Biosynthesis in vitro of tryptophanase by polyribosomes from induced cultures of Escherichia coli.

Abstract

1. Polyribosomes were isolated from Escherichia coli grown in media in which tryptophanase is induced and in which it is repressed. The polyribosomes from the induced bacteria had a small amount of tryptophanase activity associated with them. 2. A portion of the enzyme activity remained bound to polyribosomes during centrifuging in sucrose gradients. 3. Incubation of tryptophanase-containing polyribosomes with puromycin released enzyme activity. 4. The binding of the enzyme to the polyribosomes did not depend on the presence of DNA. 5. When the polyribosomes were incubated under conditions of protein synthesis with supernatant fraction obtained from repressed bacteria, a small but statistically significant increase in enzyme activity was produced. 6. When a radioactive amino acid was included in the incubation mixture for the tryptophanase system a radioactive protein was obtained whose chromatographic, electrophoretic and sedimentation properties were identical with those of tryptophanase. 7. The amount of incorporation was consistent with the amount of new enzyme synthesis predicted by the increase in enzyme activity. Both radioactive incorporation and increase in enzyme activity were shown to be energy-dependent and also negative controls were obtained by using zero-time incubations or polyribosomes isolated from either repressed cells or a mutant lacking the ability to produce tryptophanase. 8. The distribution of radioactive leucine in the carboxyl region of the newly labelled tryptophanase was examined by digesting the labelled protein with carboxypeptidases. It was shown that the radioactivity was more highly concentrated towards the carboxyl terminus when the incubation times for protein synthesis were shorter (implying that, with longer incubation times, longer lengths of polypeptide chain contained radioactive amino acid residues).