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Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor

Analytical Biochemistry, ISSN: 0003-2697, Vol: 205, Issue: 1, Page: 57-64
1992
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Article Description

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N -[fluoresceinyl]- N′ -[1-(6-(3-mercapto-2-benzyl - 1 - oxopropyl)amino - 1 - hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC 50 value of 10 n m and a good selectivity compared to that of aminopeptidase N (>100 μ m ) and angiotensin converting enzyme (32 μ m ). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 n m FTI which was completely displaced by 10 μ m HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 n m with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.

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