Binding of berberine to a membrane fraction from Coptis cells

Abstract

3H-Labelled berberine, a quaternary benzylisoquinoline alkaloid, was shown to bind specifically to a microsomal fraction prepared from cultured cells of Coptis japonica capable of accumulating the alkaloid in the vacuole. The Scatchard plot of the data suggested the existence of a single berberine-binding site with a dissociation constant ( K d) of 1.67 μM for berberine. The binding of [ 3H]berberine to the membrane fraction was reversible, the optimum pH for binding being between 9 and 10. An addition of either coptisine or jatrorrhizine, which are congeners of berberine, to the assay mixture significantly decreased [ 3H] berberine-binding, indicating that the binding is highly specific for protoberberine-type alkaloids indigenous to the C. japonica. An SH-blocking reagent, iodoacetate, as well as an excess amount of inorganic cation almost completely inhibited the binding. Further fractionation of the microsomes by sucrose density-gradient centrifugation suggested that the binding site was not associated with plasma membrane, endoplasmic reticulum or tonoplast, but with an unidentified membrane (ϱ = 1.08–1.10 g cm −3), which might be involved in the transport of berberine to the vacuole.