Polyribosomes from Peas

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Abstract
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.