Uptake of Phenylalanine into Isolated Barley Vacuoles Is Driven by Both Tonoplast Adenosine Triphosphatase and

The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent Km-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; Vmax pp was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 x 106 vacuoles). Half-maximal transport rates driven with ATP or PPi were reached at 0.5 millimolar ATP or 0.25 millimolar PPi. ATP-driven transport showed a distinct pH optimum at 7.3 while PPi-driven transport reached maximum rates at pH 7.8. Direct measurement of the H translocating enzyme activities revealed Km app values of 0.45 millimolar for ATPase (EC 3.6.1.3) and 23 micromolar for pyrophosphatase (PPase) (EC 3.6.1.1). In contrast to the coupled amino acid transport, ATPase and PPase activities had relative broad pH optima between 7 to 8 for ATPase and 8 to 9 for PPase. ATPase as well as ATP-driven transport was markedly inhibited by nitrate while PPase and PPi-coupled transport was not affected. The addition of ionophores inhibited phenylalanine transport suggesting the destruction of the electrochemical proton potential difference A ,uH while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like LTrp, L-Leu, and L-Tyr was also stimulated by ATP. They seem to compete for the same carrier system. L-Ala, L-Val, D-Phe, and DLeu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for L-amino acids having a relatively high hydrophobicity. Vacuoles are the largest organelles of differentiated plant cells occupying up to 90% of the total cell volume. As reviewed by Boller and Wiemken (4), they fulfill a series of functions. Active transport into this compartment has been demonstrated for metabolic intermediates as well as inorganic ions. Phenylalanine, the major aromatic amino acid synthesized in chloroplasts (3), is transiently stored in the vacuole. Up to 95% of all free cellular phenylalanine is found in this compartment (2; and E Martinoia, personal communication). Transport is an active. ATP consuming nrocess which is

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