Substrate specificities and properties of human phospholipases A2 in a mixed vesicle model (original) (raw)
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The Biochemical journal, 1994
Previous studies using phospholipid mixed vesicles have demonstrated that several types of phospholipase A2 (PLA2) enzymes exhibit different selectivity for fatty acids at the sn-2 position, for the type of chemical bond at the sn-1 position or for the phosphobase moiety at the sn-3 position of phospholipids. In the present study, we have utilized natural mammalian membranes from U937 monocytes to determine whether two purified 14 kDa PLA2 isoenzymes (Type I, Type II) and a partially purified 110 kDa PLA2 exhibit substrate selectivity for certain fatty acids or phospholipids. In these studies, arachidonic acid (AA) release from membranes was measured under conditions where the remodelling of AA mediated by CoA-independent transacylase (CoA-IT) activity has been eliminated. In agreement with the mixed-vesicle models, AA was the major unsaturated fatty acid hydrolysed from membranes by the 110 kDa PLA2, suggesting that this PLA2 is selective in releasing AA from natural membranes. By ...
Biochemical and Biophysical Research Communications, 1992
The action of porcine pancreatic phospholipase A2 towards fluorescent phospholipid analogs is either enhanced or suppressed by 4[~-12-O-tetradecanoylphorbol-13-acetate (TPA), depending on the chemical slructure of the substrate and the concentration of Ca 2+. In the presence of nmolar Ca 2+ concentrations increasing ITPA] enhanced by approx. 5-fold the rate of hydrolysis of the pyrene-labelled acidic alkyl-acyl phospholipid, 1-octacosany1-2-[6-(pyrene-1-yl)] hexanoyl -sn-glycero -3-phosphatidylmethanol. Maximal effect was obtained at high TPA/substrate molar ratios approaching 1:2. In the presence of 4 mM CaC12 maximal activation was reduced to -l.5fold. With the corresponding phosphatidylcholine derivative as a substrate increasing [TPA] reduced fatty acid release maximally by 90% both at low [Ca 2+] as well as in the presence of 4 mM CaC12. Essentially identical results were obtained using 4c~-TPA, a stereoisomer which does not activate protein kinase C. ® 1992 Academic Press, Inc.
Journal of Biological Chemistry, 2002
Expression of the full set of human and mouse groups I, II, V, X, and XII secreted phospholipases A(2) (sPLA(2)s) in Escherichia coli and insect cells has provided pure recombinant enzymes for detailed comparative interfacial kinetic and binding studies. The set of mammalian sPLA(2)s display dramatically different sensitivity to dithiothreitol. The specific activity for the hydrolysis of vesicles of differing phospholipid composition by these enzymes varies by up to 4 orders of magnitude, and yet all enzymes display similar catalytic site specificity toward phospholipids with different polar head groups. Discrimination between sn-2 polyunsaturated versus saturated fatty acyl chains is <6-fold. These enzymes display apparent dissociation constants for activation by calcium in the 1-225 microm range, depending on the phospholipid substrate. Analysis of the inhibition by a set of 12 active site-directed, competitive inhibitors reveals a large variation in the potency among the mammalian sPLA(2)s, with Me-Indoxam being the most generally potent sPLA(2) inhibitor. A dramatic correlation exists between the ability of the sPLA(2)s to hydrolyze phosphatidylcholine-rich vesicles efficiently in vitro and the ability to release arachidonic acid when added exogenously to mammalian cells; the group V and X sPLA(2)s are uniquely efficient in this regard.
