Isolation of ribonuclease-free intact polyribosomes from rat kidney.
High levels of RNAase present in rat kidney have prevented isolation of intact polyribosomes from this tissue. This problem has been circumvented by a thorough in situ arterial perfusion of rat kidney, coupled with homogenization of the perfused rat kidney in heparin and detergents-fortified high-speed supernatant prepared from rat liver. This procedure reduced RNAase activity in the homogenate by as much as 70%. Sedimentation of the polyribosomes from this homogenate through a layer of 2.0 M sucrose resulted in a 78--80% yield of polyribosomes from the rat kidney. The resulting polyribosomal pellet contained less than 8% of the RNAase activity present in polyribosomes from non-perfused rat kidney. The remaining RNAase activity was separated from the larger polyribomes by sucrose density gradient centrifugation. The majority of the polyribosomes were larger than tetramers. This procedure also incrased both the yield and size of polyribosomes from rat and mouse liver.
D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient / Q000379:methods; D007668:Kidney / Q000201:enzymology / Q000648:ultrastructure*; D008099:Liver / Q000648:ultrastructure; D008297:Male; D010477:Perfusion; D012335:RNA, Ribosomal / Q000032:analysis; D051381:Rats; D012260:Ribonucleases / Q000302:isolation & purification*; D012270:Ribosomes / Q000648:ultrastructure*
D012335:RNA, Ribosomal; D012260:Ribonucleases