Site-specific .epsilon.-amino monoacylation of pancreatic phospholipase A2. 2. Transformation of soluble phospholipase A2 into a highly penetrating "membrane-bound" form

Abstract

Long-chain lecithins present in bilayer structures like vesicles or membranes are only very poor substrates for pancreatic phospholipases A2. This is probably due to the fact that pancreatic phospholipases A2 cannot penetrate into the densely packed bilayer structures. To improve the weak penetrating properties of pancreatic phospholipases A2, we prepared and characterized a number of pancreatic phospholipase A2 mutants that have various long acyl chains linked covalently to Lys116 in porcine and to Lys10 in bovine phospholipase A2 [Van der Wiele, F.C., Atsma, W., Dijkman, R., Schreurs, A.M.M., Slotboom, A.J., & De Haas, G.H. (1988) Biochemistry (preceding paper in this issue)]. When monomolecular surface layers of L- and D-didecanoyllecithin were used, it was found that the introduction of caprinic, lauric, palmitic, and oleic acid at Lys116 in the porcine enzyme increases its penetrating power from 13 to about 17, 20, 32, and 22 dyn/cm, respectively, before long lag periods were obtained. Incorporation of a palmitoyl moiety at Lys10 in the bovine enzyme shifted the penetrating power from 11 to about 25 dyn/cm. Only the best penetrating mutant, viz., porcine phospholipase A2 having a palmitoyl moiety at Lys116, was able to cause complete leakage of 6-carboxyfluorescein entrapped in small unilamellar vesicles of egg lecithin under nonhydrolytic conditions. Similarly, only this latter palmitoylphospholipase A2 completely hydrolyzed all lecithin in the outer monolayer of the human erythrocyte at a rate much faster than Naja naja phospholipase A2, the most powerful penetrating snake venom enzyme presently known.

Purification of a 110-kilodalton cytosolic phospholipase A2 from the human monocytic cell line U937.

“…Pancreatic PLA2 caused the release of radioactivity from the sn-2 position (Fig. 3B) with the characteristic lag phase (25,26), and the lipase from R. arrhizus released fatty acid from the sn-i position (Fig. 3C), thus validating our methodology.…”

INTERFACIAL ENZYMOLOGY OF GLYCEROLIPID HYDROLASES: Lessons from Secreted Phospholipases A2

“…Also, interfacial activation on PC vesicles by reaction products is reversed when vesicles without product are added (186). Chemical fatty acylation of pp-PLA2 and bee venom sPLA2 with reactive esters leads to enzymes that no longer display a lag in the hydrolysis of PC mono layers (190,(192)(193)(194)(195). Thus, there seems to be general consensus that acylation increases the affinity of enzyme for zwitterionic interfaces, but the proposal of substrate-based enzyme acylation as the basis for interfacial activation seems highly unlikely when all of the evidence is considered.…”

Enhanced hydrolysis of phosphatidylcholine by human group II non-pancreatic secreted phospholipase A2 as a result of interfacial activation by specific anions. Potential role of cholesterol sulphate

“…The rate of hydrolysis of DOPC by the snake venom enzyme from N. naja was already high, and was not significantly stimulated by the presence of interfacial anions. This 'highly penetrating enzyme' is known to be effective against PC and also cell membranes [21,14].…”