Journal of Medicinal Chemistry, 1994
The substrate specificity at the active site of recombinant human synovial fluid phospholipase Az (hs-PLA2) was investigated by the preparation of a series of short-chain phospholipid analogs and measurement of their enzymatic hydrolysis at concentrations well below the critical micelle concentration. Substrates used in the study included 1,2-dihexanoylglycerophospholipids, 1,2bis(alkanoylthio)glycerophospholipids, and l-O-alkyl-2-(alkanoylthio)phospholipids. Turnover was observed for only a few of the 1,2-dihexanoylglycerophospholipids, and the rate of hydrolysis was very low, near the limit of detection of the assay. In contrast, selected 24alkanoylthio)glycerophospholipids were hydrolyzed by hs-PLAz a t much higher rates a t concentrations well below their critical micelle concentration (cmc). Thus, the 1,2-bis(hexanoylthio)glycerophosphatidylmethanol exhibits a kCatlKLI = 1800 L mol-' s-l. Over the calculated log P (cLogP) range of 3-9, cLogP and log(k,,,lK~, were linearly related for compounds with straight-chain sn-1 and sn-2 substituents. At comparable cLogP's, the sn-1 ethers and thioesters were hydrolyzed at comparable rates. A negative charge in the phosphate head group was required for enzyme activity. Unsaturation, aromaticity, and branching in the sn-2 substituent reduce turnover dramatically. The same structural modifications in the sn-1 substituent have less effect on turnover. Certain of these substrates, e.g., 1,2-bis(hexanoylthio)glycerophosphatidylmethanol, may be useful in assaying for active site inhibitors of PLAr. The structureactivity relationships established here for substrates should serve as a reference for the structure-activity relationships of substrate-based inhibitors. * Authors to whom inquiries should be directed. ' Division of Medicinal Chemistry.
Evidence for the control of the action of phospholipases A by the physical state of the substrate
Biochemistry, 1984
Abbreviations: diClzPG, 1,2-didodecanoyl-sn-glycero-3-phosphorac-glycerol; diEPG, 1,2-dihexadecyl-sn-glycero-3-phospho-rac-glycerol; diPBPG, 1,2-bis[(pyren-l-yl)butanoyl]-sn-glycero-3-phospho-racglycerol; cmc, critical micellar concentration; ffa, free fatty acid; I,, pyrene excimer emission intensity; I,, pyrene monomer emission intensity; PLA, phospholipase A; PLAI, phospholipase A,; PLA,, phospholipase A,; Tris-HC1, tris(hydroxymethy1)aminomethane hydrochloride; EDTA, ethylenediaminetetraacetic acid.
Archives of Biochemistry and Biophysics, 1997
to this artificial substrate, membranes from U937 cells were isolated and used as substrate. With these mem-Cytosolic phospholipase A 2 catalyzes the selective branes, the dephosphorylated enzyme was only 21% release of arachidonic acid from the sn-2 position of less active than the phosphorylated enzyme. In the phospholipids and is believed to play a key cellular presence of glycerol, there was no detectable differrole in the generation of arachidonic acid. The enzyence the two enzyme forms, and the rate of hydrolysis matic activity of cPLA 2 is affected by several mechawas increased by 300-390% over that measured in the nisms, including substrate presentation and the phosabsence of glycerol. These results suggest that the catphorylation state of the enzyme. Using covesicles of alytic efficiency of the phosphorylated enzyme is not 1-palmitoyl-2-arachidonoyl-[arachidonoyl-1-14 C]-snparticularly relevant to its activation in vivo. Moreglycero-3-phosphocholine and 1,2-dimyristoyl-phosover, it may be that glycerol is mimicking the effect of phatidylmethanol as substrate, the effects of phossome unidentified factor which greatly enhances the phorylation on the interfacial binding and catalytic catalytic efficiency of the enzyme. ᭧ 1997 Academic Press constants were investigated. Phosphorylated and de-Key Words: Cytosolic phospholipase A 2 ; serine phosphosphorylated enzyme forms were shown to have phorylation; glycerol; kinetic constants. identical values of 2.6 mM for K app M , an equilibrium dissociation constant which consists of the intrinsic dissociation constant from the lipid/water interface (K S ) and the dissociation constant for phospholipid from Cytosolic phospholipase A 2 (cPLA 2 ) 2 catalyzes the hythe active site (K* M ). Moreover, the values of K* M for drolysis of the sn-2 arachidonoyl ester of phospholipids phosphorylated and dephosphorylated enzyme did not (1, 2). Because of its apparent role in the generation of differ significantly (0.4 { 0.1 and 0.2 { 0.1, respecleukotrienes and prostaglandins, which are metabotively). However, dephosphorylation