Rosmarinic acid (RA) is a natural antioxidant produced by cell suspension cultures of sage (Salvia officinalis L.). The growth and production of RA by these cells can be modified by the type of culture medium. Production can be increased 10-fold to attain 6.4 g.1(-1) under optimal conditions. Investigation of kinetics showed that a change in the medium caused shifts in peaks of growth and production, and modifications of the cell metabolism. RA production can be correlated with growth or begins only when growth has stopped.

A R A D A AND M. RAIMBAULT. 1993. Extracellular amylase from Lactobacillus plantarum A6 was purified by fractionated precipitation with ammonium sulphate and by anion exchange chromatography. The homogeneity of the purified fraction was tested by polyacrylamide gel electrophoresis and showed multiple amylase forms. A major form had a n estimated molecular weight of 50 kDa. It was identified as an a-amylase, with an optimum pH of 5.5, an optimum temperature of 65°C a n d K,,, value of 2.38 g 1-' with soluble starch substrate. The enzyme was inhibited by N-bromosuccinimide, iodine and acetic acid. T h e enzyme activation energy was 30.9 kJ mol-'. I

The vacuo-lysosomes of Hevea brasiliensis (rubber tree) constitute a suitable model system for the study of active transport and energization at the level of the membrane of plant vacuoles. The pH gradient (delta pH) and the membrane potential (delta psi) of vacuo-lysosomes were determined by means of the weak base methylamine and the lipophilic cation tetraphenylphosphonium. The values obtained depended strongly on the experimental conditions such as medium pH or K+ concentration. Under experimental conditions, i.e., pH 7.5 outside and low K+, the delta pH amounts to about 0.9 unit, interior acid, and the delta psi to -120 mV, interior negative. The delta psi is presumably caused by the imposed K+ gradient, and the internal acidification might be a consequence of the passive proton inflow along the electric field. This explanation is sustained by the ineffectiveness of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in destroying the delta pH and delta psi, whereas higher K+ concentration decreased both. Under conditions existing in vivo, the membrane potential might be significantly lower. The presence of ATP increased the acidification of the intravesicular space by 0.5pH unit to a delta pH of up to 1.4 and shifts the membrane potential at least 60mV to a more positive value. The change of the protonmotive potential did not occur with ADP; the pH-dependence of the change was identical with the pH-dependence of a vacuo-lysosomal membrane-bound ATPase, and the effect of ATPase was prevented by the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The change of protonmotive potential difference, brought about by the ATPase, was at least 90 mV. This is evidence that a vacuo-lysosomal ATPase in plants can function as an electrogenic proton pump that transfers protons into the vacuo-lysosomal space.

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The relationship between the electrochemical proton gradient, Δ μH+ (-) , and citrate transport has been studied in tonoplast vesicles from Hevea brasiliensis (the rubber tree). Vesicles were generated from lyophilized samples of fresh vacuoles obtained from the latex sap. Methylamine was used to measure intravesicular pH and lipophilic ions to determine the electrical potential difference (ΔΨ) across the tonoplast. When incubated at pH 7.5 in the absence of ATP, the tonoplast vesicles showed a ΔpH of 0.6 units (interior acid) and a ΔΨ of about-100 mV (interior negative). This potential is thought to be made up of contributions from an H(+) diffusion potential, diffusion potentials from other cations and a Donnan potential arising from the presence of internal citrate. In the presence of 5 mol m(-3) MgATP the ΔpH was increased to about 1.0 unit and the ΔΨ to about-10 mV. Under these conditions the proton-motive force (Δ p Δ μH+ (-) /F) became positive and reached +50 mV. These effects were specific to MgATP (ADP and Mg(2+) having no significant effect) and were prevented by the protonophore p-trifluoromethoxycarbonylcyanidephenylhydrazone (FCCP). Citrate uptake by the vesicles was markedly stimulated by MgATP; ADP and Mg(2+) again had no effect. Nigericin greatly increased ΔpH and this was associated with a large increase in citrate accumulation. The results indicate that the vesicle membrane possesses a functional H(+)-translocating ATPase. The Δ μH+ (-) generated by this ATPase can be used to drive citrate uptake into the vesicles. The properties of the tonoplast vesicles are compared with those of the fresh latex vacuoles.