title: The effect of phospholipase A(2) immobilization upon calcium interaction: a kinetic study.
abstract: In this work we studied the effect of Ca(2+) on the ability of immobilized PLA(2) to hydrolyze phospholipid substrates either in aggregate or monomeric forms. We use a kinetic methodology for the determination of dissociation constants of soluble and immobilized PLA(2)-Ca(2+) complexes. This approach allows us to obtain the values of the dissociation constants of enzyme-Ca(2+) (K(x)) and enzyme-Ca(2+)-substrate (K'(x)) complexes from the kinetic data obtained at different substrate and Ca(2+) concentrations. Results using mixed micelles of phospholipid-Triton X-100 showed that, in most cases, productive complexes were destabilized by immobilization of PLA(2). However, a correct analysis of the interaction must be independent of the classical modes of PLA(2) action toward lipid surfaces. Thus, a substrate in monomeric form was also employed to analyze the effect of immobilization on hydrolysis rate in the absence of interfacial activation. Kinetic data showed that the immobilization affected severely the mode of PLA(2) action. The kinetic data also suggested that immobilization promoted conformational alterations in the Ca(2+)-binding site, destabilizing the productive complex enzyme-Ca(2+)-phospholipid.
authors.affiliation: Cátedra de Química Orgánica, Facultad de Ciencias Agropecuarias, Facultad de Ciencias Químicas, Universidad Nacional de Córdoba, 5000 Córdoba, Argentina.
authors.forename: R R
authors.initials: RR
authors.lastname: Madoery
authors.forename: C G
authors.initials: CG
authors.lastname: Gattone
authors.forename: G D
authors.initials: GD
authors.lastname: Fidelio
mesh_terms: D000818:Animals; D001516:Bees; D002118:Calcium / Q000378:metabolism*; D004789:Enzyme Activation; D004800:Enzymes, Immobilized; D007700:Kinetics; D010741:Phospholipases A / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism; D013329:Structure-Activity Relationship; D013499:Surface Properties
chemical_list: D004800:Enzymes, Immobilized; D010743:Phospholipids; D010741:Phospholipases A; D002118:Calcium