FLOW CYTOMETRIC DETERMINATION OF AMINOPEPTIDASE ACTIVITIES IN VIABLE CELLS USING FLUOROGENIC RHODAMINE-110 SUBSTRATES

Aminopeptidases (AP) are ubiquitously occuring, nonspecific exopeptida ses involved in protein degradation. They cleave the N-terminal amino acid of peptides and occur in practically all mammalian cells and tiss ues. Physiological and pathological processes such as metastasis of tu mors and inflammation have been thought to involve changes in AP activ ities. Determination of AP (EC 3.4.11.X) activity in viable cells by n ow cytometry was the subject of this study because of its general biol ogical and clinical interest. Different bis-substituted rhodamine 110 (R11O) peptide derivatives were synthesised and used as AP- and exopep tidase (EC 3.4.13.X-EC3.4.14X) substrates felt now cytometric measurem ents. Intracellular AP activities in viable lympho-, mono-, granule-, and thrombocytes were detected by fluorescence increase from R110 foll owing intracellular substrate cleavage, Eukaryotic-AP do not cleave D- aminoacids and hence NH2(D-Leu)(2)R110 substrate served as negative co ntrol. Specific substrate cleavage by AP is shown by complete inhibiti on of fluorescence generation following preincubation of cells with le ucine-chloromethylketone inhibitor. R110 AP- and exopeptidase substrat es are suitable indicators for coupled endopeptidase reactions due to their rapid cleavage and largely pH independent generation of intracel lular fluorescence. (C) 1995 Wiley Liss, Inc.