The Interaction of Pancreatic Phospholipase A2 with Negatively Charged Substrates — Application: The Transformation of Soluble Phospholipase A2 into a Highly Penetrating “Membrane-Bound” Form

Slotboom, Arend J.; Oort, M.G. van; Wiele, F. van der; Atsma, W.; Linde, Mona; Roelofsen, B.

doi:10.1007/978-1-4684-5212-9_16

Cited by 4 publications

(2 citation statements)

References 27 publications

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“…The p-bromophenacyl-bromide-inhibited enzyme, however, still binds micellar substrates with the same affinity as the native enzyme [23]. On the other hand de Haas et al have shown that specific palmitoylation of Lysl16 of pancreatic phospholipase A2 enables the modified enzyme to penetrate densely packed monolayers and to attack biological membranes, which is not possible with the native enzyme [24]. Similarly, we have recently shown that porcine pancreatic lipase and Rhizopus delemav lipase, when sonicated with PtdCho, became able to hydrolyze a lipoprotein substrate [25].…”

Section: Discussionmentioning

confidence: 99%

Role of a sulfhydryl group in gastric lipases

Gargouri

Moreau

Piéroni

et al. 1989

European Journal of Biochemistry

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Native human and rabbit gastric lipases (HGL and RGL, respectively) were inactivated after modification of one sulfhydryl group/enzyme molecule. HGL and RGL were covalently labeled using 5,5'-dithiobis(2-nitro-['4C]benzoic acid) and the interaction of 2-nitro-5-thio-[ ''C]benzoic-acid-labeled lipases ([14C]Nbs-lipases) with monomolecular lipid films was investigated. Our results show that [14C]Nbs-lipases bind to lipid films as efficiently as native HGL or RGL. The critical surface pressure z, and the maximal surface pressure (Azmax) of [I4C]Nbslipases were enhanced in comparison with those of native RGL and HGL. These changes in behavior were probably due to an increase in hydrophobicity brought about, directly or indirectly, by the binding of the Nbs radical. This chemical modification thus blocks the hydrolysis site and reinforces the hydrophobic character of the gastric lipases.In order to describe the kinetics of a lipolytic enzyme acting at an interface, a simple model has been proposed by Verger et al. [l]. This model consists basically of two successive equilibria. The first describes the reversible penetration of a water-soluble enzyme into an interface (E =$ E*). This is followed by a second equilibrium in which one molecule of penetrated enzyme binds a single substrate molecule, forming the enzyme-substrate complex (E*S). This is the twodimensional equivalent of the classical Michaelis-Menten equilibrium. Once the complex E*S has been formed, the catalytic steps take place, regenerating the enzyme in the form E* and liberating the lipolysis products.The interfacial binding of lipolytic enzymes is a critical step in the overall catalytic process, i. e. without interfacial adsorption no lipolysis could occur. Several examples have been reported in the literature where a lack of enzyme activity was specifically attributable to a lack of adsorption and not to any modification of the catalytic site sensu stricto. For instance, pancreatic lipase inactivation by diethyl-pnitrophenyl phosphate prevents the enzyme adsorption onto silicon-coated glass beads without affecting its ability to hydrolyze soluble esters such as p-nitrophenyl acetate [23. Chemical modification of the N-terminal alanine residue of the pancreatic phospholipase A2 does not affect the hydrolysis of monomeric synthetic short-chain phospholipids, whereas the modified enzyme becomes unable to bind and to act upon organized phospholipid micellar substrates [3]. Clostridium perfringens phospholipase C specifically requires Ca2 + for interfacial binding to occur, whereas the catalytic site is fully effective only in the presence of Zn2+ and Ca2+ ions [4]. These examples illustrate the functional differences existing between a lipid-binding site and a catalytic site in several lipolytic enzymes.In recent studies on pure HGL and RGL, we reported that the catalytic and structural characteristics of these two enzymes are very similar [5, 61. We have further shown that one sulfhydryl group was modified/molecule native HGL or RGL after incubation wit...

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Section: Discussionmentioning

confidence: 99%

Role of a sulfhydryl group in gastric lipases

Gargouri

Moreau

Piéroni

et al. 1989

European Journal of Biochemistry

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“…This modified porcine pancreatic phospholipase A 2 is able to penetrate into the outer monolayer by means of its palrnitic acid residue coupled to Lys-ll6. Recently, it was shown that this enzyme can degrade all PC present in the outer monolayer of the intact human erythrocyte at a much higher rate and with even greater efficiency than by the best penetrating Naja naja phospholipase A 2 isoenzyme [39].…”

Section: Discussionmentioning

confidence: 99%

Phospholipid asymmetry during erythropoiesis. A study on Friend erythroleukemic cells and mouse reticulocytes

Schaft

Roelofsen

Kamp

et al. 1987

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The distribution of phospholipids over the outer and inner layers of the plasma membranes of differentiated Friend erythroleukemic cells (Friend cells) and mouse reticulocytes has been determined. Phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol were found to be distributed symmetrically over both layers, sphingomyelin was found to be enriched in the outer layer (80-85%) and phosphatidylserine appeared to be present mainly in the inner layer (80-90%) of the plasma membranes of differentiated Friend cells. The outer layer of reticulocyte membranes contains 50-60% of the phosphatidylcholine, 20% of the phosphatidylethanolamine, 82-85% of the sphingomyelin and 40-42% of the phosphatidylinositol. All of the phosphatidylserine is present in the inner layer. The results show, that the asymmetric distribution of phospholipids, typical for erythrocyte membranes, is partially apparent already at an early stage of erythropoiesis, the proerythroblast, while the final organization of phospholipid distribution takes place at some stage during enucleation of the enormoblast and release of the reticulocyte into the blood stream.

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Enzymes of Lipid Metabolism II

Freysz¹,

Dreyfus²,

Massarelli³

et al. 1986

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The Interaction of Pancreatic Phospholipase A2 with Negatively Charged Substrates — Application: The Transformation of Soluble Phospholipase A2 into a Highly Penetrating “Membrane-Bound” Form

Cited by 4 publications

References 27 publications

Role of a sulfhydryl group in gastric lipases

Role of a sulfhydryl group in gastric lipases

Phospholipid asymmetry during erythropoiesis. A study on Friend erythroleukemic cells and mouse reticulocytes

Enzymes of Lipid Metabolism II

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