Studies on the transport and storage of indole alkaloids by Catharanthus roseus
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Date
1988
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Abstract
The transport and storage of indole alkaloids by mesophyll protoplasts of Catharanthus roseus (L.) G. Don was characterized. Uptake of alkaloids by mesophyll protoplasts was found to have two components: a rapid, non-specific diffusion of the alkaloid, driven by the pH gradient between the vacuole and the external medium; and a slower, energy dependent uptake which was specific for at least some of the alkaloids endogenous to C. roseus. The concentration and pH dependence for uptake of vindoline suggested the rapid phase of uptake was due to non-mediated diffusion of the neutral form of vindoline. Uptake of nicotine, which is not endogenous to Catharanthus, also conformed to the kinetics of simple diffusion. In the case of vindoline, the second sustained phase of uptake could be selectively inhibited by pretreatment of the protoplasts with NaN3 for 10 min. Metabolism of the alkaloids was ruled out as a possible cause of the observed biphasic uptake. The importance of ion-trapping of the alkaloids inside the cell was demonstrated by a rapid efflux of both vindoline and ajmalicine induced by NH4CI. There was a slow component to the efflux of ajmalicine which was not seen with vindoline, which could have been due to binding of the ajmalicine to either phenolics or counterions in the vacuole. In order to begin to extend the transport studies to the subcellular level, procedures were developed for the reliable isolation of vacuoles from mesophyll protoplasts. A combination of osmotic shock, followed by ultracentrifugation on step gradients of Ficoll was found to give a good yield of vacuoles with little contamination by non-vacuolar components. Binding of FITC-agglutinin (from Abrus precatorius) to the plasma membrane was found to give a rapid, semi-quantitative estimate of the contamination of the purified vacuoles by vacuoplasts. The plasma membrane and tonoplast ATPases were found on sucrose gradients at d=1.13-1.14 and d=1.105 respectively. No enrichment of either K+- stimulated or ionophore-stimulated pyrophosphatase activity could be found on sucrose gradients of the microsomal fraction.
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Vita
Major subject: Biochemistry
Vita
Major subject: Biochemistry
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Major biochemistry