The effect of a low molecular size (< 5 KDa) humic fraction, essentially fulvic acids, on microsomal and tonoplast ion-stimulated ATPase activity was studied. After 20 min of pre-incubation with microsomal vesicles from oat roots, humic substances at organic C concentration of up to 0.5-mu-g cm-3 increased KCl-stimulated ATPase activity, while they inhibited enzyme activity at higher concentrations. Cl--stimulated ATPase activity of tightly sealed tonoplast-enriched vesicles was similarly affected by < 5 KDa humic substances. This behaviour was not observed when gramicidin D was added to the assay medium. Proton transport by vesicles incubated up to 5 min with < 5 KDa humic molecules was affected in a concentration-dependent manner, strongly resembling that observed for ATP hydrolysis, whereas it was severely reduced when the assay conditions were close to those used for measuring ATP hydrolysis (20 min pre-incubation of vesicles with humic substances). The transmembrane electrical potential was negatively affected, irrespective of the concentration of humic molecules. Furthermore, a 15-min pre-incubation strongly reduced the formation of a potential gradient. The size and concentrations of humic substances employed make an interaction with the vacuolar membrane of root cells plausible. The results show that the main target of humic molecules is the electrical membrane potential and suggest a possible way of interference of these naturally occurring substances with the biochemical mechanisms involved in plant mineral nutrition.
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