Photoaffinity and protein modification studies on the system L and system T amino acid transporters in mammalian cells.

To gain insight into the mechanism of action of Na$\sp+$-independent transport of neutral amino acids, it will be necessary to identify and isolate the proteins responsible for this transport activity. Toward this goal, we have developed two procedures to covalently inactivate the Na$\sp+$-independent system L transporter in HTC cells. Photoaffinity labeling utilizes light-sensitive substrate analogs to specifically and irreversibly inactivate the system L transporter. Protein-modifying agents that are not specific for a particular transport system have also been used to inactivate the system L transporter. p-Azidophenylalanine is a UV-light sensitive tyrosine analog that produces a highly reactive nitrene intermediate upon photolysis. The uptake of this probe is specific for system L. Upon photolysis, azidophenylalanine inactivated system L uptake in a manner that could be protected only by system L substrates. Azidophenylalanine photolysis had no effect on other neutral amino acid transport systems. Uptake and exodus through system L was sensitive to low concentrations of organomercurial cysteine-reagents. Other cysteine-reagents did not inactivate this transporter. Inactivation by p-chloromercuribenzenesulfonate (pCMBS) was specific for system L and was diminished only by the addition of certain system L substrates. Inactivation and substrate protection occurred at 0$\sp\circ$ and 37$\sp\circ$C. pCMBS inactivation was reversed by the addition of excess dithiothreitol (DTT). In human erythrocytes, pCMBS and NEM (N-ethylmaleimide, another cysteine-reagent) inactivated uptake, but not exodus through system T. System L was unaffected. The inactivation of system T required longer time intervals and 10 times the concentration of pCMBS than required for inactivation of system L. Excess DTT did not reverse the inactivation, but it was diminished by system T substrates. System L in HTC cells and system T in erythrocytes appear to have cysteine residues near their amino acid binding sites, while system L in erythrocytes does not. The sensitivity of these transporters to organomercurial reagents and photosensitive substrate analogs can be exploited to help study and eventually isolate the proteins responsible for amino acid translocation.Ph.D.Animal PhysiologyBiochemistryBiological SciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/128347/2/8920623.pd