title: Purification and characterization of a mutant human platelet phospholipase A2 expressed in Escherichia coli. Cleavage of a fusion protein with cyanogen bromide.
abstract: Both methionine residues in phospholipase A2 (PLA2) from porcine pancreas have been replaced by leucines with retention of full enzymatic activity. The methionine-less mutant has been expressed as a Cro-LacZ fusion protein in Escherichia coli, from which a pro-PLA2 was liberated by chemical cleavage with CNBr. The general applicability of CNBr cleavage of proteins lacking methionine residue(s) was demonstrated by replacing the single Met8 in human platelet phospholipase A2 (HP-PLA2) by a leucine residue, and the introduction of a methionine at a position just preceding the HP-PLA2 sequence. This protein was expressed in E. coli as a 68-kDa Cro-LacZ fusion protein. CNBr cleavage liberated the HP-PLA2 fragment which was reoxidized in vitro. The [Met8----Leu]HP-PLA2 is monomeric in aqueous solutions, requires calcium ions in the millimolar range for enzymatic activity and has optimal activity around pH 8. p-Bromophenacyl bromide rapidly inactivates the enzyme with calcium ions having a protective effect. The highest specific activities, 2400 U/mg and 9300 U/mg, were found with pure micelles of 1,2-dioctanoyl-sn-glycero-3-phosphoglycol and with mixed micelles of taurodeoxycholate and 1,2-dioctanoyl-sn-glycero-3-phosphoglycol, respectively. In mixed micelles the activity on dioleoyl phospholipids decreases in the order phosphatidylglycerol greater than phosphatidylethanolamine much greater than phosphatidylcholine. The enzyme has low activity on monomeric 1,2-diheptanoyl-sn-glycero-3-phosphocholine as a substrate, but high activity on micelles with a distinct jump in activity at the critical micellar concentration. The binding of the HP-PLA2, porcine pancreatic PLA2 and PLA2 from Naja melanoleuca venom to lipid/water interfaces was determined with micellar solutions of the substrate analog n-hexadecylphosphocholine. The HP-PLA2 has a high apparent Kd (2 mM) compared to pancreatic (0.2 mM) and venom (0.03 mM) PLA2. In mixed micelles of taurodeoxycholate and 1,2-didodecanoyl-sn-glycero-3-phosphocholine, the competitive inhibition of HP-PLA2 by the R and S enantiomers of 2-tetradecanoylaminohexanol-1-phosphocholine, its phosphoglycol, and its phosphoethanolamine derivatives were tested. The S enantiomers are only weak inhibitors, whereas the R enantiomers are potent inhibitors. The inhibitory power depends on the nature of the polar head group and increases in the order phosphocholine much less than phosphoethanolamine less than phosphoglycol. The best inhibitor, (R)-2-tetradecanoylaminohexanol-1-phosphoglycol, binds 2200 times stronger than the substrate to the HP-PLA2 active site.
authors.affiliation: Department of Enzymology and Protein Engineering, University of Utrecht, The Netherlands.
authors.forename: P A
authors.initials: PA
authors.lastname: Franken
authors.forename: L
authors.initials: L
authors.lastname: Van den Berg
authors.forename: J
authors.initials: J
authors.lastname: Huang
authors.forename: P
authors.initials: P
authors.lastname: Gunyuzlu
authors.forename: R B
authors.initials: RB
authors.lastname: Lugtigheid
authors.forename: H M
authors.initials: HM
authors.lastname: Verheij
authors.forename: G H
authors.initials: GH
authors.lastname: De Haas
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D000818:Animals; D001792:Blood Platelets / Q000201:enzymology*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002850:Chromatography, Gel; D003435:Crotalid Venoms / Q000201:enzymology; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008823:Micelles; D009154:Mutation; D010179:Pancreas / Q000201:enzymology; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D054467:Phospholipases A2; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
chemical_list: D000098:Acetophenones; D003435:Crotalid Venoms; D008823:Micelles; D011993:Recombinant Fusion Proteins; D010741:Phospholipases A; D054467:Phospholipases A2; D003488:Cyanogen Bromide; C006821:4-bromophenacyl bromide; D002118:Calcium