex), and Km (the Michaelis constant for the catalytic step which reflects the binding of the enzyme to the substrate in the interface). The order of Vmax values was phosphatidic acid greater than phosphatidylethanolamine greater than phosphatidylcholine greater than phosphatidylserine. The order of Ks values was phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidic acid greater than phosphatidylserine; the order of Km values was phosphatidic acid greater than phosphatidylethanolamine = phosphatidylserine greater than phosphatidylcholine. When present together, phosphatidylcholine inhibited the hydrolysis of phosphatidylethanolamine but phosphatidylethanolamine did not affect the hydrolysis of phosphatidylcholine. Sphingomyelin, phosphatidylcholine plasmalogen, and phosphatidylethanolamine plasmalogen had no effect on the hydrolysis of phosphatidylethanolamine. The effects of the reaction products, lysolipids and/or fatty acids, were also considered for their influence on phosphatidylethanolamine hydrolysis catalyzed by phospholipase A1. Free fatty acid was found to inhibit, whereas lysophospholipids stimulated hydrolysis of phosphatidylethanolamine. In a mixture of 1,2- and 1,3-diacylglycerides in mixed micelles, only the acyl chain at the sn-1 position of the 1,2 compound was hydrolyzed. Surface charge did not modulate the hydrolysis of phosphatidylcholine vesicles or mixed micelles. In conclusion, it is hypothesized that steric hindrance at position 3 of the glycerol regulates substrate binding in the active site and that an acyl group in position 1 is favored over a vinyl ether linkage for binding.     

Effects of lipid fatty acyl chain structure on the activity of the (Ca2+ + Mg2+)-ATPase

The (Ca2+ + Mg2+)-ATPase purified from rabbit muscle sarcoplasmic reticulum has been reconstituted into a series of phosphatidylcholines in the liquid crystalline phase. For phosphatidylcholines containing monounsaturated fatty acyl chains, optimal activity is observed for a chain length of C18, with longer or shorter chains supporting lower activities. Phospholipids with methyl-branched chain saturated fatty acids support somewhat lower activities than the corresponding phospholipids with mono-unsaturated fatty acids. Mixed chain phospholipids support ATPase activities comparable to those shown by an unmixed chain phospholipid with the same average chain length. However, the response of the ATPase reconstituted with mixed chain phospholipids to the addition of oleyl alcohol is dominated by the longest fatty acyl chain. Based on their ability to displace brominated phospholipids, relative binding constants to the ATPase of a series of phosphatidylcholines have been determined. Binding to the ATPase is virtually unaffected by fatty acyl chain length or the presence of methyl branches.     

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.