[15] Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from porcine pancreas

Nieuwenhuizen, W.; Kunze, H.

doi:10.1016/0076-6879(74)32018-6

Cited by 226 publications

(120 citation statements)

References 14 publications

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“…The peak fractions (I, II, III, IV) were each pooled, dialyzed, lyophilized and finally redissolved in a small volume of 50 mM Tris-HCl (pH 7.5)/lmM CaC12/50% of glycerol. Each of those fractions was assayed for phospholipase A2 activity as described by Nieuwenhuizen et al [ 13 ]. Activity was recovered in the void volume peak (V0) --presumably a basic compound --and in four additional fractions (I -IV, Fig.…”

Section: Discussionmentioning

confidence: 99%

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

Roelofsen

Trip

Verheij

et al. 1980

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Summary1. Cobra venom phospholipase A2 from three different sources has been fractionated into different isoenzymes by DEAE ion-exchange chromatography. 2. Treatment of intact human erythrocytes with the various isoenzymes revealed significant differences in the degree of phosphatidylcholine hydrolysis in those cells. 3. It is argued that the plateaus observed in dose-response curves for such treatments may be caused by an increase in lateral surface pressure within the outer half of the membrane due to the production of free fatty acids and lyso-compounds.Phospholipases A2 (EC 3.1.1.4) purified from snake venoms (in particular from Naja naja venom) have been widely used as tools to study the disposition of phospholipids in biological membranes (for a recent review see Ref. 1). These enzymes have been particularly useful in elucidating the localization of phospholipids in the erythrocyte membrane [2], and the results thus obtained for the localization of phosphatidylcholine in that mem. brane could be quantitatively confirmed by studies using phosphatidylcholine exchange protein [3,4]. The reason why phospholipases could successfully be applied became apparent only recently, when analyses involving 3'P-NMR showed that even after extensive phospholipase treatments of the membrane, the residual phospholipids and products of hydrolysis remain in a bilayer configuration [5]. Indeed, fatty acids and lysophosphatidylcholine associate to form bilayers [6]. While the reliability of the results and the conclusions drawn from the above studies have been questioned by Martin et

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Section: Discussionmentioning

confidence: 99%

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

Roelofsen

Trip

Verheij

et al. 1980

Biochimica et Biophysica Acta (BBA) - Biomembranes

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“…phospholipase obtained showed no activity in an egg yolk assay [7]. Protein concentrations were determined spectrophotometrically using the absorption coefficient Ei" = 12.3 at 280 mn [7].…”

Section: Elsevierlnorth-holland Biomedical Pressmentioning

confidence: 99%

“…Protein concentrations were determined spectrophotometrically using the absorption coefficient Ei" = 12.3 at 280 mn [7]. A 0.086 M CaCl, solution was prepared by dissolving CaC03 (60% isotropically enriched in 43Ca, Oak Ridge Nat.…”

Section: Elsevierlnorth-holland Biomedical Pressmentioning

confidence: 99%

Calcium binding to porcine pancreatic prophospholipase A₂ studied by ⁴³Ca NMR

et al. 1981

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“…Routine assays of phospholipase A 2 activities were carried out as described by Nieuwenhuizen et al [23 ]. Phospholipase C activities were measured as described by Zwaal and Roelofsen [17].…”

Section: Phospholipase Assaysmentioning

confidence: 99%

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

Zwaal

Roelofsen

Comfurius

et al. 1975

Biochimica et Biophysica Acta (BBA) - Biomembranes

351

119

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SUMMARY1. The action of eight purified phospholipases on intact human erythrocytes has been investigated. Four enzymes, e.g. phospholipases A2 from pancreas and Crotalus adamanteus, phospholipase C from Bacillus cereus, and phospholipase D from cabbage produce neither haemolysis nor hydrolysis of phospholipids in intact cells. On the other hand, both phospholipases A2 from bee venom and Naja naja cause a non-haemolytic breakdown of more than 50 ~ of the lecithin, while sphingomyelinase C from Staphylococcus aureus is able to produce a non-lytic degradation of more than 80 ~o of the sphingomyelin.2. Phospholipase C from Clostridium welchii appeared to be the only lipolytic enzyme tested, which produces haemolysis of human erythrocytes. Evidence is presented that the unique properties of the enzyme itself, rather than possible contaminations in the purified preparation, are responsible for the observed haemolytic effect.3. With non-sealed ghosts, all phospholipases produce essentially complete breakdown of those phospholipids which can be considered as proper substrates for the enzymes involved.4. Due to its absolute requirement for Ca 2 ÷, pancreatic phospholipase A2 can be trapped inside resealed ghosts in the presence of EDTA, without producing phospholipid breakdown during the resealing procedure. Subsequent addition of Ca 2 ÷ stimulates phospholipase A z activity at the inside of the resealed cell, eventually leading to lysis. Before lysis occurs, however, 25 ~o of the lecithin, half of the phosphatidylethanolamine and some 65 ~ of the phosphatidylserine can be hydrolysed. This observation is explained in relation to an asymmetric phospholipid distribution in red cell membranes.Phospholipases used: phospholipase A2 (phosphatide acyl-hydrolyse, EC 3.1.1.4) from pig pancreas, bee venom, Naja naja, and Crotalus adamanteus, respectively. Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus and Clostridium welchii. Sphingomyelinase C (sphingomyelin cholinephosphohydrolase) from Staphylococcus aureus. Phospholipase D (phosphatidylcholine phosphatidohydrolase, EC 3.1.4.4) from Savoy cabbage. 84 5. It is concluded that the ability of the various phospholipases to attack the red cell membrane is dependent upon: (i) substrate specificity of the phospholipases, (ii) sidedness of the phospholipids when only one side of the membrane is exposed to phospholipase action, and (iii) compression state of the membranous lipid layer. The latter point is dealt with in more detail in the accompanying paper (Demel, R.

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[15] Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from porcine pancreas

Cited by 226 publications

References 14 publications

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

Calcium binding to porcine pancreatic prophospholipase A₂ studied by ⁴³Ca NMR

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

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[15] Phospholipase A2 (phosphatide acylhydrolase, EC 3.1.1.4) from porcine pancreas

Cited by 226 publications

References 14 publications

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

The action of cobra venom phospholipase A2 isoenzymes towards intact human erythrocytes

Calcium binding to porcine pancreatic prophospholipase A2 studied by 43Ca NMR

Organization of phospholipids in human red cell membranes as detected by the action of various purified phospholipases

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Calcium binding to porcine pancreatic prophospholipase A₂ studied by ⁴³Ca NMR