An approach to the intramolecular localization of the thiol ester bonds in the internal cavity of human alpha 2-macroglobulin based on correspondence analysis.
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.
Lambert
O
O
Laboratoire de Biochimie Fondamentale, Université François Rabelais, Tours, France.
Boisset
N
N
Pochon
F
F
Delain
E
E
Lamy
J N
JN
D000818:Animals; D002417:Cattle; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008744:Methylamines; D008854:Microscopy, Electron; D008961:Models, Structural; D009206:Myocardium / Q000378:metabolism; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000032:analysis; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure*
D003574:Cytochrome c Group; D008744:Methylamines; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; C027451:methylamine