Mammalian mitochondrial ribosomes from rat liver synthesised poly(phenyla1anine) from ['"CIPhe-tRNA in the presence of a homologous lo5 x g,, supernatent fraction. The activity depended on the addition of synthetic template and was resistant to cycloheximide. The polyanion spermidine had a stimulatory effect on peptide synthesis in vitro. In contrast to Escherichiu coli ribosomes, which also functioned with heterologous supernatant fractions, 55-S mitochondrial ribosomes were inactive when supplemented with heterologous supernatant fractions from E. coli or with purified bacterial elongation factors. EF-T slightly stimulated polyphenylalanine synthesis when added in combination with mitochondrial supernatant fractions. Two-dimensional electrophoretic analysis of the protein content of both supernatant fractions revealed considerable differences in the distribution of the species-specific proteins according to their isoelectric points. The mitochondrial superand the few acidic proteins did not co-migrate with natant proteins were in general more basic EF-Tu or EF-G from E. coli.Mammalian mitochondrial ribosomes represent a special group among organelle ribosomes. They display an unusually high protein content [l -31 and evolved fairly rapidly compared with cytoplasmic ribosomes [4]. Like all other mitochondrial ribosomes they contain binding sites for inhibitors of bacterial protein synthesis while being unaffected by antibiotics such as cycloheximide and anisomycin which interfere with eucaryotic ribosome function. Although differences have been observed between ribosomes from bacteria and mammalian mitochondria in the interaction with antibiotics binding to the ribosomal P-site, 55-S ribosomes can be classed as procaryotic by functional parameters [5].The degree of similarity between bacterial and mitochondrial mechanisms of protein synthesis can be expressed by the interchangeability of protein synthesis factors, ribosomal proteins, or even ribosomal subunits. In the bacterial system mitochondrial peptide chain elongation factors from yeast and Neurosporu were successfully substituted in place of Escherichiu coli factors [6,7], but the reverse experiment appeared to be limited by the low activity of isolated mitochondrial ribosomes in protein synthesis in vitro. Isolation of active mitochondrial ribosomes free of endogenous elongation factors therefore was a prerequisite to study the specificity of bacterial and mitochondrial peptide chain elongation factors. The present investigation reports on the ability of supernatant fractions from rat liver mitochondria and from E. coli to stimulate peptide synthesis in homologous and heterologous systems. The protein composition of both 1 O5 x g,, supernatant fractions was compared by two-dimensional gel electrophoresis applying isoelectric focusing in the first dimension.
MATERIALS AND METHODS
MateriulsRadioactive amino acids and [3H]poly(U) were obtained from Amersham, Great Britain. A11 reagents were of analytical grade. Solutions were prepared in sterilized glasswar...