To test whether we could detect a specific binding of T-toxin to mitochondria isolated from Texas material, we first prepared some 3H-labeled T-toxin. Cultures of Drechslera (=Helminthosporium) maydis race T were raised on solid medium containing 0.08 M sodium acetate (250 mci 3H-acetate/litre medium). T-toxin was extracted according to the method of Y. Kono and J. M. Daly (1979, Bioorganic Chemistry 8:391) and had an estimated specific radioactivity of 400 mCi/mole. Two separate experiments were then made with mitochondria from N and T cytoplasm material. In the first experiment mitochondria were isolated from five-day-old dark-grown shoots of inbred A188 (N and T). Using an assay of malate oxidation (DCPIP reduction at 600 nm, P. A. Peterson et al., 1974, Plant Disease Reporter 58:777) the concentration of cold or radioactive T-toxin necessary to cause total inhibition of mitochondrial activity was determined. With the Texas cytoplasm mitochondria a concentration of 90 ng/ml T-toxin was sufficient to cause complete inhibition within one minute. With the Normal cytoplasm mitochondria a hundred-fold increase in toxin concentration caused less than 10% inhibition over the same time period. For the binding studies, mitochondrial suspensions were prepared just as in the enzyme assay experiments and suspended in the same buffer solution (total volume 2.5 ml) at a concentration of 0.20 mitochondrial mg protein/ml. Samples were incubated with toxin for five minutes before they were diluted to 10 ml with ice-cold PBS and filtered through Whatman GF/C glass fibre filters. The filters were rinsed with an additional 2 x 8 ml PBS. Centrifugation of the filtrate indicated that 95% of the mitochondria remained on the filters, which were then put into scintillation vials with 10 ml of a colloidal scintillant (Instagel, Packard Inst. Co.) for determination of radioactivity.
The results showed a relatively high level of unspecific attachment of T-toxin to the mitochondrial preparations but no evidence for specific binding to the T cytoplasm mitochondria. This was confirmed in a second experiment using mitochondria from WF9 (N and T cytoplasm). The results are open to different interpretations. We favor the explanation that T-toxin does not cause the observed inhibition of mitochondrial processes through a direct association with a specific protein species, but rather acts by, for example, destabilizing an already weakened membrane (cf., B. G. Forde and C. J. Leaver, 1980, in The Plant Genome, eds. D. R. Davies and D. A. Hopwood). The presence of receptor sites cannot be ruled out, however, since some specific binding of T-toxin to Texas cytoplasm mitochondria might have been masked by the high unspecific attachment of the lipophilic T-toxin molecule to the mitochondrial membrane.
Peter Simons and Richard Brettell
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