RESUMO
The uptake of phenylalanine was studied with vacuole isolated from barley mesophyll protoplasts. The phenylalanine transport exhibited saturation kinetics with apparent K(m)-values of 1.2 to 1.4 millimolar for ATP- or PPi-driven uptake; V(max app) was 120 to 140 nanomoles Phe per milligram of chlorophyll per hour (1 milligram of chlorophyll corresponds to 5 x 10(6) 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 K(m 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 Delta muH(+) while the rate of ATP and PPi hydrolysis was stimulated. The uptake of other lipophilic amino acids like l-Trp, 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 d-Leu did not influence phenylalanine transport suggesting a stereospecificity of the carrier system for l-amino acids having a relatively high hydrophobicity.
RESUMO
The energy-dependent transport of phenylalanine into isolated vacuoles of barley (Hordeum vulgare L.) mesophyll protoplasts has been studied by silicone-layer floatation filtering. The uptake of this aromatic amino acid into the vacuolar compartment is markedly increased by MgATP, showing saturation kinetics; the K m values were 0.5 mM for MgATP and 1.2 mM for phenylalanine. V max for phenylalanine transport was estimated to 140 nmol phenylalanine·(mg·Chl)(-1)·h(-1). The transport shows a distinct pH optimum at 7.3 and is markedly inhibited by 40 mM nitrate. Azide (1 mM) and vanadate (400 µM) had no or little effect on rates of transport while p-fluorophenylalanine seemed to be an effective inhibitor, indicating a possible competition at an amino-acid carrier. Ionophores such as valinomycin, nigericin or gramicidin were strong inhibitors of phenylalanine transport, indicating that this process is coupled to both the transmembrane pH gradient (ΔpH) and the transmembrane potential (ΔΨ).