Hydrolysis of three different head groups phospholipids by chicken group V phospholipase A2 using the monomolecular film technique (original) (raw)
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Effect of the lipid hydrolysis products on the phospholipase A2 action towards lipid monolayer
Chemistry and Physics of Lipids, 1994
The effect of laurie acid (LA) and lysolauroyllecithin (LLL) on the hydrolysis of lipid in monolayer by phospholipase A2 from Bee venom was studied. It was found that LLL inhibits phospholipase action under both high (39 mN/m) and low (25 raN/m) surface pressure. On the other hand, LA inhibits phospholipase action under the low surface pressure (15 mN/m or 25 raN/m), but increases enzyme activity under high surface pressure (39 raN/m). This activating effect can be suppressed by high ionic strength of the aqueous subphase. It is suggested that an increase of the negative surface charge of the lipid monolayer, followed by an increase of the local concentrations of the positively charged enzyme and calcium near the monolayer is a coupling factor between fatty acid accumulation and phospholipase activation. Such an autocatalytie process can only occur when the substrate is organised into monolayer, bilayer or micelles, therefore it can be considered as a reason for the substrate activation and induction time before lipid hydrolysis.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1987
The effect of the phospholipid polar head-group on the porcine pancreatic phospholipase A, (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) reaction was studied using 1-palmitoyl-t-[6-(pyren-l-yl)]hexanoyl-snglycero-3-phosphatidylcholine, -ethanolamine, -glycerol, -monomethylester and -serine as substrates. Except for the monomethylester amdogue, which was maximally activated by 3.5 mM CaCl 2, maximal enhancement of hydrolysis of the other pyrenephospholipids was obtained at 2 mM Ca2+. Sodium cholate inhibited hydrolysis of the ethanolamine and serine lipids, whereas a slight (1.4-2.0-fold) activation was observed for the -choline, -glycerol and -monomethylester derivatives. Arrhenius plots of hydrolysis of pyrenephospholipids by porcine pancreatic phospholipase A, revealed no discontinuities, thus indicating the absence of phase transition for these lipids in the temperature range 15-45°C. Specific activities of porcine and bovine pancreatic, porcine intestinal and snake venom (Crotalus atrox) phospholipases A, towards pyrenephospholipid liposomes were then compared. Whereas the snake venom phospholipase A, preferred phosphatidylcholine as a substrate, the other phospholipases A, preferred acidic phospholipids in the order monomethylester 2 glycerol 2 serine.
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.
The Journal of biological chemistry, 1988
Pancreatic phospholipase A2 (PLA2)-catalyzed hydrolysis of egg yolk phosphatidylcholine (PC) in mixed PC-cholate systems depends upon composition, structure, and size of the mixed aggregates. The hydrolysis of PC-cholate-mixed micelles made of an equal number of PC and cholate molecules is consistent with a Km of about 1 mM and a turnover number of about 120 s-1. Increasing the cholate/PC ratio in the micelles results in a decreased initial velocity. Hydrolysis of cholate-containing unilamellar vesicles is very sensitive to the ratio of cholate to PC in the vesicles. The hydrolysis of vesicles with an effective cholate/PC ratio greater than 0.27 is similar to that of the mixed micelles. The time course of hydrolysis of vesicles with lower effective ratios is similar to that exhibited by pure dipalmitoyl-phosphatidylcholine (DPPC) large unilamellar vesicles in the thermotropic phase transition region. In the latter two cases, the rate of hydrolysis increases with time until substrate...
Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2005
Human groups IIA, V and X secretory phospholipases A 2 (sPLA 2 s) were incubated with human HDL 3 , total HDL and LDL over a range of enzyme and substrate concentrations and exposure times. The residual phosphatidylcholines (PtdChos) were assayed by high performance liquid chromatography with electrospray ionization mass spectrometry (LC/ESI-MS). The enzymes varied markedly in their rates of hydrolysis of the different molecular species and in the production of lysoPtdCho. The sPLA 2 s were compared at a concentration of 1 Ag/ml and an incubation time of 4 h, when all three enzymes showed significant activity. The groups V and X sPLA 2 were up to 20 times more reactive than group IIA sPLA 2. Group X sPLA 2 hydrolyzed arachidonate and linoleate containing species preferentially, while group V hydrolyzed the linoleates in preference to polyunsaturates. In all instances, the arachidonoyl and linoleoyl palmitates were hydrolyzed in preference to the corresponding stearates by group X sPLA 2. The group IIA enzyme appeared to hydrolyze randomly all diacyl molecular species. The minor alkylacyl and alkenylacyl glycerophosphocholines (GroPChos) were poor substrates for groups V and X sPLA 2 s and these phospholipids tended to accumulate. The present study demonstrates a preferential release of arachidonate from plasma lipoprotein PtdCho by group X sPLA 2 , as well as a relative resistance of polyunsaturated PtdChos to hydrolysis by group V enzyme, which had not been previously documented. The use of lipoprotein PtdCho as substrate with LC/ESI-MS identification of hydrolyzed molecular species eliminates much of the uncertainty about sPLA 2 specificity arising from past analyses of fatty acid release from unknown or ill-defined sources.
