QUANTITATIVE ISOLATION AND PROPERTIES OF NEARLY HOMOGENEOUS POPULATIONS OF UNDEGRADED FREE AND BOUND POLYSOMES FROM RAT BRAIN¹

Abstract

Abstract— A procedure is described for the preparation of free and bound polysomes from whole homogenate of rat brain tissue. Brain is homogenized in a sucrose‐polysome buffer medium high in KCl (250 mm). After a 12‐min centrifugation at 135,000 g, the free polysomes in the supernatant are decanted and saved, while the membrane bound polysomes in the pellet are resuspended in homogenizing medium, homogenized in the presence of detergent (Triton X‐100), centrifuged for 5min at 1470 g to remove nuclei, decanted, treated with deoxycholate and centrifuged for 10 min at 24,000 g to remove deoxycholate‐insoluble material. Polysomes in the two supernatants are harvested by centrifugation through sucrose gradients prepared in high KCl polysome buffer, and with or without cell sap. Free and bound polysomes prepared in this manner are undegraded, equally active in cell‐free protein synthesis, and largely free of the usual contaminants. Cross‐contamination is minimal (>10%). The recovery of polysomes is at least 95%. The distribution of ribosomes and polysomes in rat brain is 58% free and 42% membrane‐bound. The distribution of rat brain RNA is 65% ribosomal and 35% non‐ribosomal. Conditions are described for the visualization and analysis of the entire complement of free and bound ribosomes. The size fractionation procedure is rapid and reproducible, requires much less ultracentrifugation than the density‐gradient technique, and provides a nearly quantitative means of isolating undegraded free and bound polysomes of rat brain tissue.

Biochemical evidence for the association of fragile X mental retardation protein with brain polyribosomal ribonucleoparticles

“…In view of these results, we examined whether FMRP was associated with polyribosomal mRNPs or was part of different RNP complexes in brain. Because Ϸ40% of the polyribosomes are bound to membranes in rodent brain (39), the choice of the detergent was critical to release polyribosomes from these structures. Cytoplasmic extracts prepared in the presence of Nonidet P-40, a nonionic detergent, were analyzed by velocity sedimentation through linear sucrose density gradients.…”

“…We also tested a different procedure for the preparation of free and bound polyribosomes in the presence of a combination of nonionic and ionic detergents (1% Triton X-100 plus 1% DOC) that has previously been used for rat brain tissue (39). This approach allowed us to successfully isolate polyribosomes; however, under these conditions, FMRP could not be detected at the level of polyribosomes, but was instead present in the upper part of the sucrose gradients (data not shown).…”

In Vitro Synthesis of the Four Mouse Myelin Basic Proteins: Evidence for the Lack of a Metabolic Relationship

“…Twelve milliliters of this supernatant were layered over a discontinuous sucrose density gradient consisting of 5 ml each of 0.7 M sucrose over 2.0 M sucrose in the homogenization buffer and was centrifuged at 214,000 x g for 4.5 h. The free polysome pellet was rinsed several times with buffer A and stored as a pellet at -80°C. (b) For the polysome size distribution studies and for the isolation of mRNA, free polysomes were isolated by a modification of the procedure of Ramsey and Steele (1977). Mouse brains were homogenized in 9 volumes of 0.25 M sucrose in H buffer [50 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), pH 7.4, 250 mM potassium chloride, 5 mM magnesium acetate, 100 p d m l of heparin], and then centrifuged for 2 min at 720 X g and for 13 rnin at 122,000 x g, as described by Rarnsey and Steele (1977).…”

“…Freshly prepared free polyribosomes were separated into different size classes by centrifugation at 122,000 x g for 110 min on a 15-27% sucrose density gradient prepared in 10 mM HEPES, pH 7.4, 75 mM KC1, 5 mM MgC12, 0.5 mM EDTA, and 40 p d m l of heparin (Ramsey and Steele, 1977). Fractions corresponding to appropriate ribosomal peaks were pooled and diluted with the same buffer without sucrose and centrifuged at 214,000 X g for 4.5 h to recover the ribosomal aggregates.…”

Synthesis of S100 Protein on Free and Membrane‐Bound Polysomes of the Rabbit Brain

“…Free and membrane-bound polysomes were isolated from the cerebral hemispheres and Cerebellum of young adult New Zealand white rabbits (1.5-1.8 kg body weight) according to the procedure of Ramsey and Steele (1977), with the following modifications. Cerebellum or cerebral hemispheres were homogenized in 7 volumes of homogenization buffer (TKMD: 0.25 M sucrose, 50 mM Tris-HCI, pH 7.4, 250 mM KCI, 5 mM MgClp, and 2 mM dithiothreitol) with six passes of a motor-driven Teflon pestle (measured clearance 0.15 mm) followed by two additional passes with a tighter Teflon pestle (measured clearance 0.06 mm).…”