Presence of sterol-binding sites in the cytosol of French-bean (Phaseolus vulgaris) roots

Abstract

Vitamin D3 (cholecalciferol) and stigmasterol have been shown to stimulate Ca2+ uptake and to induce calmodulin synthesis in cultured French-bean (Phaseolus vulgaris) roots. In addition, the appearance of calmodulin in the cultures in response to vitamin D3 could be prevented by RNA-synthesis inhibitors. To investigate the possibility that the sterols affect root DNA transcription through a receptor-mediated mechanism, the existence of sterol-binding sites in P. vulgaris roots was investigated. Specific binding of [3H]vitamin D3 could be demonstrated with intact tissue and the cytosolic fraction obtained therefrom. Equilibrium in the binding reaction with cytosol was attained after 4 h of incubation at 0 degrees C. The [3H]vitamin D3 was reversibly bound, since it could be displaced by an excess of unlabelled sterol. An equilibrium binding constant (KD) of (3.48 +/- 0.09) x 10(-9) M and a maximum binding-site concentration (nmax) of 32 +/- 2.54 (3) pmol/mg of protein could be calculated by Scatchard [(1949) Ann. N.Y. Acad. Sci. 51, 660-672] analysis. In addition to vitamin D3, stigmasterol and sitosterol were effectively able to compete with [3H]vitamin D3 for binding to root cytosol. Cortisol, oestradiol and progesterone displaced bound labelled vitamin D3 to a lesser extent, whereas 5 beta-dihydrotestosterone, lanosterol and diosgenin were ineffective. The affinity and specificity of the root sterol-binding sites are in agreement with the characteristics of tissue responses to the sterols (Ca2+ uptake and calmodulin synthesis).

Perception of Fungal Sterols in Plants (Subnanomolar Concentrations of Ergosterol Elicit Extracellular Alkalinization in Tomato Cells)

“…Binding sites for vitamin D, and the endogenous plant sterols p-sitosterol and stigmasterol have been observed in French bean roots (Vega and Boland, 1988), but the biological role of these sterol-binding sites and their specificity with respect to "self" and "nonself" sterols remain to be examined. Sterol-or steroid-binding proteins are also present in different yeasts (Loose et al, 1981;Feldman et al, 1982) and in some water molds (Riehl and Toft, 1984), and their biological significance remains an open question, too.…”

Presence of Estrogens and Estrogen Receptor-like Proteins in Solanum glaucophyllum

“…A 500-fold molar excess of unlabelled 17␤-estradiol or DES was included in additional paired samples (non-specific binding). The samples were incubated at 4°C for at least 4 h. To separate bound from free sterol, 250 l of a 0.5% dextran/5% charcoal suspension (pH 7.4) was added to each tube (18). The samples were further incubated for 5 min at 4°C and centrifuged at 1,200 ϫ g (5 min) to pellet the charcoal.…”

Presence of estrogen receptor (ER)-like proteins and endogenous ligands for ER in solanaceae

“…Saturation analysis was typically measured by incubating 0.25-0.3 mg protein in 0.4 ml complete TEKM buffer with increasing concentrations of [ 3 H]17␤-estradiol in the absence or presence of a 100-fold molar excess of unlabeled 17␤-estradiol. The samples were incubated at 4 • C for at least 4 h. To separate bound from free sterol, 100 l of a 0.5% dextran/5% charcoal suspension (pH 7.2) was added to each tube [25]. The samples were further incubated for 20 min at 4 • C by gently stirring and then centrifuged at 1200 × g (5 min) to pellet the charcoal.…”