Activation Of Porcine Pancreatic Phospholipase A2 Research Articles

Effects of antiflammins on transglutaminase and phospholipase A2 activation by transglutaminase

Two anti-inflammatory peptides, named antiflammins (AFs), corresponding to a region with high amino acid similarity between lipocortin-1 and uteroglobin were tested for their ability to inhibit transglutaminase (TG) and low-molecular-mass phospholipase A2 (PLA2). Porcine pancreatic PLA2 activity and guinea pig hepatic TG activity were determined by arachidonyl release from arachidonyl-phosphatidylcholine and by the incorporation of putrescine into succinylated casein, respectively. AFs inhibited TG activity but did not affect PLA2 activity. Moreover, porcine pancreatic PLA2 was activated by TG and AFs decreased porcine pancreatic PLA2 activation induced by TG. Taken together, our results support the hypothesis that the anti-inflammatory effects of AFs are, at least in part, due to the action of AFs on TG activity.

Structural characterization of a biologically active human lipocortin 1 expressed in Escherichia coli.

Lipocortin or annexin 1 is a calcium-dependent phospholipid-binding protein which probably acts as a glucocorticoid- regulated anti-inflammatory factor. cDNA for human lipocortin 1 was cloned in the pT7.7 expression plasmid under the control of the inducible bacteriophage T7 RNA polymerase promoter. Upon induction with isopropyl thio-beta-D-galactoside, large amounts of the protein were produced and accumulated in Escherichia coli in a soluble form. The recombinant protein was purified to homogeneity by means of two subsequent ion-exchange chromatographic steps. The final yield was about 30 mg/l bacterial culture. Electrospray mass spectrometric analysis of the purified protein demonstrated that the recombinant product corresponds to the native human lipocortin 1, without the initial methionine and with a free N-terminal alanine; tryptic peptide mapping by fast-atom-bombardment mass spectrometry showed that the recombinant protein contains cysteine residues at positions 263 and 324 with free thiol groups, whereas Cys270 and Cys343 are probably involved in an intrachain disulfide bridge. Recombinant human lipocortin 1 reduces the carrageenin-induced paw oedema in rat in vivo and inhibits porcine pancreatic phospholipase A2 activity in vitro; in both cases, a dose-related response is observed.

Bilayer packing stress and defects in mixed dilinoleoylphosphatidylethanolamine and palmitoyloleoylphosphatidylcholine and their susceptibility to phospholipase A2

The hydrolysis of mixed dilinoleoylphosphatidylethanolamine (DiLinPE) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) dispersions by porcine phospholipase A2, under conditions leading to the bilayer-to-nonbilayer phase transition, has been studied. Two structurally distinct forms of the dispersions were used, multilamellar vesicles (MLV) and supercritical large unilamellar vesicles (SCLUV). In MLV, maximum free fatty acid was produced in dispersions containing 85 mol % DiLinPE. The peak in the fatty acid release is found at the onset of appearance of the nonbilayer defects reported earlier. DiLinPE was found to be preferentially hydrolyzed as compared to POPC. When cholesterol was added to the mixed DiLinPE/POPC MLV, the onset of the observable appearance of nonbilayer defects, the positions of the peaks for total hydrolysis, and the preferential hydrolysis of DiLinPE were all shifted toward lower DiLinPE concentrations. In SCLUV, where the appearance of nonbilayer structures is prevented by constraining the lipids in bilayer configuration, the hydrolysis by PLA2 increases with increasing DiLinPE as predicted from the increase in the calculated monolayer bending energy. The results are interpreted to be related to the pretransition molecular-packing stress and defects at the onset of the bilayer-to-nonbilayer transition. Results indicate that the porcine pancreatic phospholipase A2 activity is controlled by bilayer-packing stress, which may cause structural defects of the substrate, among other factors. Results also indicate a preferential localization of PE at stress-related defect regions.