Release of Calcium from Suspension-Cultured Glycine max Cells by Chitosan, Other Polycations, and Polyamines in Relation to Effects on Membrane Permeability

Abstract

Treatment with chitosan of suspension-cultured Glycine max cells labeled with 45Ca2" caused a rapid release of calcium, which was complete much earlier than the chitosan-induced leakage of intracellular electrolytes and probably reflects calcium loss primarily from the cell wall and/ or plasma membrane. A linear correlation was found between calcium release from chitosan-treated whole cells or isolated cell walls and the amount of chitosan bound. Other polycations (poly-L-lysine, histone, DEAE-dextran, and protamine sulfate), low molecular weight polyamines (spermine, spermidine, and putrescine) and polyanions (polygalacturonate and poly-L-aspartate, which act as chelating agents) also released calcium from whole cells and isolated cell walls; however, only the polycations increased membrane permeability. Poly-L-lysines of differing molecular weight showed a similar ability to release calcium, but their effect on membrane permeability increased with increasing molecular weight. The results suggest that the effect of polycations on permeability is not the direct result of calcium displacement from the cell surface but is probably due to cross-linking of surface components. The order of effectiveness of inorganic cations in displacing calcium from whole cells and isolated cell walls was Ca2l, Ba2 , Sr2+ > Mg2'> K+, Na+.Chitosan (8-1,4-linked glucosamine) has been implicated as an important regulatory component in host-fungal interactions (10) based on histochemical and immunochemical evidence of its production at the host-pathogen interface during infection (7-9), and its many physiological effects on plant and fungal cells (1,7,12,14,19,20 branes (5).In this report, we compare the abilities ofdifferent polycations, polyamines, and other molecules to release Ca2" from Glycine max cells with their effects on membrane permeability, and discuss the results in relation to the mechanism by which basic polymers increase permeability. In addition, the possible significance of Ca2" displacement by chitosan during fungal infection is considered. MATERIALS AND METHODS Cell Culture. Cell suspension cultures of Glycine max cvHarosoy 63 were grown as described previously (20) for 6 d in B5 medium (6), modified by using 50 ,M FeSO4-EDTA as the source of iron (3). 45Ca Labeling of Cells. Cells were washed three times in an incubation medium consisting of 1 mm NaH2PO4 and 2% sucrose (w/v), adjusted to pH 5.5 with 0.1 N NaOH, by centrifugation at 1 5OOg for 5 min. After settling in a measuring cylinder for 20 min, the cells were resuspended in incubation medium to give a 4:1 ratio of total volume to settled volume. [45Ca]C12 (100 mM) in incubation medium was added to give a final concentration of 2 mm containing 0.4 gCi/ml, and the suspension was shaken in an Erlenmeyer flask on a 1.5-cm rotary shaker at 120 rpm for 30 min at 26°C. The cells were then washed and resuspended in incubation medium as above.Uniform labeling of cells was achieved by growing cultures for 6 d in culture medium containing 0.2 ,Ci 45Ca/ml (the medi...

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