Cells separated by
enzyme treatment of the R3230AC mammary
carcinoma were used to characterize the entry of
proline. These cells showed minimal changes in cell viability and intracellular volume and were found to be suitable for transport studies, since the vi of
proline was maintained for at least 4 h when cells were stored at 37 or 4 degrees C, or when transport was measured in the presence or absence of Na+.
Proline was acitvely transported by these
tumor cells, reaching a distribution ratio ([
proline] intracellular/[
proline] extracellular) of 20 after 2 h.
Proline entry consisted of two processes, one saturable (carrier mediated) and the other, non-saturable. The carrier-mediated entry, Km - 0.83 mM and V = 151.10(-5) mumol/min per 5.10(6) cells, was Na+-dependent, sensitive to pH and metabolic inhibitors, and completely inhibited by alpha-(methylamino)-
isobutyric acid (Ki = 0.34 mM).
Proline entry in the absence of Na+ was 20% that in the presence of Na+ and was found to be due to a non-saturable process, since (a) vi of
proline uptake in the absence of Na+ increases linearly with increasing
proline concentration and (b) was not suppressed by either 20 mM alpha-(methyl-amino)-
isobutyric acid, 50 mM
glycine +20 mM
phenylalanine, or 50 mM
serine +20 mM
phenylalanine when
proline uptake was measured in the presence or absence of Na+. Therefore, under the conditions studied, we conclude that
proline transport appears to be restricted to the A (
alanine-preferring) system. Furthermore, these cells should provide a suitable model to study the effect of hormonal manipulations on the
amino acid transport process.