engleski

Aminopeptidase-N (APN) enzyme activity in the absence of membrane CD13 on mouse T cell line

Membrane peptidases, aminopeptidase N (APN ; CD13) and neutral endopeptidase (NEP ; CD10) play important role in degradation of endogenous opioid pentapeptides enkephalins, by inhibiting their binding to  and  class of the opioid receptors. On cells of nervous system APN also degrades the dynorphin-related peptides, endogenous ligands for  opioid receptors, due to their Leu-enkephalin sequence at the N-terminal site (1). R1.1 is a T cell line, which originates from C58Bl mouse thymoma. It is characterised by constitutive selective expression of   opioid receptors (2). We hypothesised that, in addition to the receptors for  opioid ligands, R1.1 cells might express the enzyme which cleaves these peptides. Therefore we tested: (a) the APN enzyme activity of R1.1 cells ; (b) membrane expression of CD13 on R1.1 cells and (c) the possibility that the endogenous  opioid Dynorphin-A1-17 would be a substrate for APN. APN enzyme activity was determined spectrophotometrically by using the specific substrate L-Ala- -naphtylamid (L-Ala-  NA). Membrane expression of CD13 was tested on FACS by using PE-conjugated anti-mouse CD13 (clone R3-242, Pharmingen). Quality of the antibody was checked by analysing the bone-marrow cells of C57Bl mouse. The role of APN in cleavage of Dynorphin-A1-17 was tested by preincubation of R1.1 cells with Dynorphin-A1-17 before addition of the substrate. The results obtained have shown that R1.1 cells exhibit enzymatic activity, which on the basis of substrate and inhibitor (bestatin ; probestin) specificity, corresponds to APN. However, CD13 could not be detected on the membrane of R1.1 cells. Preincubation with Dynorphin-A1-17 mildly inhibited APN of R1.1 cells. In conclusion, R1.1 cells do not express CD13, but exhibit APN enzyme activity, which is inhibited by Dynorphin-A1-17, suggesting the possibility that Dynorphin-A1-17 may be a substrate for APN. Thus, R1.1 cells in addition to the receptors for  ligands, express the enzyme that cleaves them. The absence of membrane CD13 on R1.1 cells is in accordance with their T cell origin, since APN and CD13 association was reported for cells of myelo-monocytic origin, but not for cells of T lymphocytic origin. References 1. Safavi A, Hersh LB, J. Neurochem., 65: 389-395, 1995. 2. Bidlack, JM, Saripalli LD, and Lawrence, DMP, Europ. J. Pharmacol., 227: 257-265, 1992. 3. Hansen AS, Noren O, Sjostrom H, Werdelin O, Eur. J. Immunol., 23:2358-2364.

T lymphocytes - APN - CD13

nije evidentirano