Isolated vacuoles from ajmalicine-producing cell suspensions of Catharanthus roseus accumulated the alkaloid ajmalicine. Dissipation of the transtonoplast pH gradient with nigericin abolished ajmalicine accumulation, whereas dissipation of the transtonoplast potential with valinomycin had no effect. Addition of Mg-ATP resulted in a higher ajmalicine accumulation. Serpentine produced by the cells was largely recovered in isolated vacuoles; in contrast, ajmalicine was lost. Ajmalicine was converted in vitro into serpentine by horseradish basic peroxidases (EC 1.11.1.7). In cultured cells there was a striking conformity between the time course of serpentine content and that of the activity of basic peroxidases. Ajmalicine was converted efficiently into serpentine by basic peroxidases extracted from vacuoles and by intact isolated vacuoles. The results are consistent with the hypothesis that ajmalicine accumulates by an ion-trap mechanism and that the accumulated ajmalicine is converted into serpentine inside the vacuoles. By the transformation of ajmalicine into the charged serpentine a trap is created to retain the alkaloids more efficiently in the vacuole.

The influence of initial concentrations of two of the major medium components, sucrose and nitrate, on the growth and the production of alkaloids and anthraquinones in cell suspension cultures of Onchona ledgeriana Moens was studied. It was found that maximum growth and maximum alkaloid yield were obtained with a B smedium containing the normal level of nitrate and 4% sucrose, whereas the anthraquinone yield was maximum at 8% sucrose at the normal level of nitrate. Maximum contents of secondary metabolites, expressed as tag. g DW -t , were found using a medium containing 2% sucrose, but four times the normal nitrate concentration.

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A procedure is described for the extraction of both anthraquinones and alkaloids from tissue culture material of CINCHONA species. Using this extraction method, it is possible to quantitatively determine the two groups of secondary metabolites produced in CINCHONA tissue cultures.