Interaction of crotoxin and its isolated subunits with spin-labeled fatty acids.
We have investigated the interaction of crotoxin (component A-component B complex) and of its isolated phospholipase subunit (component B) with hydrophobic compounds by ESR, using spin-labeled fatty acids as probes. The phospholipase subunit alone (component B) binds more than three labeled fatty acid molecules/molecule with different affinities, the highest corresponding to a Kd of 10 microM in the case of 5-doxyl palmitic acid. In contrast, the noncatalytic subunit (component A) and the crotoxin complex do not bind fatty acids. ESR studies of the component B-fatty acid complex reveal a strong immobilization of the whole length of the fatty acid chain, strong spin-spin interactions between bound fatty acids, and nonaccessibility of the bound paramagnetic probe to Ni2+ ions. This suggests that the phospholipase component B possesses a hydrophobic cleft which may contain one or two fatty acids. This hydrophobic cleft is not accessible to spin-labeled fatty acids in the crotoxin complex. An overall rotational correlation time of about 200 ns of the phospholipase component B was determined by saturation transfer ESR. This high value is incompatible with the diffusion of a polypeptide of 14,500 molecular weight. The hydrodynamic analysis of the fatty acid-component B complex led us to estimate an apparent molecular weight of 95,000 which reveals that fatty acids induce the formation of polymers (most probably octamers) of component B. We propose a model in which the phospholipase component B exists in two conformational states which differ by their hydrophobicity.
Radvanyi
F
F
Rousselet
A
A
Devaux
P
P
Bon
C
C
D003435:Crotalid Venoms / Q000032:analysis / Q000494:pharmacology; D003439:Crotoxin / Q000032:analysis / Q000494:pharmacology; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D005227:Fatty Acids; D009589:Nitrogen Oxides; D010740:Phospholipases / Q000494:pharmacology; D011487:Protein Conformation; D012399:Rotation
D003435:Crotalid Venoms; D005227:Fatty Acids; D009589:Nitrogen Oxides; D003439:Crotoxin; D010740:Phospholipases