Immunochemical identification of the cell surface bound leucine aminopeptidase, the target enzyme for the immunostimulant bestatin.
The microbial product bestatin is known to inhibit soluble microsomal- and cytosolic leucine aminopeptidase (Leu-APm and Leu-APc) as well as aminopeptidase B (AP-B). To clarify which of these enzymes is the target for bestatin on the cell surface, indirect immunofluorescence studies with antisera raised against purified Leu-APm and AP-B were performed. These antibodies (anti-Leu-APm and anti-AP-B) were found to react with intracellularly localized Leu-APm and AP-B of ethanol-treated L5178y cells. Using non-treated L5178y cells fluorescence was detected only on the cell surface after incubation with anti-Leu-APm. To confirm the supposition that only Leu-APm is present on the cell surface, the AP from the cell membrane was solubilized and analyzed electrophoretically. Based on relative migration data it could be shown, that the cell surface is charged with Leu-APm and not with detectable amounts of Leu-APc or AP-B. Moreover, it could be demonstrated that the solubilized Leu-APm binds to [3H]bestatin.
D000276:Adjuvants, Immunologic / Q000494:pharmacology*; D000818:Animals; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D007930:Leucine / Q000031:analogs & derivatives* / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000276:immunology / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D051379:Mice; D011485:Protein Binding
D000276:Adjuvants, Immunologic; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex