Showing papers by "F. Van Leuven published in 1982"
TL;DR: The results show that the internal thioesters in alpha 2M are necessary for the conformational change producing sterically inhibited endoproteinase complexes, but do not participate as such in receptor-mediated endocytosis of these complexes.
Abstract: The unique steric type of inhibition of endopeptidases by human alpha 2-macroglobulin (alpha 2-M) and the inactivation of the latter by methylamine were examined in relation to the internal thioesters in alpha 2M The present results confirm our previous findings that disruption of the internal thioesters, is not in itself sufficient to cause the conformational change of alpha 2M typical of alpha 2-M-proteinase complexes; the electrophoretically slow form of alpha 2M with [14C]methylamine incorporated was isolated Moreover, this group is stabilized by derivatization of the exposed cysteine thiol groups Cyanylation with 2,4-dinitrophenyl thiocyanate during the methylamine reaction was the most effective procedure, yielding essentially only slow-form alpha 2M Other thiol-specific reagents were less effective When allowed to react with trypsin the cyanylated derivative (slow-form alpha 2M with thioesters broken) produced anomalous complexes; only half the expected amount of trypsin was bound, whereas the complexes were fully inhibited by soya-bean trypsin inhibitor and were proteolytically active Despite this, the anomalous complexes were recognized by two highly specific probes: the fibroblast alpha 2M-complex receptor and the monoclonal antibody (F2B2) directed against the receptor-recognition site on alpha 2M complexes The results show that the internal thioesters in alpha 2M are necessary for the conformational change producing sterically inhibited endoproteinase complexes, but do not participate as such in receptor-mediated endocytosis of these complexes
60 citations
Journal Article•
TL;DR: The results clearly indicate that the spreading of human fibroblasts can be modulated by the nature of the substratum and that, by using quantitative methods, these differences in behaviour can be measured accurately.
Abstract: In contrast to established cell lines, normal human skin fibroblasts spread on their own fibronectin. The present investigation has examined whether human fibroblasts, like established cell lines, would be capable of spreading on substrata coated with proteins with different reactivity towards the cell surface. Coverslips were coated with human serum, fibronectin, alpha 2 macroglobulin-trypsin, a polyspecific anti-fibroblast antibody and a polyspecific anti-calf-serum antibody. The attachment of the cells to these substrate was of the same extent. Spreading was examined qualitatively using phase, interference contrast and reflection contrast optics on live cells, as well as scanning electron microscopy on fixed cells. To quantitate the maximum degree of cell spreading a semi-automated system was used, which measured the cell perimeter on a large number of cells. The distributions of the degree of cell spreading on the five substrata were compared statistically. The qualitative and quantitative differences observed on the various substrata could be further differentiated by adding various amines to the cells during 60 min spreading or during a 30 min preincubation before spreading. No strict correlation could be found between the effect of the amines on attachment or on spreading and their presumed effects on cellular transglutaminases. The results clearly indicate that the spreading of human fibroblasts can be modulated by the nature of the substratum and that, by using quantitative methods, these differences in behaviour can be measured accurately.
6 citations
TL;DR: The production of alpha 2M by human endothelial cells must, therefore, be lower than 3 ng/10(6) cells/48 h, the lower limit of the assay.
Abstract: The presence of an α2-macroglobulin (α2M) receptor, the receptor-mediated endocytosis of α2M-protease complexes, and the secretion of α2M by cultured endoth
5 citations
TL;DR: These clonal variants derived from a human osteosarcoma cell line provide a powerful system in the study of the function of these cell surface markers, compared to diploid fibroblasts.
1 citations
TL;DR: Results suggested a close relation between the B2 antigenic sites and the receptor recognition sites on α2M complexes.
Abstract: Hybridoma antibodies to α2 Macroglobulin (α2M) were screened for inhibition of receptor-mediated endocytosis of 125I- α2M-trypsin by normal human fibroblasts. One strongly inhibiting clone, B2, detected a neo-antigen present on α2M-proteinase complexes. Purified antibodies of a non-inhibiting clone, D9, recognized antigenic sites on both native and complex α2M. Purified B2 antibodies and F(ab) fragments of B2 completely inhibited endocytosis of 125I-α2M-trypsin, in contrast to D9 antibodies. These results suggested a close relation between the B2 antigenic sites and the receptor recognition sites on α2M complexes.