Photoinhibition of 2-Amino-2-carboxybicyclo[2,2,1]heptane Transport by O-Diazoacetyl-L-serine: An Initial Step in Identifying the L-system Amino Acid Transporter

Abstract

Neutral amino acid uptake into mammalian cells occurs predominantly through the L, A, and ASC carrier-mediated transport systems. The proteins responsible for transport by these systems have not been isolated, and the three pathways presently are defined by their amino acid specificity and physiologic parameters. We have found that the amino acid derivative, O-diazoacetyl-L-serine (azaserine), is a potentially useful probe for identification of the L-(leucine-favoring) system transporter in human T-lymphocytes. Uptake of azaserine competitively inhibits the uptake of the prototype L-system amino acid, 2-amino-2-carboxybicycloheptane (BCH). Azaserine undergoes photolytic cleavage with 365 nm incident light to yield a highly reactive carbene intermediate and free N2. Following photolysis of [14C]azaserine in a suspension of lymphocytes, the 14C label is detectable within a crude cytoplasmic membrane preparation, and this process is inhibited by a 50-fold excess of unlabeled azaserine or 2-amino-2-carboxybicycloheptane, suggesting that the 14C-product is associated with the membranes at or near the L-system transport site. Furthermore, photolysis of azaserine in the presence of lymphocytes results in specific irreversible inhibition of L-system transport. Thus, photolysis of azaserine provides an initial step toward the identification of the L-system transporter.