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An immunoradiometric assay for endopeptidase-24.11 shows it to be a widely distributed enzyme in pig tissues.

ABSTRACT: An immunoradiometric assay for endopeptidase-24.11, which depended on the absorption by tissues of a monoclonal antibody, GK7C2, was established. The optimum conditions for the assay were defined and its correlation with an enzymic assay determined. The immunoassay was used to survey the endopeptidase in crude homogenates of various tissues of the pig. Detergent treatment decreased the sensitivity of the assay but did not invalidate it. Although the endopeptidase was found in many tissues, it was neither uniformly nor ubiquitously distributed. Kidney cortex was confirmed as the major location of the endopeptidase, containing 5000 ng/mg of protein. Lymph nodes were also very active (1370 ng/mg), followed by chondrocytes from articular cartilage (650 ng/mg). In the gut, the endopeptidase was concentrated mainly in the jejunum (130 ng/mg). Various glands (salivary, adrenal, anterior pituitary and pancreas) also contained the antigen in the range 20-55 ng/mg of protein. Lung contained only 5 ng/mg of protein and, in other tissues examined, little or none was detectable. In particular, other lymphoid tissues (spleen, thymus, tonsillar tissues) were relatively poor sources, and none was detectable in peripheral-blood leucocytes or in peritoneal macrophages.

SUBMITTER: Gee NS

PROVIDER: S-EPMC1144960 | biostudies-other | 1985 May

REPOSITORIES: biostudies-other

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Project description:Endopeptidase-24.11 (EC 3.4.24.11), a widely distributed cell-surface endopeptidase in pig tissues, was purified by immunoaffinity chromatography from its second most abundant source, lymph nodes. The detergent-solubilized enzyme is a glycoprotein with an apparent subunit Mr of 91,000, by electrophoresis in the presence of SDS. This value is intermediate between those observed in preparations from kidney and intestine. The specific activity (125I-labelled insulin B-chain as substrate) was similar to that prepared from other sources. Immuno-peroxidase and immunofluorescent cytochemical methods with either a monoclonal antibody, GK7C2, or an affinity-purified polyclonal antiserum, RP109, were used to establish the distribution and localization of the antigen in lymph nodes. Examination of many nodes confirmed the variability of endopeptidase-24.11 content from node to node. Pig lymph nodes are composed of functionally discrete nodelets and are anatomically inverted, with medulla being located peripheral to the cortex. Endopeptidase-24.11 was present in medulla, paracortex and cortex. The medulla, containing relatively few lymphocytes, stained more intensely than other zones. Lymphocyte-rich areas stained only weakly, but antigen was detectable in the centres of follicles and more strongly in a band surrounding them. The pattern of staining was reticular in appearance in all zones. In primary cell cultures, set up after enzymic disruption of nodes, the immuno-positive cells were found to be adherent to glass or plastic and to exhibit a fibroblastic morphology. Diffuse surface immunofluorescence and brighter intracellular immunofluorescence in granules were observed in these cells in the first few days of culture, but by the fourth day no immuno-positive cells remained and the fibroblasts that grew to confluence were somewhat different in morphology. The cells expressing the endopeptidase-24.11 antigen did not express Ia antigen and were clearly distinct from antigen-presenting dendritic cells. In appearance and properties they belong to the group described as reticular cells.

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An immunoradiometric assay for endopeptidase-24.11 shows it to be a widely distributed enzyme in pig tissues.

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