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Requirement for Extraction of Polyribosomes from Barley Tissue ¹

^a Departments of Station Biochemistry and Plant Science, South Dakota State University, Brookings, South Dakota 57006

The isolation of barley (Hordeum vulgare L.) polyribosomes, showing minimal degradation effects of endogenous RNase, required a buffer adjusted to pH 8.0 and containing 0.40 M KCl in addition to common extraction components. The extracted polyribosomes were characterized in sucrose gradients by their conversion to monosomes when incubated with pancreatic RNase and by their dependence on adequate amounts of Mg²⁺ during extraction and analysis. Factors which contributed to polyribosome stability were evaluated by the relative sedimentation rates of aggregates in sucrose gradients. Tissue extraction at KCl concentrations less than 0.40 M and below pH 8.0 resulted in an appearance of larger amounts of ribosomes in the less dense region of the sucrose gradient after centrifugation. The addition of 10 mM dithiothreitol was partially effective in preventing the loss of higher polymerized states of polyribosomes at KCl concentrations below 0.40 M. Extractions conducted at KCl concentrations greater than 0.40 M and at pH 8.0 reduced the amount of ribosomes obtained from the tissue. The monosome portion of the polyribosomal profile was partially dissociated into subunits when the tissue was extracted in 0.60 M KCl. A similar effect on monosomes was obtained when polyribosomes were incubated with cycloheximide and 0.40 M KCl, a result not observed by use of a combination of 0.10 M KCl and the drug or 0.40 M KCl alone.

¹ Approved for publication by the Director, Agricultural Experiment Station, South Dakota State University, Brookings, S. D. as Journal series No. 1039. This investigation is a portion of a thesis submitted to the Graduate School of South Dakota State University by the senior author in partial fulfillment of requirements for a M.S. degree.