Intrinsic differences in the perturbing ability of alkanols in bilayer: Action of phospholipase A2 on the alkanol-modified phospholipid bilayer

The effects of tumour promoters, namely phorbol esters and teleocidin, on the activity of porcine pancreatic phospholipase A2 (PLA2) was investigated by using a system of small unilamellar vesicles composed of dipalmitoyl-phosphatidylcholine (DPPC). DPPC vesicles encapsulating Quin 2 (Quin 2/DPPC vesicles) were suspended in a medium containing Ca2+. The addition of PLA2 to Quin 2/DPPC vesicles increased the fluorescence intensity of Quin 2. This increase was due to chelation of Quin 2 with Ca2+, which resulted from an increase in the permeability of the phospholipid bilayer caused by the hydrolytic activity of PLA2. The tumour promoters phorbol 12-myristate 13-acetate (PMA) and teleocidin, at low concentrations, enhanced PLA2 activity at temperatures below the phase-transition temperature of the membrane, but, in contrast, high concentrations of the tumour promoters suppressed PLA2 activity. Phorbol 12-myristate (PM) also had a similar effect on PLA2 activity. PMA and PM disturbed the membrane structure markedly, which was indicated by the enhanced leakage of carboxyfluorescein (CF) from DPPC vesicles encapsulating CF. On the other hand, phorbol 12,13-didecanoate and 4 alpha-phorbol 12,13-didecanoate, which did not disturb the membrane structure to the same extent, had an insignificant effect on PLA2 activity. It is therefore concluded that PLA2 catalyses the hydrolysis of phospholipids in bilayer vesicles which contain a moderate degree of structural defects. However, the effects of tumour promoters on PLA2 activity was not related to their potencies as inflammatory and tumour-promoting agents.

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ISOLATION OF A SOLUBLE PHOSPHOLIPASE A2 FROM HUMAN PLATELETS ACTIVE AGAINST 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE

10.1055/s-0038-1644628 ◽

1987 ◽

Author(s):

A D Purdon ◽

J B Smith

Keyword(s):

Phospholipase A2 ◽

Specific Activity ◽

Deae Cellulose ◽

Human Platelets ◽

Phospholipid Bilayer ◽

Ionophore A23187 ◽

Pla2 Activity ◽

Enzyme Substrate ◽

Membrane Bound ◽

Layer Chromatography

Previously, we have shown that 1-acyl-2-arachidonoyl glycero-phosphocholine (GPC) is the main source of arachidonic acid in thrombin-stimulated (5 U/ml) human platelets. Thus 1-acyl-2-3H-arachidonoyl GPC was dispersed in Tris buffer, 0.01 M, pH 7.5, 0.01 M CaCl2 for use a substrate for the assay of phospholipase A2 activity in human platelets. The released 3H-arachidonate(AA) was isolated by thin layer chromatography following Bligh and Dyer extraction of the enzyme-substrate incubate. Phospholipase A2 (PLA2) specific for this phospholipid was thought to be membrane bound and of low activity when solubilized, however, we have found, that provided resting platelets are gently sonicated while suspended in tyrode's buffer in the presence of suitable concentrations of protease inhibitors and metal chelators (EGTA, EDTA), a large amount of soluble PLA2 activity can be isolated following centrifugation to remove membranes. The enzyme required calcium for activity and was inactive in the presence of EGTA. No activity was found in the secretate from thrombin-stimulated cells, indicating that the PLA2 assayed at pH 7.5 was not lysosomal. PLA2 was further purified by DEAE cellulose chromatography where a 5 times increase in specific activity was achieved. It is known that OAG (1-oleoyl-2-acetyle-glycerol) augments deacylation of 1,2 diradyl GPC in platelets stimulated with suboptimal levels of ionophore A23187. Thus the effect of OAG stimulation of platelets on the distribution of soluble PLA2 was studied. Platelets (109 cells/ml) suspended in tyrode's buffer and stimulated with 100 ug/ml OAG or 5 U/ml thrombin (10 min, 37°C., 10 min, without stirring), showed a considerable decrease in soluble PLA2 activity suggesting a partitioning of soluble PLA2 into the membrane bilayer. Thus a model for PLA2 action is suggested in which binding of the cytosolic enzyme to its site of hydrolysis is induced by diglyceride-perturbation of the membrane, phospholipid, bilayer phase.

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Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A2

Biochemical Journal ◽

10.1042/bj20020092 ◽

2002 ◽

Vol 366 (3) ◽

pp. 971-976 ◽

Cited By ~ 53

Author(s):

Lucimara CHIOATO ◽

Arthur H.C. de OLIVEIRA ◽

Roberto RULLER ◽

Juliana M. SÁ ◽

Richard J. WARD

Keyword(s):

Phospholipase A2 ◽

Lipid Membrane ◽

Terminal Region ◽

Site Directed Mutagenesis ◽

Phospholipid Bilayer ◽

Directed Mutagenesis ◽

Aromatic Residues ◽

Loop Region ◽

Terminal Loop ◽

Arginine Residues

Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 from the venom of Bothrops jararacussu which demonstrates both myotoxic and Ca2+-independent membrane-damaging activities. The structural determinants of these activities are poorly defined, therefore site-directed mutagenesis has been used to substitute all cationic and aromatic residues between positions 115 and 129 in the C-terminal loop region of the protein. Substitution of lysine and arginine residues with alanine in the region 117—122 resulted in a significant reduction of myotoxic activity of the recombinant BthTx-I. With the exception of Lys122, these same substitutions did not significantly alter the Ca2+-independent membrane-damaging activity. In contrast, substitution of the positively-charged residues at positions 115, 116 and 122 resulted in reduced Ca2+-independent membrane-damaging activity but, with the exception of Lys122, had no effect on myotoxicity. These results indicate that the two activities are independent and are determined by discrete yet partially overlapping motifs in the C-terminal loop. Results from site-directed mutagenesis of the aromatic residues in the same part of the protein suggest that a region including residues 115—119 interacts superficially with the membrane interface and that the residues around position 125 partially insert into the lipid membrane. These results represent the first detailed mapping of a myotoxic site in a phospholipase A2, and support a model of a Ca2+-independent membrane-damaging mechanism in which the C-terminal region of BthTx-I interacts with and contributes to the perturbation of the phospholipid bilayer.

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