Radioactive amino acids were incorporated into isolated mitochondria from Neurospora crassa. Then the mitochondrial ribosomes were isolated and submitted to density gradient centrifugation. A preferential labelling of polysomes was observed. However, when the mitochondrial suspension was treated with puromycin after amino acid incorporation, no radioactivity could be detected in either the monosomes or the polysomes. The conclusion is drawn that isolated mitochondria under these conditions do not incorporate significant amounts of amino acids into proteins of their ribosomes.Mitochondrial ribosomes from Neurospora crassa have been isolated and characterized by Kuntzel and No11 [I] and by Rifkin et al. [2]. Radioactivity has been found in these ribosomes after labelling of isolated mitochondria by incorporation of [l4CC]-leucine [l]. However, no distinction was made between labelling of the proteins of the ribosomes and the peptide chains being actually synthesized. Puromycin, which is known to release the growing peptide chain from the ribosome, was used to examine this question. MATERIALS AND METHODSHyphae of Neurospora crassa (wild type 74A) were grown for 18 h on a reciprocal shaker from an inoculum of 2 x 106 conidia/ml in 50 ml portions of Vogel's minimal medium supplemented with 2 Olio sucrose. They were harvested by filtration.Mitochondria were prepared by a slight modification of the method described by Luck [3]. Cells were mixed with 3 volumes (with respect to wet weight) of isolation fluid (0.44 M sucrose, 10 mM Tris, 2 mM EDTA, p H 7.2) and ground with sand (0.7 g per g of wet weight) for 8 min. The suspension, diluted with 6 additional volumes of isolation fluid, was passed through a filter cloth. The residue was ground again under the same conditions for 5min. For isolation of mitochondria the combined filtrates were successively centrifuged in the following way : 10 rnin at 1500 xg, the resulting supernatant 30 rnin a t 11 000 x g, the resuspended pellet 10 min a t 550 x g, the resulting supernatant 20 min a t 17000 xg, and the resulting supernatant again 20 rnin a t 17000 x g.Unusual Abbreviation. ATeo unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm when measured in a 1 cm path length cell. One portion served as a control. It was immediately cooled to 0". To the other portion puromycin hydrochloride (Nutritional Biochemicals) was added to a final concentration of 0.4 mM. The incubation was continued for additional 7 min. This part of the suspension was then also cooled to 0". From this point on control and puromycin treated mitochondria were handled in the same way. 3 volumes of isolation fluid containing 5 mM each of unlabelled L-leucine, L-isoleucine and L-phenylalanine were added. The mitochondria were centrifuged for 20 rnin a t I7 000 x g.The pellet was resuspended in isolation fluid containing amino acids. The suspension was centrifuged again for 20 min a t 17 000 x g.For further purification mitochondria were resuspended in isolation fl...
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