Energetic mechanism of system A amino acid transport in normal and transformed mouse fibroblasts.
Ouabain treatment (0.4 mM) of normal and transformed C3H-10T1/2 cells caused a progressive increase in 2-aminoisobutyrate (AIB) transport reaching a maximum after 16 to 18 h exposure. There was a virtually complete blockage of this stimulated rate when 3 microM cycloheximide (CHX) was added together with ouabain at T = 0. In the transformed cell, addition of CHX after 14 h had no effect; in the normal cell, it inhibited (ca. 50%) the final AIB transport rate achieved after 24 h. The t1/2 for reaching maximal activity (insensitive to CHX exposure) was thus shifted from 8 h in the transformed cell to 15 h in the normal cell. Since the rate of achieving maximal activity in the absence of CHX was about the same in the two cells, the shift in t1/2 in the presence of CHX suggests that the rate of degradation is more rapid in the normal cell. Following ouabain treatment, the apparent Km for Na+ was decreased in both cells. The Km returned to the basal level 1 h after ouabain removal in the normal cell, but remained low in the transformed cell during this time period. The stimulation of AIB transport following ouabain removal was largely abolished by a proton ionophore (1799), a lipophilic cation (tetraphenyl-phosphonium), or ouabain. These results suggest that, under the conditions of ouabain stress, there is a switch in the bioenergetic mechanism. The Na+/K+ pump and System A transporter appear to be linked and the membrane potential generated by the Na+/K+ pump activity becomes a major driving force for AIB uptake.
Leister
K J
KJ
Section of Biochemistry, Molecular, and Cell Biology, Cornell University, Ithaca, New York 14853.
Schenerman
M A
MA
Racker
E
E
D000596:Amino Acids / Q000378:metabolism*; D000621:Aminoisobutyric Acids / Q000378:metabolism; D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D007700:Kinetics; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D010042:Ouabain / Q000494:pharmacology
D000596:Amino Acids; D000621:Aminoisobutyric Acids; C100049:2-aminoisobutyric acid; D008748:Methylcholanthrene; D010042:Ouabain; D003513:Cycloheximide