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The structure of alpha 2-macroglobulin-methylamine after papain digestion as determined by electron microscopy
I M Hussaini1, N L Figler, S L Gonias
1Department of Pathology, University of Virginia Health Sciences Center, Charlottesville 22908.
Abstract:
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.
Insights
Papain digestion of alpha 2-Macroglobulin-methylamine (alpha 2M-CH3NH2) yielded a 600 kDa fragment retaining the hollow cylinder shape. This fragment
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Medicine
Background:
- Alpha 2-Macroglobulin (α2M) is a large plasma proteinase inhibitor.
- Understanding α2M structure is crucial for its biological functions and therapeutic potential.
Purpose of the Study:
- To characterize the structural and functional properties of a major papain-digested fragment of alpha 2-Macroglobulin-methylamine (α2M-CH3NH2).
- To investigate the impact of digestion on the molecular structure and receptor-binding capabilities.
Main Methods:
- Papain digestion of α2M-CH3NH2 at pH 5.0.
- Purification of the 600 kDa fragment using molecular-exclusion chromatography.
- Analysis by non-denaturing gel electrophoresis, transmission electron microscopy (negative staining).
- Assessment of cellular uptake in primary rat hepatocyte cultures.
Main Results:
- A major 600 kDa fragment was isolated, maintaining a similar migration rate and elution volume to intact α2M-CH3NH2.
- Electron microscopy revealed the 600 kDa fragment retains the overall 'hollow cylinder' shape with intact paddle structures.
- Lateral walls of the fragment were reduced in length by approximately 19%, causing paddle structures to be closer to the molecule's apices.
- Cellular uptake of 125I-α2M-CH3NH2 was unaffected by the 600 kDa fragment.
Conclusions:
- The 600 kDa fragment preserves the core structural integrity of α2M-CH3NH2.
- The papain digestion likely removes receptor-binding sites located near the apices of the lateral walls.
- These findings support a model where α2M receptor-binding sites are situated at the lateral wall apices.