Enzymatic properties of rat group IIA and V phospholipases A(2) compared.
Group IIA and V phospholipases A(2) (PLA(2)s) are known to play a role in inflammatory responses. We have constructed a bacterial expression vector for rat group IIA and V PLA(2)s, over-expressed, folded and purified the proteins with the aim to study and compare the properties of the enzymes in detail. For zwitterionic phospholipid micelles, both enzymes display optimum activity at pH 8. 0 and absolutely require Ca(2+) for enzymatic activity. In the presence of substrate, group V PLA(2) has a high affinity for Ca(2+) (K(Ca2+)=90 microM) while K(Ca2+) of group IIA PLA(2) was found to be 1.6 mM. The absence of substrate only marginally influences the Ca(2+) affinities. In contrast to group IIA PLA(2), group V PLA(2) does not show a jump in the activity profile at substrate concentrations around the critical micelle concentration. Direct binding studies using n-alkylphosphocholines indicate that group V PLA(2) forms protein-lipid aggregates at pre-micellar lipid concentrations in a cooperative and Ca(2+)-dependent manner. This behavior, which is comparable to that observed for the PLA(2) from Naja melanoleuca snake venom, reflects the high affinity of this enzyme for zwitterionic phospholipids. Competitive inhibition by the substrate analogues (R)-2-dodecanoylaminohexanol-1-phosphocholine and its phosphoglycol derivative was tested on zwitterionic micelles as substrate. Group IIA PLA(2) shows a preference for the phosphoglycol inhibitor whereas the phosphocholine inhibitor binds stronger to the active site of group V PLA(2). The enzymatic activity was also measured on zwitterionic liposomes which appear to be much better substrates for group V PLA(2) than for group IIA PLA(2). The overall results suggest that group V PLA(2) is better suited for action on biological membranes than group IIA PLA(2).
Janssen
M J
MJ
Department of Enzymology and Protein Engineering, Centre for Biomembranes and Lipid Enzymology (Institute of Biomembranes), Faculty of Chemistry, Utrecht University, P.O. Box 80.054, 3508 TB, Utrecht, The Netherlands.
Vermeulen
L
L
Van der Helm
H A
HA
Aarsman
A J
AJ
Slotboom
A J
AJ
Egmond
M R
MR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D008081:Liposomes; D008823:Micelles; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D007527:Isoenzymes; D008081:Liposomes; D008823:Micelles; D010743:Phospholipids; D011994:Recombinant Proteins; D010741:Phospholipases A; D002118:Calcium