Photoaffinity labeling of a cell surface polyamine binding protein

Abstract

Intracellular polyamine pools are partially maintained by an active transport apparatus that is specific for and regulated by polyamines. Although mammalian transport activity has been characterized by kinetic studies, the actual protein itself has yet to be identified, purified, or cloned. As one approach to this problem, we attempted photoaffinity labeling of plasma membrane proteins using two specifically designed and synthesized polyamine conjugates as photoprobes. The first is a spermidine conjugate bearing the photoreactive moiety 4-azidosalicylic acid at the N4 position via an alkyl linkage, and the second is a norspermine conjugate with 4- azidosalicylic acid at the N1 position via an acyl linkage. Labeling of murine L1210 lymphocytic leukemia cells was carried out at 4 °C to promote selective alkylation of cell surface proteins. Separation of plasma membrane proteins from cells cross-linked with the N4-spermidine conjugate by SDS- polyacrylamide gel electrophoresis revealed two heavily labeled proteins at ~118 and ~50 kDa (designated p118 and p50, respectively). Band p118 was more well defined and much more intensely labeled. Analogous proteins were also observed in human U937 lymphoma cells. Specificity of labeling was strongly suggested by competition with polyamines and analogs during labeling and further indicated by the nearly identical labeling of the same protein by the N1-norspermine photoprobe but not by the unconjugated photoreagent. Neuraminidase pretreatment of L1210 cells increased mobility of the p118, suggesting that it was glycosylated and, thus, of plasma membrane origin. In transport-deficient L1210 cells, p118 and p50 were found to have a slightly higher molecular mass and were accompanied by a less distinct protein band (~100 kDa). These findings indicate the presence of a polyamine binding protein at the surface of murine and human leukemia cells, which could be directly or indirectly related to the polyamine transport apparatus.