Tritium-labeled toxin analogs were prepared by reduction with NaB3H4 of either the toxin from Helminthosporium maydis race T or a toxin component from Phyllosticta maydis. These reduced analogs had high radiochemical specific activities, high biological activities, and plant specificities identical to the native toxins. A filtration assay was developed to test the binding of these labeled analogs to isolated-mitochondria. Binding was not energy dependent nor (3,4,10,24). These toxins have been characterized and each consists ofa family ofrelated compounds, shown in Figure 1 (6,13,14). HmT-toxin consists ofcompounds with a linear (3-oxydioxo polyketol structure and chain lengths which vary from C35 to C45. The PM-toxin family of compounds have shorter chain lengths, C33 to C35, and a linear oxy-oxo polyketol structure.The toxins appear to affect various physiological processes, but their effects on mitochondrial function have been studied extensively and shown to be specific for cms-T-maize mitochondria (cms-T-mitochondria) (6,18 cide, bears little structural resemblance to these toxins but demonstrates selective toxicity with a much lower potency (1,12).These toxins, their analogs and methomyl stimulate NADH oxidation but inhibit malate oxidation in isolated cms-T-mitochondria (1, 6, 12, 18). HmT-toxin and methomyl have been shown to cause leakage of NAD+ and coenzyme A from mitochondria (1, 9, 17). HmT-toxin also behaves as a Ca2" ionophore in cms-T-mitochondria but not in N-maize-mitochondria (Nmitochondria) or in cms-T-maize derived microsomes (9) and can form cation-selective channels in artificial phospholipid bilayers (8). Thus, cms-T-mitochondria are selectively affected; but the molecular site(s) of action of these toxins has not been established. Consequently, binding studies were undertaken to investigate possible mitochondrial receptors for these toxins.
MATERIALS AND METHODSPreparation of Reduced Toxin Analogs. Component C of PMtoxin (PM[C]-toxin) was prepared as previously described (6) and HmT-toxin was prepared by the methods of Kono and Daly (13). Either toxin (about 15 mg) was dissolved in a minimum of anhydrous, reagent grade methanol (5-10 ml) and then reduced with sodium borohydride (5-10 mg) at room temperature (25°C). The reaction was stopped by the addition of two to four drops of 1 N HCI. The resulting mixture was fractionated on a Sephadex LH-20 (Pharmacia) column (30 x 1 cm), which was eluted with methanol at a flow rate of 0.6 ml/min. Fractions (2 ml) containing reduced toxin (normally fractions 6-10) were combined, and the volume was reduced below 2 ml by gentle heating under a stream of N2. Acetone was added dropwise to the warm, concentrated solution until the solution became turbid. The solution was then stored at -20°C for 24 to 40 h to precipitate the reduced toxin analogs. The precipitated analogs were recovered by centrifugation and then dried under vacuum at room temperature. Stock solutions and dilutions were made in DMSO (Aldrich Chemical Co.) and stored at 4°C.
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