Sedimentation Analyses of Polyribosomes
I. Band Sedimentation Analyses of Rat Liver Polyribosomes
1969 Volume 21 Issue 4 Pages 138-151
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1969 Volume 21 Issue 4 Pages 138-151
This manuscript reports a new method of band sedimentation analyses for rat liver polyribosomes.
(1) Don-Ryu rats,150-200g body weight, were fasted for 9-12 hr before use. Livers were homogenized in the two times volume (w/v) of 0.25M-Sucrose TKM buffer (0.25MSucrose containing 0.05M-Tris-HCl, pH 7.5 at 20°C; 0.025M-KCl; 0.005M-MgCl2). The postmitochondrial supernatant was prepared by centrifuging the homogenate for 15 min at 10,000g (average) at 0°C. DOC (deoxycholate) was added to the supernatant at a final concentration of 1%. Band sedimentation analyses were carrid out at 4°C, by a Spinco Model E analytical centrifuge, using a ultra-violet absorption optical system.
(2) When 0.5M-Sucrose TKM buffer was used as a solvent for the band se d imentation analyses, no sedimentation band was observed. When 1.0M-Sucrose TKM buffer was used, only a monomer (80S) band appeared. Perhaps in these cases, polysomes were spun down to the bottom of the band forming cell. When polysomes were analysed in 1.5M-Sucrose TKM buffer at a rotor speed of 50,740 rev. /min, several bands of polyribosomes appeared. When the sedimentation velocity was increased to 59,780 rev. /min or decreased to 39,460rev. /min, the polysomes bands decreased in number and became obscure. When 2.0MSucrose TKM buffer was used as a solvent and polysomes were centrifuged at 59,780rev. /min for 68 min, polysomes bands was not observed.
(3) From these results, it was concluded that the ba n d sedimentation at a rotor speed of 50,740 rev. /min in 1.5M-Sucrose TKM buffer is the best condition for the analyses of rat liver polyribosomes.
This band sedimentation method is superior to the sucrose density gradient method by the following reasons: In the first place, much smaller amount of sample (only 10μl) is needed for the analyses; in the second, it is possible to observe successively the intermediate state of sedimentation of the polyribosomes bands, and finally, it is possible to determine the blank level (UV absorption due to the Sucrose TKM buffer) rather precisely.
