Abstract
Abstract The functional and structural properties of sodium deoxycholate-liberated bound and free ribosomes from rat spleen have been compared in vitro. The endogenous amino acid-incorporating activity of bound ribosomes relative to that of free ribosomes (B:F) varies only slightly (71 to 103%) under a variety of isolation and incubation conditions. However, the net exogenous activity for 14C-phenylalanine with the use of polyuridylic acid (poly U) as messenger varies widely (B:F, 11 to 75%) and is greatly dependent on experimental conditions. Minimal translation of poly U and the lowest B:F ratio are seen when the source of supernatant factors is a spleen pH 5 fraction. Maximal translation of poly U and the highest B:F ratio are achieved with a liver pH 5 fraction, isolation of ribosomes in the presence of a rat liver supernatant, the omission of centrifugation through 2 m sucrose, and the addition of a 0.5 m NH4Cl wash. Relative to the minimal system, incorporation is enhanced 54-fold for bound ribosomes and 8-fold for free ribosomes when these optimal conditions are used. Rat spleen bound and free ribosomes are immunologically identical, have similar sedimentation coefficients, similar contents of RNA and nucleotide bases, and virtually identical patterns of polyribosomes and structural and rapidly labeled RNAs. These results caution that different ribosome populations may seem more or less alike functionally, depending upon the particular conditions of isolation and incubation.
Highlights
Maximal translation of poly U and the highest B:F ratio are achieved with a liver pH 5 fraction, isolation of ribosomes in the presence of a rat liver supematant, the omission of centrifugation through 2 M sucrose, and the addition of a
Under cell-free conditions, we previously found that ribosomcs liberated from membranes with deoxycholate were more sensitive to the inhibitory action of chloramphenicol t.han treated free ribosomes [18]
The net counts per min incorporated are derived after subtraction of the counts per min incorporated without poly U
Summary
The functional and structural properties of sodium deoxycholate-liberated bound and free ribosomes from rat spleen have been compared in vitro. The endogenous amino acid-incorporating activity of bound ribosomes relative to that of free ribosomes (B:F) varies only slightly (71 to 103%) under a variety of isolation and incubation conditions. Maximal translation of poly U and the highest B:F ratio are achieved with a liver pH 5 fraction, isolation of ribosomes in the presence of a rat liver supematant, the omission of centrifugation through 2 M sucrose, and the addition of a. Relative to the minimal system, incorporation is enhanced 54-fold for bound ribosomes and 8-. Rat spleen homogenates contain membrane-bound and free ribosomes, and both arc active in protein synthrsis [17]. Further studies of the structural and functional properties of deoxycholate-liberated bound and free ribosomes of rat splcerl arc presented in this report
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