Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin

Abstract

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.1 1, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. (1) In both membrane preparations, endopeptidase 24.1 1 was responsible for the formation of neurotensin-(1 -11) and contributed to the formation of neurotensin-( 1 -10) ; a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(I -10). (2) A carboxypeptidase-like activity hydrolysed neurotensin at the Ile1'-Leu13 peptide bond, leading to the formation of neurotensin-(I -12). (3) Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(I -7) and neurotensin-(I -8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. (1) In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(I -10) into neurotensin-(I -8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(I 1 -13) by bestatin-sensitive aminopeptidases. (2) A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11 -13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.Neurotensin is a 13-amino-acid peptide (Glp-Leu-TyrGlu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) first isolated from hypothalami [l] and later from the intestine of bovine [2]. In the periphery, neurotensin-like immunoreactivity was reported to be essentially distributed in the jejunum and in the distal part of small intestine of several mammalian species [3,4]. Immunofluorescence studies showed that neurotensin was mainly located in discrete cells of the mucosa corresponding to the lower portion of dog ileum [5]. In addition to these studies, specific neurotensin receptors were

Tissue distribution of a novel neurotensin-degrading metallopeptidase. An immunological approach using monospecific polyclonal antibodies

“…Western blot analysis not shown). This was apparently in contradiction with the fact that the neurotensin-degrading peptidase activity was previously detected in plasma membranes from circular and longitudinal dog ileum smooth muscle (Checler et al, 1987) as well as in the nervous layers innervating these tissues (Barelli, H., Checler, F. & Vincent, J.-P., unpublished data). These discrepancies led us to examine the species-specificity of the immune purified IgG fraction.…”

“…6). This restricted specificity could explain the discrepancies observed between the biochemical detection of the peptidase in the human adenocarcinoma HT29 cell line (Checler et al, 1 986c) as well as in the circular and longitudinal dog ileum smooth muscle plasma membranes (Checler et al, 1987), and the total lack of staining obtained after exposure of these preparations to the immune IgG-fraction ( Fig. 5; other results not shown).…”