Ophiobolin A. A natural product inhibitor of calmodulin

Ophiobolin A, a Fungal metabolite and a phytotoxin which can stimulate the net leakage of electrolytes and glucose from maize seedling roots (Tipton, C. L., Paulsen, P. V., and Betts, R. E. (1977) Plant Physiol. 59, 907-910) was found to be a potent inhibitor of calmodulin-activated cyclic nucleotide phosphodiesterase. The physiologically less active analogue, 3-anhydro-ophiobolin A, was found to be less inhibitory than ophiobolin A in the phosphodiesterase assay. The direct interaction between ophiobolin A and Calmodulin has been demonstrated by changes in fluorescence of the protein and by the effect of ophiobolin A on Calmodulin activity upon preincubation. Addition of ophiobolin A to Calmodulin solutions resulted in an instantaneous quenching of the intrinsic tyrosine fluorescence followed by a time-dependent quenching. The instantaneous quenching is probably due to the inner filtering effect of ophiobolin A. The time-dependent fluorescence quenching was correlated with a time-dependent inhibition of Calmodulin upon preincubation with ophiobolin A. The inhibition of Calmodulin by ophiobolin A could not be reversed by dialysis, dilution, nor denaturation by urea in the presence of methanol followed by renaturation, and was much more pronounced in solutions containing Ca2+ than in those containing EGTA. Ophiobolin A also was shown to inhibit spinach Calmodulin. The results of the present study suggest that Calmodulin may be one of the target proteins of the phytotoxic action of ophiobolin A and that the interaction of ophiobolin A with Calmodulin may involve a covalent modification of the protein by the Fungal metabolite.