Properties and specificity of binding sites for the immunomodulator bestatin on the surface of mammalian cells

The binding of the immunomodulator bestatin, an inhibitor of cell surface bound leucine amino-peptidase and aminopeptidase B, to mammalian cells of varying origin has been studied. The specific binding of [³H] bestatin was a rapid and saturable process exhibiting one affinity, characterized by an association constant of 0.8×10⁵ M^-1, as determined in the L5178y mouse lymphoma system. Optimal binding was observed at 37°C. L-leucine and L-leucine-β-naphthylamide prevented the binding, suggesting that the complex was formed between leucine aminopeptidase and bestatin. The protein nature of the bestatin-"receptor" was suggested by its susceptibility to trypsin. Under the conditions used here intracellular translocation of bestatin appeared to be negligible. A maximum of about 2.2 × 10⁶ bestatin molecules could bind to L5178y mouse lymphoma cells. Under identical conditions by far the highest amount of bestatin was bound to macrophages from mice. Lower levels were measured with T-lymphocytes; very low binding capacity was observed with B-lymphocytes. Experiments with synchronized L5178y cells revealed a cell cycle dependent change of binding capacity for bestatin; the highest level was observed during the transition from S-to G₂ phase and the lowest during G₁- and early S phase. These data lend further support to the assumption that the immuno-potentiating activity of bestatin is due to a stimulation of T-lymphocyte proliferation probably mediated through the activation of macrophages.