Cosedimentation of pea root polysomes with the cytoskeleton

Area of Science:

• Plant molecular biology
• Cell biology
• Biochemistry

Background:

• Polysome isolation is crucial for studying protein synthesis.
• Conventional methods using high ionic strength buffers yield low polysome recovery from pea roots.
• Endogenous RNase activity poses a significant challenge during polysome extraction.

Purpose of the Study:

• To optimize polysome isolation from pea roots.
• To identify buffer conditions that prevent RNase degradation while maintaining polysome integrity and yield.
• To investigate the role of the cytoskeleton in polysome retention.

Main Methods:

• Comparison of high and low ionic strength buffers for polysome isolation.
• Testing various RNase inhibitors and buffer components (e.g., Tris-HCl, heparin, KCl).
• Cosedimentation assays using radiolabeled polysomes to assess non-specific binding.

Main Results:

• High ionic strength buffers resulted in ~10% polysome retention and no RNase degradation.
• Low ionic strength buffers increased retention to 60% but caused severe polysome degradation.
• Tris-HCl buffer at 15-25 mM prevented RNase degradation without polysome release.
• Cosedimentation experiments ruled out artifactual adsorption or trapping of polysomes to the cytoskeleton.

Conclusions:

• Tris-HCl buffer in the 15-25 mM range is optimal for pea root polysome isolation.
• The study demonstrates that polysome association with the cytoskeleton is specific and not due to non-specific binding.
• Optimized conditions enhance the study of plant translation machinery.