Abstract
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.
Highlights
(componentA-component B complex)and of its isolated 5)
This suggests that the of component B by an active site-directed reagent, p-bromophospholipase component B possesses a hydrophobic phenacyl bromide (9, 10). This alkylating agent only cleft which may contain one or two fatty acids. This reacts with a dimeric form of component B which is in hydrophobic cleft is not accessible to spin-labeled fatty equilibrium with a nonreactive and predominant monomeric acids in the crotoxin complex
The hy- brings about the appearance of a hydrophobic area on the drodynamic analysis of the fatty acid-component B phospholipase subunit which may be responsible for its adcomplex led us toestimate an apparent molecular weight of 95,000 which reveals that fattyacids induce the formation of polymers of component B
Summary
(componentA-component B complex)and of its isolated 5). In thepresence of membranes, crotoxin splits into itstwo phospholipase subunit (component B) with hydropho- components, the noncatalytic subunit being released in solubic compounds by ESR, using spin-labeled fatty acids tion (6, 7). This reacts with a dimeric form of component B which is in hydrophobic cleft is not accessible to spin-labeled fatty equilibrium with a nonreactive and predominant monomeric acids in the crotoxin complex. Fig. 1shows that the spin-labeled fatty acid completely inhibits the phospholipase activity of component B with an apparent Ki of 15 pM.
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