Date of Award
Doctor of Philosophy
Biochemistry, Biophysics and Molecular Biology
Ophiobolin A, a fungal phytotoxin which can stimulate the net leakage of electrolytes and glucose from maize seedling roots, is a potent inhibitor of calmodulin. The inhibition at pH 7.0 is time-dependent and could not be reversed by dialysis, dilution, nor denaturation, and was more pronounced in the presence than in the absence of Ca('2+). The direct interaction between ophiobolin A and calmodulin is shown by the quenching of the intrinsic tyrosine fluorescence of bovine brain calmodulin upon the addition of ophiobolin A. Maize calmodulin, reacted with ophiobolin A, has a smaller electrophoretic mobility than untreated calmodulin. Ophiobolin A reacts with primary amino groups to give conjugated enamine products with (lamda)max from 270-285 nm. With the (epsilon)-amino group of N-(alpha)-acetyl-L-lysine, the (lamda)max is 272 nm. The same (lamda)max is also observed when bovine brain calmodulin reacts with ophiobolin A. The above suggests that ophiobolin A reacts with the (epsilon)-amino group of lysine residues in calmodulin to form a conjugated enamine product. By using UV measurements, one calmodulin molecule reacts with two molecules of ophiobolin A. The reaction with one molecule of ophiobolin A, however, completely inhibits the calmodulin. Calmodulin treated with ophiobolin A is resistant to tryptic cleavage at lysine 77. Dictyostelium discoideum calmodulin, which has glutamine instead of lysine at residue 77, is inhibited equally by ophiobolin A in the phosphodiesterase assay. Thus apparently, lysine 77 in the brain calmodulin is not essential for the interaction with ophiobolin A and that lysine 75 is the more probable site of reaction. Several similarities exist between the inhibition of maize calmodulin by ophiobolin A in vitro and the effects of ophiobolin A on excised roots. Both are irreversible and time-dependent. The concentration of ophiobolin A for half-maximal inhibition of calmodulin in the phosphodiesterase assay is similar to that for phytotoxicity. In both cases, ophiobolin A derivatives behave similarly, i.e., 18-bromo-19-methoxyophiobolin A is as potent as ophiobolin A, whereas 3-anhydro-ophiobolin A and 6-epi-ophiobolin A are less potent. A smaller amount of active calmodulin was measured in the extract from roots treated with ophiobolin A than in those from untreated roots. These similarities suggest that calmodulin is a target protein for the phytotoxicity of ophiobolin A.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Pak Chow Leung
Leung, Pak Chow, "Interaction of calmodulin and ophiobolin A, a fungal phytotoxin " (1986). Retrospective Theses and Dissertations. 8016.