Carbodiimide modification enhances activity of pig pancreatic phospholipase A2.
Pig phospholipase A2, pig iso-phospholipase A2 and bovine pancreatic phospholipase A2 were reacted in solution with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, in the presence of N-hydroxysulfosuccinimide, at pH 7. The influence of micellar protectants was analyzed. In the presence of n-hexadecylphosphocholine, the losses of activity in micellar diheptanoyl-lecithin were 80, 35, and 10% in bovine phospholipase A2, pig iso-phospholipase A2, and pig phospholipase A2, respectively. With 1-oleoylglycerophosphocholine, the bovine enzyme lost 40% activity, but the pig enzyme was activated sevenfold. The modified pig enzyme showed pre-micellar activation on monomeric diheptanoyl-lecithin, and either reduced or increased activities on mixed micelles of bile salt with egg phosphatidylcholine, depending on the composition of the micelles. This activation is consistent with previous protein-engineering studies of pig pancreatic phospholipase A2. In this study, we present new information concerning the specificity and interfacial recognition behaviour of this enzyme in relation to this activation.
Ferreira
J P
JP
Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge 02139.
Sasisekharan
R
R
Louie
O
O
Langer
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D005022:Ethyldimethylaminopropyl Carbodiimide / Q000737:chemistry / Q000494:pharmacology*; D005997:Glycerylphosphorylcholine / Q000031:analogs & derivatives / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008244:Lysophosphatidylcholines; D008823:Micelles; D010179:Pancreas / Q000201:enzymology*; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D054467:Phospholipases A2; D013379:Substrate Specificity; D013388:Succinimides / Q000494:pharmacology; D013552:Swine
D008244:Lysophosphatidylcholines; D008823:Micelles; D013388:Succinimides; C082823:1-oleoyl lysophosphatidylcholine; D005997:Glycerylphosphorylcholine; C035761:N-hydroxysulfosuccinimide; D010741:Phospholipases A; D054467:Phospholipases A2; D005022:Ethyldimethylaminopropyl Carbodiimide; D002118:Calcium