The structure of alpha 2-macroglobulin-methylamine after papain digestion as determined by electron microscopy.
alpha 2-Macroglobulin-methylamine (alpha 2M-CH3NH2) was digested with papain at pH 5.0. The major 600 kDa fragment was purified by molecular-exclusion chromatography. In a non-denaturing gel-electrophoresis system, the 600 kDa fragment migrated in a single band at a rate that was comparable with that for the untreated alpha 2M-CH3NH2. The elution volume of the 600 kDa fragment on Superose-6 was slightly increased. In primary cultures of rat hepatocytes, cellular uptake of 125I-alpha 2M-CH3NH2 was not affected by the 600 kDa fragment, confirming the results of other investigators. The 600 kDa fragment was negatively stained with uranyl formate and analysed by transmission electron microscopy. The major structural characteristics of the parent protein (alpha 2M-CH3NH2) remained intact. The most common image included prominent lateral walls and two centrally located regions of stain exclusion termed 'paddle structures'. The distance between the paddle structures was equivalent in alpha 2M-CH3NH2 and the 600 kDa fragment [approximately 13.5 nm (135 A)]. By contrast, the lateral walls in the 600 kDa fragment were decreased in length by approximately 0.37 nm (37 A) (19%). It is proposed that the 600 kDa structure retains the 'hollow cylinder' shape of alpha 2M-CH3NH2. The structure of the cylinder is formed by the lateral walls and four paddle structures (only two are imaged, owing to overlapping). The paddle structures in the 600 kDa fragment are intact and relatively closer to the apices of the molecule, owing to the decrease in lateral wall length. Since the alpha 2M receptor-binding sites are removed by papain digestion, the studies presented here support the location of the receptor-binding sites near the apices of the lateral walls.
Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
I M
IM
Hussaini
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Figler
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Gonias
D000818:Animals; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism; D008854:Microscopy, Electron; D008970:Molecular Weight; D010206:Papain / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D000511:alpha-Macroglobulins / Q000378:metabolism*
D010446:Peptide Fragments; D000511:alpha-Macroglobulins; C065661:alpha2-macroglobulin-methylamine; D010206:Papain