Purification and reconstitution of an intestinal Na(+)-dependent neutral L-alpha-amino acid transporter.
Using an improved reconstitution method, we have purified an Na(+)-dependent neutral L-alpha-amino acid transporter from rabbit small intestine to apparent homogeneity. The preparation solubilized with octaethylene glycol dodecyl ether (C12E8) was purified by successive chromatographies on DEAE-Toyopearl and lentil lectin-Sepharose 4B columns. The transport activity was assayed by reconstitution of the protein into liposomes. The specific activity of the final preparation was 1364-fold that of brush border membrane vesicles. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the active fractions gave one band of 90 kDa. Kinetic analysis using proteoliposomes reconstituted with the purified fraction showed that alanine transport was mediated by high affinity system with Kt value of 0.19 mM and Jmax value of 2.8 nmol/mg protein/s. Analysis of the amino acid composition of the purified transporter revealed that the transporter is very hydrophobic protein. From its specific activities for transport of individual amino acids this transporter was concluded to possess broad specificity for neutral L-alpha-amino acids. Furthermore, inhibition study of other amino acids allowed us to identify this transport pathway as the intestinal system B.
Nakanishi
M
M
Department of Biochemistry, Jichi Medical School, Tochigi, Japan.
Kagawa
Y
Y
Narita
Y
Y
Hirata
H
H
D026905:Amino Acid Transport Systems; D000596:Amino Acids / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D007421:Intestine, Small / Q000737:chemistry*; D007700:Kinetics; D008871:Microvilli / Q000737:chemistry; D011817:Rabbits; D012964:Sodium / Q000378:metabolism; D013379:Substrate Specificity
D026905:Amino Acid Transport Systems; D000596:Amino Acids; D002352:Carrier Proteins; D012964:Sodium