The catabolism of alanine in isolated rat liver cells was studied using a perifusion system in order to titrate the cells with different concentrations of alanine.The Concentrations of the components of the alanine aminotransferase reaction in the cytosolic and mitochondrial compartments were measured after digitonin fractionation of samples of the cell suspension taken during each steady state. Comparison of the mass-action ratios with the equilibrium constant indicated that the cytosolic enzyme is poised towards pyruvate formation at all concentrations of alanine used, whereas the mitochondrial enzyme is always poised towards alanine formation. The calculated flux through cytosolic alanine aminotransferase, using literature data for the kinetic parameters of the enzyme and experimental data for the concentration of metabolites, was in close agreement with the measured carbon flux. It is concluded that the transamination step in alanine catabolism occurs in the cytosol.The concentration of the components of the glutamate dehydrogenase and 3-hydroxybutyrate dehydrogenase reactions in the mitochondrial compartment during each steady state were also measured. The dC of the combined reactions did not change appreciably on increasing the extracellular alanine concentration to 0.6 mM. At higher alanine concentrations AG became slightly more negative. As the extracellular Concentration of alanine was increased, the concentration of glutamate in both the cytosolic and the mitochondrial compartments increased. However, the cytosolic alanine concentration was always lower than that in the extracellular compartment even at the highest concentration of alanine (6 mM) infused. When increasing concentrations of pyruvate were infused together with a fixed concentration of alanine (0.6 mM), the cytosolic concentration of alanine increased to levels even higher than that in the perifusate. However, there was no significant effect in the rate of formation of nitrogenous products.It is concluded that the transport of alanine across the plasma membrane is the only rate-controlling step in alanine catabolism under the conditions used.Alanine plays an important role as a carrier of amino groups in the mammalian circulation. Thus amino nitrogen formed during muscle metabolism is transported to the liver mainly in the form of alanine (see [1,2] for reviews). It is, therefore, evident that the metabolism of alanine in the liver is a physiologically important process.The first step in alanine metabolism in liver is the translocation of the amino acid across the plasma membrane of the parenchymal cells. Our previous studies [3] have demonstrated that the transport step is rate-controlling in alanine degradation. Indeed, the agreement between the kinetics of alanine transport and that of alanine metabolism [3] suggested that at physiological concentrations of alanine in plasma (0.2-0.5 mM) the transport of the amino acid across the plasma membrane is the only rate-controlling step for its metabolism. These observations were confirmed by ...

L-Alanine transport was studied in plasma-membrane vesicles from rat liver. A gradient of NaSCN, but not of KSCN, stimulated alanine uptake. Monensin plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone abolished the observed overshoot in uptake. After equilibration of alanine, NaSCN induced uphill transport.

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Plasma-membrane vesicles, isolated from rat liver, catalyze active transport of L-alanine. The transient accumulation of L-alanine requires the presence of a Na' concentration gradient (outside > inside). The alanine-Na' symport is an electrogenic process, since it is stimulated under conditions that allow compensatory ion movements : both co-transported anions as well as counter-transported cations influence the rate of alanine-Na' symport. However, no uptake is observed in the presence of a membrane potential, when no Na+ concentration gradient is present. Sodium-gradient-stimulated alanine uptake is dependent on temperature and pH, stereospecific, and is affected by the presence of other amino acids. The activity of the L-alanine transport system is influenced both by the Na' and by the L-alanine concentration. In the presence of 100 mM Na', an apparent K, for alanine of 2 mM is observed; lowering the Na' concentration results in an increase in the apparent K,, and a decrease in the apparent V. The apparent K, for Na' is 34 mM in the presence of 0.2 mM L-alanine. Increasing the L-alanine concentration also results in a lower apparent K, for Na', and a higher V.The passage of amino acids across the hepatocyte plasma membrane into the cytosol is an important parameter in liver metabolism : gluconeogenesis and ureogenesis are largely dependent on intracellular amino acid pools [l]. The uptake of amino acids has been studied with the use of different experimental systems : perfused liver [2,3], liver slices [4,5] and also isolated hepatocytes [6 -13,381. From these studies, our understanding of amino acid transport across the hepatocyte plasma membrane remains ambiguous : for instance, both a free permeability of the membrane for amino acids [6,7], and the presence of specific transport systems [ 101 have been claimed.A major obstacle in elucidating the mechanism of solute passage across cell membranes has been the difficulty in differentiating between intracellular metabolic conversions, and processes at the cell membrane level. Isolated plasma membrane vesicles which have retained transport activity can provide a simplified system, in which transport processes can be studied dissociated from intracellular components and under well defined conditions. Moreover, translocation of naturally occurring compounds can be studied without the necessity of using non-metabolizable analogs.Vesicular plasma-membrane preparations have been employed in the study of amino acid transport in animal cells : membrane vesicles from tissues, e. g. kidney [I 4 -181, intestine [19,20], and pancreas [21,22], as well as from free-occurring mammalian cells, e. g. cultured fibroblasts [23 -271 and Ehrlich ascites tumor cells [28] have been isolated.Recently, we described the isolation of a plasmamembrane preparation from rat liver [29], which has retained transport capacity for amino acids. Such a preparation can supply essential data on amino acid translocation across the hepatocyte plasma membrane.In this paper, the transport of the me...

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