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The 72-kDa microtubule-associated protein from porcine brain
M Takeuchi1, S Hisanaga, T Umeyama
1Department of Anatomy and Cell Biology, Faculty of Medicine, University of Tokyo, Japan.
Abstract:
A microtubule-associated protein (MAP) with a molecular mass of 72-kDa that was purified from porcine brain by using its property of heat stability in a low pH buffer was characterized. Low-angle rotary shadowing revealed that the 72-kDa protein was a rodlike protein approximately 55-75 nm long. The 72-kDa protein bound to microtubules polymerized from phosphocellulose column-purified tubulin (PC-tubulin) with taxol and promoted the polymerization of PC-tubulin in the absence of taxol. Microtubules polymerized by the 72-kDa protein showed a tendency to form bundles of several microtubules. Quick-freeze, deep-etch electron microscopy revealed that the 72-kDa protein formed short crossbridges between microtubules. We performed peptide mapping to analyze the relationship of the 72-kDa protein to other heat-stable MAPs, and the results showed some resemblance of the 72-kDa protein to MAP2. Cross-reactivity with a monoclonal anti-MAP2 antibody further suggested that the 72-kDa protein and MAP2 are immunologically related. To study the relationship between the 72-kDa protein and MAP2C, a smaller molecular form of MAP2 identified in juvenile rat brain, we prepared the 72-kDa protein from rat brain by the same method as that used for porcine brain. The fact that the 72-kDa protein from juvenile rat brain was also stained with our monoclonal anti-MAP2 antibody also suggested that the 72-kDa protein is an MAP2C homologue of the porcine brain.
Insights
This study characterizes a 72-kDa heat-stable microtubule-associated protein (MAP) from porcine brain. The protein promotes microtubule polymerization and bundling, showing immunological relation to MAP2 and suggesting it is a MAP2C homologue.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Microtubule-associated proteins (MAPs) are crucial for microtubule dynamics and cellular structure.
- Heat-stable MAPs offer unique biochemical properties for purification and characterization.
- MAP2, a major MAP, plays significant roles in neuronal development and function.
Purpose of the Study:
- To characterize a 72-kDa heat-stable microtubule-associated protein (MAP) purified from porcine brain.
- To investigate the protein's structural and functional properties, including its interaction with microtubules.
- To determine the relationship of this 72-kDa MAP to known MAPs, particularly MAP2 and its isoforms.
Main Methods:
- Purification of the 72-kDa protein from porcine brain using heat stability in a low pH buffer.
- Low-angle rotary shadowing and quick-freeze, deep-etch electron microscopy for structural analysis.
- Microtubule polymerization assays with taxol and without taxol.
- Peptide mapping and immunological cross-reactivity assays using a monoclonal anti-MAP2 antibody.
Main Results:
- The 72-kDa protein is rodlike (55-75 nm) and promotes microtubule polymerization and bundling.
- It forms crossbridges between microtubules, influencing their organization.
- Peptide mapping and antibody cross-reactivity indicate a relationship to MAP2, suggesting it is an MAP2C homologue.
Conclusions:
- The 72-kDa heat-stable MAP from porcine brain exhibits structural and functional similarities to MAP2C.
- This protein plays a role in microtubule organization, promoting polymerization and bundle formation.
- The findings contribute to understanding the diversity and function of MAP2 family members in the brain.