Abstract
We have characterized a T lymphocyte endopeptidase activity that hydrolyses succinyl-alanine-alanine-phenylalanine-paranitroanilide (Suc-Ala-Ala-Phe-pNa). Hydrolysis of this substrate by intact Jurkat T cells was markedly enhanced when exogenous aminopeptidase N was added to the incubation medium. It thus appears that the release of paranitroaniline from Suc-Ala-Ala-Phe-pNA results from the combination of two distinct enzymatic activities: (i) an endopeptidase activity that cleaves the substrate at the alanyl bond and (ii) an aminopeptidase activity that ultimately cleaves the phenylalanyl bond. This cleavage was further confirmed by HPLC analysis. Specific endopeptidase 24.11 inhibitors were shown to inhibit the endopeptidase activity. These features are reminiscent of the characteristics of neutral endopeptidase (NEP, also known as endopeptidase 24.11, CALLA or CD10). Anti-CD10 monoclonal antibodies (mAbs) recognized the CD10+ B cell line Raji, but not Jurkat cells as assessed by FACS analysis. This is probably due to a lack of sensitivity of this method, the level of NEP activity in Jurkat T cells being 3-5% of that measured in B cell lines. Anti-CD10 mAbs immunoprecipitated endopeptidase 24.11 activities in both Jurkat T cells and Raji B cells, demonstrating that T lymphocytes express a CALLA-related endopeptidase. We also demonstrate that T and B cell endopeptidases have the same molecular weight, that T cells express less functional CALLA mRNA than B cells and that there are at least two shorter transcripts (1.8 and 0.8 kb) in both T and B cells.(ABSTRACT TRUNCATED AT 250 WORDS)