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Isolation and partial characterization of active polysomes from calcified and matrix-containing tissues
Archivos De Investigacion Medica
|January 1, 1981
Summary
This study presents a novel method for isolating active polysomes from challenging calcified tissues, crucial for understanding protein synthesis, particularly collagen production, in these tissues.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Calcified and matrix-rich tissues present unique challenges for polysome isolation.
- Understanding polysome activity is key to elucidating protein synthesis pathways.
Purpose of the Study:
- To develop and validate a method for isolating active polysomes from calcified and matrix-containing tissues.
- To characterize the protein synthetic activity of these isolated polysomes, with a focus on collagen synthesis.
Main Methods:
- Tissue swelling in hypotonic buffer with heparin and cycloheximide.
- Homogenization followed by differential centrifugation to separate polysomes.
- Rehomogenization with detergent and potassium ions, followed by sucrose cushion centrifugation.
Main Results:
- Isolated polysomes (total, non-bound, membrane-bound) demonstrated equal protein synthesizing activity in a cell-free system.
- Collagen synthesis was a major activity, evidenced by preferential incorporation of 3H-proline and collagenase digestion.
- The method successfully yielded active polysomes from difficult tissue sources.
Conclusions:
- The developed method effectively isolates active polysomes from calcified and matrix-containing tissues.
- This technique facilitates the study of protein synthesis, especially collagen, in these tissues.
- The findings provide a valuable tool for research in bone and cartilage biology.