of the enzyme relized from arachidonate, cPLA 2 has received considerduced the value of k cat by 39%. The phosphorylation able medicinal interest. state of the enzyme had no effect on either the coopera-The cPLA 2 is a calcium-dependent enzyme which is tivity shown by this enzyme or the thermal stability of present in a number of different tissues and cells inthe enzyme. Surprisingly, the presence of glycerol (4 M) masks the effect of phosphorylation on k cat . Instead, cluding monocytes, neutrophils, and platelets (3-5). glycerol increased the value of k cat by 440% for the The enzyme is normally located in the cytosol, but phosphorylated enzyme and by 760% for the dephosphorylated form. Moreover, addition of glycerol had 2 Abbreviations are: BSA, bovine serum albumin; CaI-PLA 2 , calonly small effects on K app M . The increase in the k cat upon cium-independent phospholipase A 2 ; [ 14 C]PAPC, 1-palmitoyl-2-araaddition of glycerol results from a substantial dechidonoyl-[arachidonoyl-1-14 C]-sn-glycero-3-phosphocholine; cPLA 2 , crease in the activation energy from 29.4 to 14.8 kcalr cytosolic phospholipase A 2 ; DMPM, 1,2-dimyristoyl-sn-glycero-3mol 01 . To determine whether the effects of phosphoryphosphomethanol; EDTA, ethylenedinitroilotetraacetic acid; EGTA, lation of the enzyme or addition of glycerol are unique ethylene bis(oxyethylenenitrilo)tetraacetic acid; HBSS, Hanks' buffered salt solution;
Purification and Characterization of Ca2+-Dependent Phospholipases A2from Rat Kidney
Archives of Biochemistry and Biophysics, 1996
Phospholipase A 2 (phosphatide 2-acyl hydrolase, EC 3.1.1.4.) (PLA 2) 5 specifically hydrolyzes the fatty acyl Three phospholipase A 2 (PLA 2) activities were ester bonds at the sn-2 position of sn-3 phosphoglyceridentified in rat kidney. In the particulate fraction a ides. Ca 2/-dependent PLA 2 is a heterogeneous family PLA 2 activity was present which was cross-reactive with polyclonal antibodies against the 14-kDa group of enzymes that is widely distributed in nature (1). II PLA 2. This PLA 2 was partially solubilized and puri-High concentrations of this enzyme are found in panfied to near homogeneity. The amino acid sequence creatic juice and in the venoms of snakes and bees (2). at the N-terminus of the purified enzyme was identi-Additional PLA 2 activities are found in trace amounts cal to that of the 14-kDa rat group II PLA 2 from rat in almost every cell type and with a diverse subcellular liver mitochondria, platelet, and spleen. The cytolocalization, including mitochondria, Golgi memsolic fraction of rat kidney contained at least two branes, plasma membranes, secretory granules, and PLA 2 activities which could be separated on a Mono cytosol (3). These intracellular PLA 2 s are involved in Q column. Upon gel filtration the activity that eluted the turnover of phospholipids and in the deacylationfrom the anion-exchange column in the salt gradient reacylation cycle, participating in the biosynthesis of behaved as a high molecular mass PLA 2 , exhibited a phospholipids with a specific acyl chain composition. preference for arachidonic acid at the sn-2 position Moreover these phospholipases can liberate arachiof glycerophospholipids, and was already optimally donic acid from the sn-2 position of glycerophospholipactive at submillimolar Ca 2/ concentrations. The cyids. This generation of free arachidonic acid is thought tosolic PLA 2 activity that did not bind to the anionto be the rate-limiting step in the biosynthesis of a exchange column was purified by gel filtration, imvariety of biologically active lipids like prostaglandins, munoaffinity chromatography using immobilized leukotrienes, thromboxane, and lipoxins (3-5). polyclonal antibodies to group I PLA 2 , and C18 re-Based on the molecular mass, phospholipases A 2 can versed-phase chromatography. Immunological propbe divided into two classes: the low molecular mass erties and N-terminal sequence analysis identified enzyme (14 kDa) (3) and the high molecular mass enthis enzyme as rat group I PLA 2. Rat glomerular meszyme (85 kDa) or cytosolic phospholipase A 2 (cPLA 2) angial cells contained only group II and high molecu-(6-8). The low molecular mass enzymes can be divided lar mass PLA 2 enzymes. ᭧ 1996 Academic Press, Inc. into group I (pancreatic-type) and group II (non-pancre-Key Words: calcium; phospholipase A 2 ; rat; kidney. atic-type) phospholipases A 2 (9) and both enzymes require millimolar concentrations of Ca 2/ in the catalytic step (1, 10). On the other hand, cPLA 2 needs micromolar concentrations of the divalent cation for its translo