Modulation of phospholipase A2 activity by neutral and anionic glycosphingolipids in monolayers
The Biochemical journal, 1989
The effect of neutral (galactocerebroside and asialo-ganglioside GM1) or anionic (sulphatide and gangliosides GM1, GD1a and GT1b) glycosphingolipids on the activity of phospholipase A2 from pig pancreas was studied in mixed monolayers of dilauroyl phosphatidylcholine with the glycosphingolipids in different molar fractions at various constant surface pressures. The activity of the enzyme depends on the proportion and type of glycosphingolipid in the interface. Sulphatide activates the enzyme at all proportions, whereas galactocerebroside shows inhibition or activation depending on its proportion in the film. Asialo-ganglioside GM1 and gangliosides GM1, GD1a and GT1b can strongly inhibit the enzyme at relatively low molar fractions in the film in the following order: asialo-ganglioside GM1 less than ganglioside GM1 less than ganglioside GT1b less than ganglioside GD1a. The changes of activity are not due to a direct action of the lipids on the active centre or interfacial recognition...
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2015
Surface pressure Isochoric method Bothrops diporus sPLA 2 Bothrops asper myotoxin We present an analysis of lipid monolayer hydrolysis at a constant area to assess the optimal lateral surface pressure value (Π opt) and thus, the surface packing density of the lipid, at which the activity of a given lipolytic enzyme is maximal. This isochoric method consists of a measurement of the decrease down to zero of the Π opt of phospholipid substrate monolayer due to continuous hydrolysis using only one reaction compartment. We performed the comparison of both approaches using several commercially available and literature-evaluated sPLA 2 s. Also, we characterized for the first time the profile of hydrolysis of DLPC monolayers catalyzed by a sPLA 2 from Streptomyces violaceoruber and isoenzymes purified from Bothrops diporus venom. One of these viper venom enzymes is a new isoenzyme, partially sequenced by a mass spectrometry approach. We also included the basic myotoxin sPLA 2-III from Bothrops asper. Results obtained with the isochoric method and the standard isobaric one produced quite similar values of Π opt , validating the proposal. In addition, we propose a new classification parameter, a lipolytic ratio of hydrolysis at two lateral pressures, 20 mN•m −1 and 10 mN•m −1 , termed here as LR 20/10 index. This index differentiates quite well "high surface pressure" from "low surface pressure" sPLA 2 s and, by extension; it can be used as a functional criterion for the quality of a certain enzyme. Also, this index could be added to the grouping systematic criteria for the superfamily proposed for phospholipase A 2 .
Hydrolysis by phospholipase D of phospholipids
Phospholipases D (PLD) catalyse hydrolysis and transphosphatidylation reactions in phospholipids. In the present study, the hydrolytic activity for cabbage PLD was investigated with five different substrates (dipalmitoylphosphatidylethanolamine (DPPE), dipalmitoylphosphatidylcholine (DPPC), didecanoylphosphatidylcholine (DDPC), 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine and lyso-phosphatidylcholine (lyso-PC)) in solution or adsorbed on a silica matrix. In the specific buffer solutions, where the substrates were proved to form large multilamellar polydisperse aggregates, PLD showed preference for DPPC > DPPE > DDPC > 1alkyl-2-acetyl-sn-glycero-3-phosphocholine > lyso-PC. When the substrates were adsorbed on the silica matrix, PLD hydrolysed 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine and lyso-PC, DDPC, but not DPPC or DPPE. Theoretical studies of the simplest possible adducts between the phospholipids and the silica matrix were performed. Examination of local geometries of DPPC showed a significant blocking of the P-O-X bond-prone to hydrolysis, which could possibly block the access of PLD. Immobilization of phospholipids could be applied for improving the yield of reactions catalysed by PLD as well as for performing a targeted production of short-chain length phosphatidic acid analogs.