Electron microscopy studies of alpha 2-macroglobulin subunit association after limited reduction with dithiothreitol.
The subunits of human alpha 2-macroglobulin (alpha 2M) were dissociated by treatment with reductant (0.5 mM dithiothreitol) under mild conditions. Intact tetramers, half-molecules (subunit dimers), and monomers were identified by chromatography on Superose-6. These products were not in rapidly reversible equilibrium since purified half-molecules were completely stable for up to 18 h. Monomers slowly associated to form some half-molecules in the same time period. Negatively stained preparations of monomers and half-molecules demonstrated significant heterogeneity by electron microscopy. This heterogeneity probably reflected structural differences as well as variation in projection and staining. After reaction with methylamine or proteinase, half-molecules and purified monomers reassociated. The principal product was an intact tetramer displaying an "H-like" image which was visually indistinguishable from the structure of intact tetrameric alpha 2M after conformational change. Incompletely reassociated alpha 2M species were also identified after performing chromatography to increase the fraction of these products. Images resembling one-half of the intact tetrameric alpha 2M structure (epsilon-image) or three-quarters of the intact tetramer (chair-image) were observed. These studies demonstrate that reassociation of reductant-dissociated alpha 2M subunits occurs by the formation of appropriate interactions, so that the resulting tetramers are equivalent to nontreated alpha 2M-trypsin. Unlike native alpha 2M, the structure of conformationally transformed alpha 2M is relatively insensitive to the loss of interchain disulfide bonds. We propose that reassembly of alpha 2M subunits occurs by the binding of two half-molecules or by the addition of individual subunits to half-molecules or trimers.
Gonias
S L
SL
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Marshall
L B
LB
Figler
N L
NL
Hussaini
I M
IM
Moncino
M D
MD
Pizzo
S V
SV
D002850:Chromatography, Gel; D004229:Dithiothreitol / Q000494:pharmacology*; D006801:Humans; D046911:Macromolecular Substances; D008744:Methylamines / Q000494:pharmacology; D008854:Microscopy, Electron; D014357:Trypsin / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000737:chemistry* / Q000648:ultrastructure
D046911:Macromolecular Substances; D008744:Methylamines; D000511:alpha-Macroglobulins; C027451:methylamine; D014357:Trypsin; D004229:Dithiothreitol