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POLYSOMES OF THE SEA URCHIN EMBRYO: AN IMPROVED METHOD FOR EXTRACTION OF INTEGRATED POLYSOMES
Minoru Hirama1, Yoshitake Mano1
1Department of Biochemistry Faculty of Medicine University of Tokyo, Tokyo 113, Japan.
Abstract:
Suitable conditions for extracting integrated polysomes from embryos of the sea urchins, Hemicentrotus pulcherrimus and Pseudocentrotus depressus were investigated. Integrated polysomes could not be extracted under the conditions reported by other investigators. It was found, however, that use of 5 mm MgCl2 , 0.30 m KCl, 0.5 mm EDTA and 2 mm cycloheximide was effective for maintaining the integrity of polysomes. At higher concentrations of Mg2+ , and even at higher concentrations of K+ , monosomes and polysomes aggregated to form polysome-like particles which had sedimentation patterns with a small amount of nascent peptide. Thus, a medium consisting of 0.05 m Tris-HCl buffer, pH 7.5, 0.30 m KCl. 5 mm MgCl2 , 0.5 mm EDTA-2K, 2 mm cycloheximide, 5 mm mercaptoethanol and 0.5% (v/v) Nonidet P-40 is concluded to be the most suitable for extraction of sea urchin polysomes. Under the conditions used EDTA did not suppress polysome degradation completely and their degradation was linear with time.
Insights
Researchers optimized conditions for extracting intact polysomes from sea urchin embryos. The study identified specific concentrations of magnesium, potassium, and other agents crucial for preserving polysome integrity during extraction.
Area of Science:
- Molecular Biology
- Developmental Biology
- Marine Biology
Background:
- Polysomes are essential for protein synthesis.
- Extracting intact polysomes from sea urchin embryos presents challenges.
- Previous methods were insufficient for maintaining polysome integrity.
Purpose of the Study:
- To investigate optimal conditions for extracting integrated polysomes from sea urchin embryos.
- To identify key reagents and concentrations for preserving polysome structure.
- To overcome limitations of existing polysome extraction protocols.
Main Methods:
- Investigated various concentrations of MgCl2, KCl, EDTA, and cycloheximide.
- Utilized Tris-HCl buffer, mercaptoethanol, and Nonidet P-40.
- Assessed polysome integrity through sedimentation patterns and nascent peptide analysis.
Main Results:
- Established an effective extraction medium: 0.05 m Tris-HCl (pH 7.5), 0.30 m KCl, 5 mm MgCl2, 0.5 mm EDTA-2K, 2 mm cycloheximide, 5 mm mercaptoethanol, and 0.5% Nonidet P-40.
- Identified specific ion concentrations (MgCl2, KCl) critical for preventing polysome aggregation.
- Observed that EDTA did not completely inhibit polysome degradation, which occurred linearly over time.
Conclusions:
- The defined medium is optimal for extracting intact polysomes from Hemicentrotus pulcherrimus and Pseudocentrotus depressus embryos.
- Understanding ion effects is crucial for preventing artifactual polysome formation.
- Further research may be needed to fully inhibit polysome degradation during extraction.
