Biomedical subjects
Publications and source records attributed to E Delain.
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The plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) can trap small proteins including cytokines. With the four internal thiol ester bonds being involved in the covalent binding of proteinases and other ligands, it was of interest to precisely localize these active groups in the alpha 2M molecule. This was approached by comparing methylamine-transformed human alpha 2M (alpha 2M-MA) and alpha 2M-MA chemically coupled to cytochrome c (Cyt c). This enzyme was chosen because of its size, which is close to that of cytokines, and because of the easy quantification of the reaction stoichiometry. The two molecular species were first mixed in a single sample that was deposited on a carbon-coated grid, negatively stained, and imaged in the electron microscope. The aligned images of the two macromolecular species were then separated by correspondence analysis and hierarchical ascendant classification and compared through the calculation of a subtraction image. The interest of this approach is that prior to the separation of the images, the two molecular species are subjected to rigorously identical treatments. The subtraction images between the average images of alpha 2M-MA and Cyt c-alpha 2M-MA allowed an unambiguous localization of the Cyt c molecules in the internal cavity of the alpha 2M molecule. The internal cavity is gradually filled when the Cyt c/alpha 2M ratio increases. However, it is not yet clear whether the thiol groups are located in the median portion of the wall or in the interwall (paddle) structure.
In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed alpha 2M, with and without activated PLA2, were determined by image processing and compared. A localization of the PLA2 was achieved by subtracting the average image of alpha 2M transformed by methylamine from that containing PLA2. The results are consistent with previous work showing the central localization of chymotrypsin trapped in alpha 2M. They also suggest that the four thiol esters are located near the center of the alpha 2M molecule.
Cysteine 949 and glutamine 952 are known to be part of the thiol ester site of each of the four subunits of human alpha 2-macroglobulin (alpha 2M). The hydrolysis of this thiol ester bound to methylamine results in the incorporation of the amine and liberation of a free sulfhydryl group that can be specifically labeled. Therefore, a high-resolution marker specific for the sulfhydryl groups, the monomaleimido Nanogold (Au1.4nm) cluster was used to bind this amino acid. After cryoelectron microscopy, a three-dimensional reconstruction of the alpha 2M-Nanogold conjugates (alpha 2M-Au1.4nm) was achieved, revealing the internal location of the thiol ester sites in the transformed alpha 2M molecules. From this study we propose three possible locations for the cysteine 949.
Steroid hormone-receptor complexes regulate the transcription of specific genes. Recent studies of high-affinity interactions between the receptors and discrete regions of DNA, together with gene-transfer experiments, have led to the precise mapping of hormone regulatory elements. Nothing is known, however, about the mechanisms whereby DNA-bound receptors modulate gene transcription. At the start of transcription in prokaryotes two oligomeric molecules of several regulatory proteins must bind to two specific DNA sites and interact with one another to regulate the binding of RNA polymerase to DNA. Using electron microscopy to observe progesterone receptor binding to regulatory regions of uteroglobin and mouse mammary tumour virus genes, we demonstrate a similar binding between receptor oligomers at two DNA sites. DNA loops are formed when the hormone regulatory elements are at a distance from one another. Thus, in common with certain prokaryotic systems, protein-protein interactions may be important in steroid hormone regulation of gene transcription.
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