Amino Acid Transport in Plasma‐Membrane Vesicles from Rat Liver

Abstract

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...

Sensitivity of system A and ASC transport activities to thiol-group-modifying reagents in rat liver plasma-membrane vesicles. Evidence for a direct binding of N-ethylmaleimide and iodoacetamide on A and ASC carriers

“…Under our conditions, alanine uptake mediated by system ASC accounted for 52 + 20% of total Na+-dependent alanine uptake (n = 125), whereas alanine uptake mediated by system A represented 48 % of total Na+-dependent alanine transport. This agrees with previous observations carried out in isolated rat hepatocytes (Edmonson et al, 1979) and in rat liver plasma-membrane vesicles (Sips et al, 1980).…”

“…1. After 10 s it had increased more than 10-fold in the presence of a NaSCN gradient as compared with that obtained in the presence of a KSCN gradient, in agreement with previous observations (Van Amelsvoort et al, 1978;Sips et al, 1980). This accumulation of L-alanine in the plasma-membrane vesicles was transient and, after 3 min of uptake, a state of equilibrium was attained ( Fig.…”

“…In the presence of 10 mMMeAIB (50-fold excess compared with L-alanine), L-alanine transport after 10 s was substantially diminished owing to the blocking of system A L-alanine uptake; under these conditions, Na+-dependent alanine uptake is a reflection of system ASC activity Pastor-Anglada et al, 1987). In keeping with previous data (Van Amelsvoort et al, 1978;Sips et al, 1980) the final amount of L-alanine associated with membrane vesicles after 30 min of uptake was independent of the presence of Na+.…”

“…An identical protection pattern was observed in experiments in which vesicles were exposed to NEM for 5 min (results not shown). To assess whether this protection was L-alanine-specific, we performed experiments similar to those specified above in which vesicles were preincubated in the presence of 10 mM-Lphenylalanine, an amino acid that is mainly transported by system L (Na+-independent) and which does not inhibit Na+-dependent L-alanine transport in rat liver plasma-membrane vesicles (Sips et al, 1980;. Results shown in Table 3 indicate that 10 mM-L-phenylalanine did not protect Na+-dependent L-alanine transport against inactivation induced by 0.1 mM-NEM.…”

Na+-Dependent Alanine Transport in Plasma Membrane Vesicles from Late-Pregnant Rat Livers

“…Animals were caged individually and maintained under controlled conditions of light (12 h Transport assays. The procedure used was adapted from that of Sips et al (16). Briefly, 10 pL of plasma membrane preparations were mixed with 40 pL of the incubation mixture to give the following concentrations: 0.25 M sucrose, 0.2 mM CaC12, 10 mM MgC12, 10 mM HEPES/KOH, pH 7.4, 100 mM either Naor K-sulfocyanate and L-(2,3)-3H-alanine (Amersham, Bucks, U.K.) from 0.1 to 20 mM.…”