Self-association and active enzyme forms of Naja naja naja and Crotalus atrox phospholipase A2 studied by analytical ultracentrifugation

Abstract

The dimerization of phospholipase A2 (PLPA2) from Naja naja naja (Pakistani cobra) and Crotalus atrox (Western Diamondback rattlesnake) has been studied from pH 2.5 to 11 at 20 degrees C in 1 mM CaCl2, 0.21 M ionic strength. For the C. atrox enzyme, it was found necessary to use a combination of sedimentation equilibrium and fluorescence yield data to analyze the association. Sedimentation equilibrium in the analytical ultracentrifuge sufficed for the study of the N. naja PLPA2. In the region of enzymatic activity at pH 8, the dimerization association constants found were k2 = 2.8 X 10(6) L/mol and k2 = 6.9 X 10(4) L/mol for the C. atrox and N. naja enzymes, respectively. Analytical linked functions are presented which describe the data. Because the associations are linked to Ca2+ as well as the hydrogen ion, no attempt was made to interpret the ionization of residues in terms of the molecular structure. Active-enzyme sedimentation velocity experiments have been used to study the relation between enzymatic activity and association for both the C. atrox and N. naja enzymes. The substrate 1,2-dibutyryl-sn-glycero-3-phosphocholine (diC4PC) did not dissociate the C. atrox PLPA2. The substrate 1,2-dihexanoyl-sn-glycero-3-phosphocholine (diC6PC) at 7.5 mM dissociated the C. atrox PLPA2 when monitored either as the active enzyme or as the Sr2+-inhibited enzyme. At low enzyme concentrations, 40 mM diC4PC had no effect on N. naja PLPA2 dimerization. However, the sedimentation coefficients observed at enzyme concentrations above 0.2 mg/mL in active-enzyme sedimentation velocity experiments were larger than the values predicted from the thermodynamic studies. Sedimentation coefficients observed for the N. naja PLPA2 acting on diC6PC were larger than those of the monomeric protein, which was the form layered on this substrate. The dissociation of the C. atrox PLPA2 effected by diC6PC was analyzed by the thermodynamics of association and the kinetic Michaelis constant. The analysis suffices to account for the observed sedimentation coefficient. The sedimentation behavior of the N. naja PLPA2 acting on diC6PC substrate was analyzed in terms of a protein-lipid complex. With this model, 68 +/- 16 phospholipid molecules per protein monomer were determined. It is proposed that this enzyme has two micelle nucleation sites per monomer. These putative sites promote micelle formation of the substrate on the enzyme below the critical micelle concentration of the lipid alone.

A pH-Induced Dissociation of the Dimeric Form of a Lysine 49-Phospholipase A2Abolishes Ca2+-Independent Membrane Damaging Activity

“…Although these results were corroborated by chemical modification (49) and analytical centrifugation techniques (50), further investigation concluded that the active form might be either a monomer or a dimer (46). Nevertheless, the crystal structure of the PLA 2 from C. atrox presents a dimer conformation in which the substrate binding and catalytic sites are shielded by the dimer interface, and a quaternary structure transition of the membrane-bound enzyme has been proposed which would permit access of substrate and Ca 2+ ions to these regions (51).…”

“…Previous studies have indicated that dimerization may play a functional role in the catalytically active Asp49-PLA 2 s. The stability of the Crotalus atrox PLA 2 dimer is favored at pH 7.0 (45,46), and kinetic studies of substrate hydrolysis of both this protein (47) and the closely related PLA 2 from Crotalus adamanteus (48) have indicated that the membrane active form is dimeric. Although these results were corroborated by chemical modification (49) and analytical centrifugation techniques (50), further investigation concluded that the active form might be either a monomer or a dimer (46).…”

INTERFACIAL ENZYMOLOGY OF GLYCEROLIPID HYDROLASES: Lessons from Secreted Phospholipases A2

“…The analysis of sPLArcatalyzed hydrolysis of short-chain phospholipids dis persed as solitary monomers in aqueous solution has been clouded by obser vations that pre-micellar protein-lipid microaggregates form (see e.g. 127,157,169,[196][197][198]. Such aggregation may be due to the segregation of short-chain phospholipids in contact with the i-face.…”

Solution and Interface Aggregation States of Crotalus atrox Venom Phospholipase A2 by Two-Photon Excitation Fluorescence Correlation Spectroscopy

“…The secreted PLA 2 from C. atrox is a dimer when free in solution (20) with a reported K d in the nanomolar range (24). Using chromatographic profiles, Myatt et al also studied the effect of Ca 2+ on the K d , and they report a 8-fold decrease of the dissociation constant when the cofactor is present.…”