Abstract
Na(+)-independent hepatic transport of branched-chain amino acids occurs via at least two distinct transport processes. System L1, characterized by micromolar Km values, predominates in hepatoma and fetal hepatocytes, whereas System L2, distinguished by Km values in the millimolar range and sensitivity to inhibition by N-ethylmaleimide (NEM), predominates in adult hepatocytes. To determine the plasma-membrane domain localization and ontogeny of System L activity in the rat, we prepared membrane vesicles from the livers of suckling (10 days old) and adult rats enriched for either basolateral (BLMV) or canalicular (CMV) domains. The initial rate of [3H]leucine uptake into BLMV and CMV derived from adult liver was significantly inhibited by the addition of 5 mM NEM; transport into BLMV and CMV derived from 10-day-old rat liver was not affected. Michaelis-Menten kinetic parameters estimated in BLMV derived from adult liver were consistent with System L2 (Km = 2.16 +/- 0.62 mM, Vmax. = 781 +/- 109 pmol/5 s per mg of protein), as were those estimated in adult CMV (Km = 0.83 +/- 0.21 mM, Vmax. = 385 +/- 38 pmol/5 s per mg of protein). Conversely, kinetic parameters estimated in BLMV derived from livers of suckling rats were consistent with System L1 (Km = 0.041 +/- 0.024 mM, Vmax. = 8.8 +/- 1.5 pmol/5 s per mg of protein), as were those from CMV of suckling rats (Km = 0.023 +/- 0.09 mM, Vmax. = 28.1 +/- 2.1 pmol/5 s per mg of protein). We conclude that NEM-inhibitable Na(+)-independent leucine transport activity consistent with System L2 is present in both BLMV and CMV derived from adult rat liver, whereas System L1 predominates in 10-day-old rat liver tissue.