Identification and properties of the cell membrane bound leucine aminopeptidase interacting with the potential immunostimulant and chemotherapeutic agent bestatin.

Bestatin was found to be a competitive inhibitor (with respect to the Leu-NA substrate) not only of the isolated microsomal and cytosolic leucine aminopeptidases (Leu-APm and Leu-APc) but also of the aminopeptidases (APs) present in membrane preparations (from mouse liver) and on the cell surface of L5178Y cells. Kinetic parameters indicate that cellular AP is identical to Leu-APm. To rule out the possibility that AP-B is involved in the inhibition reactions, comparable studies with amastatin were performed. Electrophoretical studies revealed the solubilized cell membrane bound AP to co-migrate with Leu-APm in polyacrylamide gels. The activity of the separated membrane AP was inhibited by bestatin in situ. The cell membrane bound AP activity was found to be lowest in lymphocytes, higher in tumor cells and highest in bone marrow cells and macrophages. Using synchronized L5178Y cells, the AP activity changes during the cell division cycle; the lowest activity was determined during the G1-phase and 35% higher values were measured during the S/G2-phase. The fluctuation of the cell surface associated AP activity parallels with changes in the number of binding sites for bestatin.