Aflatoxin-induced alterations in Glycine max,cv. ‘essex’ root cell membrane protein content

Aflatoxins (AFTs) are hepatocarcinogens, mutagens, teratogens and toxins. Isolates of AFTs-producing strains of Aspergillus flavus can grow upon both autoclaved soybeans and to a lesser extent field-grown beans. Besides inhibiting the elongation of excised, in vitro cultured soybean roots, the AFTs can impair the roots' ability to remove ¹⁴C]-leucine from a culture medium and to incorporate the amino acid into acidinsoluble, cytoplasmic protein. In this connection, exogenous AFTs once taken-up and compartmentalized by the excised roots can reduce the acid-insoluble protein content of what appears to be a non-enriched plasmalemma fraction. Here, we report a combined biochemical and microscopical attempt to determine whether the protein reduction occurs throughout the excised, in vitro-cultured root and whether the plasmalemma proteins which are affected by AFTs can be both solubilized and characterized. Histochemistry of Carnoy-fixed roots revealed a reduction in Ninhydrin-Schiff-stainability throughout the AFTs-treated root. Transmission electron microscopy of 80 000 × g pellets derived from homogenates of both non-treated (NT) and treated (T) roots provided further evidence to our previously reported marker enzyme analyses for the occurrence of the plasmalemma in the 80 000 × g pellet. Treatment of the latter with the Laemmli SDS procedure released >85% of the protein associated with the pellets obtained from homogenates of either NT or T roots. Gel permeation chromatography on Biogel-100 of released proteins from 80 000 × g pellets of both NT and T roots yielded both void volume and retarded peaks. Both the amplitude and the quantities of protein recovered within the void volume peak were less within the void volume of the T than the NT root. Polyacrylamide tube gel electrophoresis of G-100 void volume peaks of chromatographed, SDS-released proteins from the pellets revealed quantitative but not qualitative differences in Coomassie Blue-gel staining patterns between T and NT roots. These results suggest that the SDS methods which we employed could solubilize the 80 000 × g pellet proteins but that combined gel permeation chromatography and tube gel electrophoresis were not sensitive enough to reveal whether AFTs could alter the types of proteins associated with the 80 000 × g pellet (purported plasmalemma).