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Purification and partial characterization of fibrillin, a cysteine-rich structural component of connective tissue
L Y Sakai1, D R Keene, R W Glanville
1Shriners Hospital for Crippled Children, Portland, Oregon 97201.
Abstract:
Fibrillin, a connective tissue macromolecule (Mr = 350,000) which is normally insoluble in its tissue form, has been purified from the medium of human skin fibroblast and ligament cells in culture. Analysis of the amino acid composition indicates that fibrillin contains approximately 14% cysteine, of which one-third appears to be in the free reactive sulfhydryl form. Electron microscopic images of fibrillin reveal an extended, flexible molecule approximately 148 nm long and 2.2 nm wide. These length measurements are consistent with shape calculations based upon velocity sedimentation data. It is likely that the material we have purified from cell culture medium represents monomeric fibrillin consisting of a single polypeptide chain. Additional ultrastructural immunohistochemical data presented here suggest a model for the parallel, head-to-tail alignment of fibrillin molecules in microfibrils.
Insights
Researchers purified fibrillin, a key connective tissue protein, from cell cultures. This study characterizes fibrillin
Area of Science:
- Biochemistry
- Cell Biology
- Connective Tissue Research
Background:
- Fibrillin is a large (Mr = 350,000) macromolecule crucial for connective tissue structure.
- In its native state, fibrillin is insoluble within the tissue.
- Understanding fibrillin's properties is vital for comprehending connective tissue disorders.
Purpose of the Study:
- To purify and characterize fibrillin from cultured human cells.
- To investigate the molecular properties and structural organization of fibrillin.
- To propose a model for fibrillin assembly into microfibrils.
Main Methods:
- Purification of fibrillin from the culture medium of human skin fibroblast and ligament cells.
- Amino acid composition analysis, focusing on cysteine content.
- Electron microscopy for molecular imaging and length determination.
- Velocity sedimentation analysis for shape calculations.
- Ultrastructural immunohistochemistry for molecular alignment studies.
Main Results:
- Fibrillin was successfully purified from cell culture medium, representing monomeric form.
- Amino acid analysis revealed approximately 14% cysteine, with one-third in sulfhydryl form.
- Electron microscopy showed extended, flexible molecules (approx. 148 nm length, 2.2 nm width).
- Sedimentation data supported the molecular shape and length.
- Immunohistochemical data suggested a parallel, head-to-tail alignment model for fibrillin in microfibrils.
Conclusions:
- The purified material is likely monomeric fibrillin, characterized by its unique amino acid profile and physical dimensions.
- The study provides insights into fibrillin's molecular structure and its role in forming microfibrils.
- A model for fibrillin's self-assembly into microfibrils was proposed based on ultrastructural evidence.