Solubilization and reconstitution of high-and low-affinity Na+-dependent neutral l-α-amino acid transporters from rabbit small intestine

High- and low-affinity Na+-dependent neutral l-α-amino acid transporters were solubilized with 0.25% octaethylene glycol dodecyl ether (C12E8) after removal of the proteins from the brush-border membrane vesicles with 2% CHAPS and 4 M urea. When the CHAPS-insoluble protein was treated with papain before its solubilization with C12E8, a substantial amount of protein was removed without any decrease of the transport activities. The solubilized transporters were reconstituted into proteo-liposomes after removal of C12E8 with Bio-Beads SM2. Several parameters proved to be important for optimal reconstitution efficiency: (a) the type of detergent, and (b) the phospholipid/protein and detergent/protein ratio during reconstitution, and (c) the salt concentration during reconstitution. Reconstituted proteoliposomes showed rapid uptake of neutral l-α-amino acids but not imino acid, basic or acidic amino acids driven by an electrochemical potential of Na+ (out > in). The uptakes under low-and high-substrate condition were further augmented by an artificial membrane potential introduced by K+ diffusion via valinomycin (negative interior). Kinetic analysis revealed that both the brush-border membranes and the solubilized fraction involved two carrier-mediated pathways for alanine transport. The kinetic parameters were determined by curve fitting with a computer to be Kn  0.28 mM (0.21 mM) and Kt2  43.2 mM (28.4 mM), respectively (those with brush-border membrane vesicles in parentheses). Studies on the specific activities for transport of individual amino acids under low or high substrate concentration and the cross-inhibitory effects of various amino acids on alanine uptake (low concentration) revealed that these transporters possess broad specificity for neutral l-α-amino acids.