An isolated ribosome preparation from cardiac muscle which retains effects of insulin pretreatment [proceedings].

Protein synthesis is very active in cardiac muscle, and the isolated perfused rat heart has been shown to be sensitive to hormone administration (Morgan et al., 1971; Mowbray er al., 1975). We describe here a preparation of purified ribosomes in improved yield and in which the effects of insulin pretreatment have been preserved. Preparations of Flyribosomes from muscle generally involve attempts to solubilize membrane-bound polyribosomes by addition of detergents to postmitochondrial supernatants (Earl & Morgan, 1968; Manchester, 1974). However, there is evidence that up to 20% of rat liver cellular RNA may be lost in the nuclear pellet (Blobel & Potter, 1967; Lewis & Tata, 1973). Hearts were therefore minced with scissors, then homogenized in 9ml of ice-cold buffer A [Tris/HCl, pH7.4, 2 0 m ~ ; KCI, 2 5 0 m ~ ; MgCI,, 2 m ~ ; glycerol, 10% (v/v); dithiothreitol, 1 m ~ ; Triton X-100, 1% (w/v)l in a Virtis 45 instrument (3 x lOs, speed 8; 1 x 30s, speed 41, with cooling between each homogenization. Washings (1 ml of buffer A) from the vessel were added to the homogenate, which was centrifuged at 8000g for lOmin at 4OC. After incubation on ice for l h the supernatant was clarified, if necessary, by a short re-centrifugation. divided into two portions, and each was layered over 3ml of 1.0M-sucrose in 20m~-Tris/HCI (pH 7.4)/150m~KC1/2m~-MgCl,. After centrifugation at l05000g for 2 h at 4OC, the supernatant was removed and the ribosome pellets were washed twice with. buffer B (Tris/HCl, pH7.4, 50mM; KCI, 100mM; MgCl,, 2 m ~ ; dithiothreitol, 1 mM; sucrose, 250m~) . Each pellet was carefully resuspended in 1 5 0 ~ 1 of buffer B and the ribosome solutions were pooled. Non-ionic detergents such as Triton X-100 are generally preferable to ionic detergents such as sodium deoxycholate, as deoxycholate may cause disaggregation of polyribosomes (Olsnes et al., 1972). A K+ concentration of 2 5 0 m ~ is recommended (Heywood er al., 1967) to avoid co-precipitation of polyribosomes with myosin. The total RNA content of these hearts, measured aAer extraction (Munro & Fleck, 1969), was 1.39 f 0.06 1 (n = 11) mg/g wet wt. of tissue (C. M. Mackie, unpublished work) and the yield of ribosomes, measured by an ethidium bromide fluorescence assay (Mackie er al., 1979), ranged between 32.2 and 63.4% (mean 45 t 4 % ; n = 8). This appears to be a substantial improvement on the preparation of Earl & Morgan (1968). When buffer A (without Triton X-100, but with 5 mM-MgCI,) was used, and the postmitochondrial supernatant made 1% with respect to the detergent Lubrol WX before the incubation on ice, the mean yield was 29f7% (n= 5) . Homogenization in the presence of Triton X-100 gave a significantly better yield (P < 0.005). In addition, although density-gradient profiles of the ribosomes were indistinguishable, Triton-prepared ribosomes were more active at amino acid incorporation in oitro than Lubrol-prepared ones (results not shown). It may be that this preparation does not provide a representative sample of cell polyribosomes. However, perfusion in the absence of hormones increased the proportion of subunits, monomers and dimers within 30min (results not shown). Moreover, perfusion with insulin (4jig/ml) reversed this effect, decreasing the percentage of these ‘free’ particles (see Fig. 1) by 18.5 +2.6% (paired t test, P<O.005; n = 4: