| Abstract: | Adaptive regulatory control of System A activity was investigated using MDCK cells and a chemically induced, oncogenic transformant of MDCK cells, MDCK-T1. Within 7 hours after transfer to an amino-acid-deficient medium, A activity of subconfluent MDCK cells had maximally derepressed, but this activity in confluent MDCK cells and in subconfluent transformed cells showed little capacity for derepression. Amino-acid-starved, subconfluent MDCK cells were used to study trans-inhibition and repression of A activity by individual amino acids. Trans-inhibition and repression were defined as the cycloheximide-insensitive and cycloheximide-sensitive components, respectively, of the total inhibition. Trans-inhibition correlated well with substrate affinity, but repression did not. Trans-inhibition and repression were further characterized using alpha-(methylamino) isobutyric acid (mAIB), a trans-inhibitor, and glutamate, an effective repressor. The apparent initial T 1/2 for inhibition by mAIB in the presence of cycloheximide was 0.5 hours, while that for repression by glutamate was 4.7 hours. Half-maximal inhibition by mAIB and repression by glutamate occurred at approximately 0.02 mM and 0.07 mM, respectively. Reversal of trans-inhibition by methionine occurred in the presence of cycloheximide within 1-4 hours after removal of methionine. The A system of the transformed MDCK-T1 cells showed elevated activity, little capacity for derepression, resistance to repression by amino acids, but retention of sensitivity to trans-inhibition. Kinetic analysis of mAIB uptake indicated that the A system of MDCK-T1 cells has become kinetically more complex in a manner which resembled amino-acid-starved rather than amino-acid-fed MDCK cells. These results suggest that the A system of MDCK-T1 cells has become resistant to adaptive regulatory control. |
