Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.
Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.
Campbell
G S
GS
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109-0606.
Yu
J H
JH
Oxender
D L
DL
D002731:4-Chloromercuribenzenesulfonate / Q000494:pharmacology*; D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D016466:CHO Cells; D006224:Cricetinae; D006863:Hydrogen-Ion Concentration; D007700:Kinetics
D000596:Amino Acids; D002731:4-Chloromercuribenzenesulfonate