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Isolation of monkey aortic lysosomes using Percoll density gradient.
G S Dhaunsi1, S Majumdar, R N Chakravarti
1Department of Experimental Medicine, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Biochemistry International
|May 1, 1987
Summary
Researchers developed a new method using Percoll density gradients to isolate lysosomes from aortic tissue. This technique successfully purified lysosomes, confirmed by enzyme activity, ensuring minimal contamination for further cellular research.
Area of Science:
- Biochemistry
- Cell Biology
- Physiology
Background:
- Lysosomes are crucial cellular organelles involved in degradation.
- Accurate isolation of lysosomes is essential for studying their function.
- Existing methods may lack specificity or introduce contaminants.
Purpose of the Study:
- To develop and validate a novel technique for lysosome isolation from aortic tissue.
- To assess the purity and integrity of isolated lysosomes.
Main Methods:
- Utilized Percoll density gradient centrifugation.
- Employed 0.01M phosphate buffer for isolation.
- Assessed lysosome purity using marker enzymes: acid phosphatase and N-acetyl-beta-D-glucosaminidase.
- Evaluated latent activities of lysosomal hydrolases.
Main Results:
- A novel technique for lysosome isolation from aortic tissue was established.
- The heavier fraction (density 1.08) demonstrated high specific activities of lysosomal hydrolases.
- Isolated lysosomes exhibited marked latency of enzyme activity.
- The lysosome-rich fraction was confirmed to be free of subcellular contaminants.
Conclusions:
- The Percoll density gradient method provides an effective means for isolating pure lysosomes from aortic tissue.
- This technique enhances the reliability of lysosome-based biochemical and cellular studies.
- The isolated lysosomes are suitable for investigating lysosomal hydrolase activity and function.