Hydrolysis of monomeric substrates by porcine pancreatic (pro)phospholipase A₂. The use of a spectrophotometric assay

Abstract

Abstract. In the course of searching for specific chromogenic substrates which might be useful for the assay of phospholipase A, at monomeric substrate concentrations, we tested short-chain lecithin analogues in which the oxygen ester bonds were replaced by thio ester. Spectrophotometric measurement of the hydrolysis of the thio ester in the presence of 5,5'-dithiobis(2-nitrobenzoic acid) or di-4-pyridyl disulfide provides a continuous and sensitive assay. Using this assay it was found that the zymogen expressed considerably higher enzymatic activities than phospholipase A, which reinforces the conclusions of Piererson et al. [Biochemistry 13, 1455[Biochemistry 13, (1974l that the zymogen, like the phospholipase, has a fully functional active site and that the fundamental difference between phospholipase A, and the zymogen only becomes evident if the substrate is present in an organized lipid water interface. The use of the spectrophotometric assay allowed a determination of the pH-rate profile of both phospholipase A, and its precursor. Phospholipase A, shows an optimum at pH 8.5 whereas the maximal activity of the precursor remains constant between pH 6 and 9. Initial velocity patterns as a function of Ca2+ and substrate are consistent with a random addition of substrate and cofactor to the enzyme.

Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2

“…1,2-Dioctanoyl-sn-glycero-3-phosphocholine was prepared as described by Cubero Robles & van den Berg (1 969). 2,3-Bis(hexanoylthio)propylphosphocholine was synthesized as described by Volwerk et al (1979). All other chemicals were of the highest purity available.…”

The Lipase from Staphylococcus aureus

“…were synthesised as described before (Volwerk et al, 1979;Aarsman et al, 1976). Tripentanoylglycerol and trihexanoylglycerol were obtained by acylation of glycerol with pentanoyl and hexanoyl chloride, respectively, in dry methylene chloride and pyridine.…”

Modification of Arginine Residues in Porcine Pancreatic Phospholipase A2

“…anoyl-sn-glycero-3-phosphocholine and 2,3-bis(hexanoyl-14C radioactivity was determined in a Searle-Isocap 300 [6,21]. All other chemicals were of the highest quality available and used without further purification.…”

“…In kinetic analysis on micellar substrate 1,2-dioctanoyl-sn-glycero-3-phosphocholine was used, according to De Haas et al [4]. For kinetic studies on monomeric substrates 2.3-bis(hexanoylthio)propylphosphocholine was used as described by Volwerk et al [6].…”

“…The enzyme has an absolute requirement of Ca" and reaches its highest activity when bound to aggregated substrates [4]. It has been shown that the snake venom enzymes bind Caz+ and substrates via an ordered mechanism in which Ca2+ is bound first [ 5 ] , while for porcine pancreatic enzyme a random mechanism has been proposed [4,6]. Investigations of the binding of pancreatic phospholipase Az to aggregated substrate and substrate analogs, have led to the proposition of a micelle-binding site on the porcine enzyme, which is topologically distinct from the active centre [7,8].…”