Estradiol-induced synthesis of vitellogenin. IV. The isolation of non-degraded polysomes from avian liver using an endogenous ribonuclease inhibitor

Abstract

A procedure allowing the isolation of intact polysomes from rooster liver is described. Good recovery of polysomes is achieved by the presence of Triton X-100 in the homogenization and centrifugation steps since the detergent prevents the sedimentation of microsomes with the nuclear fraction. This sedimentation of microsomes leads to considerable losses of polysomes, especially the larger ones. In the detergent-treated homogenate the integrity of the polysomes is threatened by various ribonucleases, some of which can be effectively inhibited by the addition of both heparin and yeast RNA. The remaining nuclease activity is counteracted by the endogenous ribonuclease inhibitor of the liver. In estradiol-treated roosters, sufficient endogenous inhibitor is present to inhibit its specific ribonuclease, but in control roosters there is not. This difference is due to a hormone-mediated increase in inhibitor level and decrease in nuclease level. Consequently, for an estrogenized rooster, the addition of both heparin and yeast RNA to the homogenate suffices to stabilize the polysomes, whereas control rooster liver homogenate needs supplementation with endogenous ribonuclease inhibitor. The cytosol of estrogenized rooster liver can be used as a crude inhibitor preparation. Rat liver cytosol is only partially effective; this may indicate a certain degree of species specificity of the inhibitor. The isolation procedure described also yields large polysomes from the livers of duck and Xenopus.