A STUDY ON THE FUNCTIONAL SUBUNITS OF PHOSPHOLIPASES A(2) BY ENZYME IMMOBILIZATION

Pancreatic and venom phospholipases A(2) have complex and distinct oli gomerization behaviour. Pancreatic enzymes are monomeric in solution, but their quaternary structure at interfaces is unknown. On the other hand, certain crotalid venom phospholipases A(2) are dimeric in soluti on, and different reports have proposed either the monomer or the dime r as the catalytically functional subunit. In this study, enzyme immob ilization was used as a tool for determining the functional subunits o f these enzymes. The dimeric Crotalus atrox phospholipase A(2) was cov alently attached to agarose beads, via either the amine or the carboxy lic groups of the protein. In the first case immobilization led to an 80% loss of activity as compared with the soluble form, and measured b y using micellar diheptanoylphosphocholine. Inclusion of micellar prot ectants in the coupling media did not improve the activity. Enzyme imm obilized via carboxylic groups was 2-3-fold more active than the amine -coupled form. In a second approach, Crotalus atrox enzyme was immobil ized with single-subunit attachment. The removal, with denaturating wa shes, of the non-covalently bound units involved in monomer-monomer in teractions, caused a large decrease in specific activity of the suppor t-bound enzyme. This suggests the dimeric form as the fully active one . Similar procedures were also carried out with pig pancreatic and Naj a naja phospholipases A(2). The results indicated that these enzymes a re active as monomers.