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Polyribosomes from Peas

III. Stimulation of Polysome Degradation by Exogenous and Endogenous Calcium ¹

^a Department of Botany, University of Nebraska, Lincoln, Nebraska 68508

Attempts were made to isolate microsomes from Pisum sativum L. var. Alaska by low speed centrifugation of a postmitochondrial supernatant made 8 mM in Ca²⁺. However, the addition of Ca²⁺ in concentrations as low as 1 mM to the postmitochondrial supernatant resulted in extensive polysome degradation. Degradation was dependent on both Ca²⁺ concentration and the duration of incubation. Resuspension of isolated polysomes in Ca²⁺-containing buffer did not result in degradation, whereas resuspension in Ca²⁺-containing postpolysomal supernatant did. Both Ca²⁺ and a heat-labile factor in the supernatant were required for polysome degradation. The degradation in the homogenate with or without added Ca²⁺ could be reduced by (a) dilution with larger volumes of grinding buffer, (b) increasing the concentration of tris-HCl in the grinding buffer, (c) adding diethylpyrocarbonate or ethyleneglycol-bis (2-aminoethylether) tetraacetic acid (a specific calcium chelator) prior to homogenization or immediately after the addition of Ca²⁺. Endogenous Ca²⁺ can increase the destruction of polysomes during their isolation in this tissue, presumably by activating a ribonuclease. Addition of Ca²⁺ is not a useful technique for separating undegraded free and membrane-bound polyribosomes.

¹ This research was supported in part by a Biomedical Sciences Support Grant RT-07055 through the University of Nebraska Research Council and a National Science Foundation Traineeship to B.A.L.