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

Abstract

A monospecific polyclonal antiserum was raised against a recently purified rat brain neurotensin-degrading metallopeptidase. The purified IgG fraction immunoprecipitated the peptidase and inhibited its proteolytic activity. Western blot analyses revealed that the immune fraction recognizes only one protein in rat brain homogenates, and this corresponds closely to the purified enzyme. The IgG displayed a restricted specificity towards the peptidase from murine origin. In the rat, the neurotensin-degrading enzyme was widely distributed throughout peripheral organs with the noticeable exception of the duodenum. In addition, the peptidase was detected in various cell lines or membrane preparations of neural or extraneural origin in which it had been previously characterized by means of biochemical methods. In light of this widespread distribution, the putative role of the peptidase in the metabolism of neuropeptides is discussed.

Rat kidney endopeptidase 24.16

“…Proteins were transferred onto nitrocellulose sheets and allowed to hybridize to the IgG-purified fraction of a polyclonal antiserum developed against brain endopeptidase 24.1 6 (dilution 1 : 1500). Protein-IgG complexes were revealed with goat anti-(rabbit immunoglobulins) coupled to peroxidase with 4-chloro-1-naphthol as described previously (Checler et al, 1989). tently affected by dithiothreitol (ICso w 0.26 mM) while EDTA appeared markedly less potent (IC50 w 5.5 mM) (Fig. 4).…”

“…The stability of endopeptidase 24.16 in the final pellet fraction prepared from rat kidney homogenate was checked by its electrophoretic pattern after SDSjPAGE fractionation and immunolabelling of the peptidase with the IgG-purified fraction of a monospecific polyclonal antiserum developed against rat brain endopeptidase 24.16 (Checler et al, 1989). Rat kidneys (about 2 g) were homogeneized in 20 ml20 mM Tris/HCl pH 7.5 in the absence or presence of a mixture of protease inhibitors including o-phenanthroline (1 mM), phenylmethylsulfonyl fluoride (1 mM), iodoacetamide (1 mM) and pepstatin (1 pM).…”

“…After various days of storage at 4"C, proteins of the last pellets (about 200 pg) were diluted in 100 p160 M sodium phosphate pH 7.5 containing 2% SDS and 5% 2-mercaptoethanol. Samples were boiled and electrophoresed in 8% acrylamide gels as previously described (Checler et al, 1989). Proteins were then blotted onto nitrocellulose sheets as described by Towbin et al (1979).…”

“…Proteins were then blotted onto nitrocellulose sheets as described by Towbin et al (1979). Hybridization with antibodies and revelation of the peptidase-IgG complex were performed as previously described (Checler et al, 1989) except that the IgG fraction was used at a 1 : 1500 dilution.…”

Distinct Properties of Neuronal and Astrocytic Endopeptidase 3.4.24.16: A Study on Differentiation, Subcellular Distribution, and Secretion Processes

“…Fourday-old plated neurons and 15-d-old plated astrocytes were immunolabeled for endopeptidase 3.4.24.16 as described (Chabry et al, 1990) using the IgG-purified fraction of a monospecific rabbit polyclonal antiserum directed toward the rat brain purified enzyme (Checler et al, 1989). Briefly, cells were rinsed twice in 50 mM Tris-HCl containing 140 mM NaCl (buffer A) and fixed with glutaraldehyde 3.5% for 1 hr at 4ЊC.…”