Characterization of threonine transport into a kidney epithelial cell line (BSC-1). Evidence for the presence of Na(+)-independent system asc [corrected].
The transport routes for threonine in a primate kidney epithelial cell line (BSC-1) grown as monolayer in continuous cell culture were studied. We discovered at least four different transport systems for threonine uptake. The Na(+)-dependent route shows biphasic kinetics with a low and high affinity parameter. The apparent kinetic constants for Km1 and Km2 were 0.3 and 36 mM with apparent Vmax values of 6.3 and 90 nmol/mg protein/min, respectively. The high affinity, low Km component resembles system ASC activity, with respect to substrate selectivity. The Na(+)-independent route also exhibits biphasic kinetics. A high affinity component (apparent Km of 1.0 mM, and apparent Vmax of 7.2 nmol/mg protein/min) is sensitive to inhibition by leucine and the aminoendolevo-rotatory isomer of 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid, suggesting participation by system L. The low affinity component (apparent Km of 10.2 mM, and apparent Vmax of 71 nmol/mg protein/min) was specifically inhibited by threonine, serine, and alanine and could be assigned to system asc. The discrimination between system L and asc is based upon differences in pH sensitivity, trans stimulation, and Ki values. In addition, the effects of harmaline, a suspected sodium transport site inhibitor, have been studied. Harmaline noncompetitively inhibited Na(+)-dependent threonine uptake but had no effect on Na(+)-independent transport of threonine. This report is the first to present evidence for the presence of system asc in renal epithelial cells. The physiological and biochemical significance of our findings are discussed.
Kuhlmann
M K
MK
Department of Medicine, Harbor-UCLA Medical Center, UCLA School of Medicine, Torrance 90509.
Vadgama
J V
JV
D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D006246:Harmaline / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D011323:Primates; D012964:Sodium / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*
D013912:Threonine; D012964:Sodium; D006246:Harmaline; D007930:Leucine