Solubilization and reconstitution of high- and low-affinity Na(+)-dependent neutral L-alpha-amino acid transporters from rabbit small intestine.
High- and low-affinity Na(+)-dependent neutral L-alpha-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 proteoliposomes 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-alpha-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 Kt1 = 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-alpha-amino acids.
Department of Biochemistry, Jichi Medical School, Tochigi, Japan.
M
M
Nakanishi
T
T
Tetsuka
Y
Y
Kagawa
A
A
Moriyama
M
M
Sasaki
H
H
Hirata
D000409:Alanine / Q000378:metabolism; D026905:Amino Acid Transport Systems; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002793:Cholic Acids; D007421:Intestine, Small / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008871:Microvilli / Q000378:metabolism; D011510:Proteolipids / Q000378:metabolism; D011817:Rabbits; D012964:Sodium / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity
D026905:Amino Acid Transport Systems; D002352:Carrier Proteins; D002793:Cholic Acids; D011510:Proteolipids; C020485:proteoliposomes; D012964:Sodium; D000409:Alanine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate