File(s) under permanent embargo
Reason: Restricted by author. A copy can be supplied under Section 51(2) of the Australian Copyright Act 1968 by submitting a document delivery request through your library or by emailing firstname.lastname@example.org
Some characteristics of the mammalian mitochondrial protein synthesising system
thesisposted on 08.02.2017, 03:49 by Towers, Neale R.
Phylogenetic differences between the mitochondrial protein synthesising systems of yeast and that of higher organisms have been studied using as probes a variety of antibiotics known to inhibit bacterial protein synthesis. While most of the experiments were performed on mammalian mitochondria (rat liver, kidney, heart and brain) some characteristics of trout liver, chick brain, potato, sweet potato and corn root mitochondria were also investigated. It was confirmed that intact and sonicated mitochondria from several rat tissues all had a restricted antibiotic sensitivity spectrum in comparison to that of yeast mitochondria, being resistant to the 50S acting antibiotics erythromycin, oleandomycin, vernamycin B and virideogrisein and to paromomycin, neomycin, spectinomycin and kasugamycin, which are 30S binding antibiotics. Protein synthesis by rat liver mitochondria was found to be inhibited by chloramphenicol, carbomycin, spiramycin, mikamycin complex, vernamycin A, thiostrepton and sparsomycin - all of which inhibit reactions of the large subunit of bacterial ribosomes - and also by pactamycin, which affects the smaller subunit. In view of a report which appeared early in the course of these experiments that the phylogenetic difference in sensitivity to erythromycin and lincomycin results from a selective permeability barrier to these antibiotics in rat liver mitochondria, an extensive study was made of the effects of erythromycin, lincomycin and paromomycin under a variety of experimental conditions designed to overcome any permeability barrier. This included an intensive study of the access of antibiotics to the mitochondrial matrix based on the observation that high concentrations of paromomycin inhibited respiration by fragmented mitochondria but not that by intact mitochondria. When the effects of the three antibiotics on mitochondrial protein synthesis were measured under a variety of conditions (several different hypotonic swelling and digitonin treatment procedures, including conditions under which paromomycin at least could be demonstrated to have access to the mitochondrial matrix) none of the antibiotics inhibited 14C-leucine incorporation when the antibiotic concentration was less than 250 ~g/ml and when incorporation depended on exogenously supplied ATP. Definitive proof of the access of erythromycin and lincomycin to the mitochondrial ribosome under conditions of protein synthesis was achived by showing that these two antibiotics competed with other antibiotics for binding sites on the mitochondrial ribosome. Both erythromycin and lincomycin reduced the level of inhibition of 14c-leucine incorporation obtained with any given concentration of either carbomycin or vernamycin A without themselves inhibiting protein synthesis, demonstrating that erythromycin and lincomycin can bind to the mammalian mitochondrial ribosome in a manner which excludes simultaneous binding of carbomycin or vernamycin A. The binding of erythromycin and carbomycin was competitive and reversible, depending on the relative concentrations of the two drugs but being independent of the order in which they were added. Extension of the antibiotic sensitivity studies to other than mammalian mitochondria showed that protein synthesis by mitochondria from a variety of higher organisms was inhibited by a similarly restricted range of antibiotics. There is thus a very clear and strong case for the existence of phylogenetic differences between the mitochondria of higher organisms and the lower eukaryotes (as represented by yeast). Another aspect of mitochondrial function studied was the ability of the membrane to influence the activity of the ribosome. The possibility of ribosome-membrane interactions in mitochondria and the physical association of the mitochondrial ribosome with the membrane was raised by investigators studying mitochondriogenesis in this laboratory. Evidence identifying the majority of mitochondrial ribosomes as being membrane-bound has been obtained by studying the effects of temperature on the kinetics of 14c-leucine incorporation. Initial studies using cytoplasmic ribosomes as a model system showed that the kinetics of protein synthesis by membrane-bound ribosomes (rough endoplasmic reticulum) gave non-linear Arrhenius plots, with a marked discontinuity at a characteristic temperature (22°), below which a second and higher Arrhenius activation energy was found. This is not a characteristic of all ribosomes, as free ribosomes obeyed the predictions of the Arrhenius equation and gave a simple linear plot. The importance of the membrane-ribosome association in the genesis of the discontinuity was demonstrated in "reconstitution" experiments. Studies on the effects of temperature on the kinetics of mitochondrial protein synthesis revealed Arrhenius plots similar to those for rough endoplasmic reticulum, with a discontinuity at a characteristic temperature for a poikilotherm (rat liver:23°) and for temperature sensitive plants (sweet potato, corn: 10 - 13°) but not for the temperature insensitive potato. These results correlate with the nature of the membrane lipids, and it is therefore concluded that the mitochondrial ribosome is in association with the inner mitochondrial membrane in a manner analogous to the association of the ribosomes and membranes of the rough endoplasmic reticulum.