Molecular Pharmacology, Vol 8, 111-127, Copyright © 1972 by the American Society for Pharmacology and Experimental Therapeutics

Effects of Aurintricarboxylic Acid on Ribosomes and the Biosynthesis of Globin in Rabbit Reticulocytes

¹ Departments of Pharmacology, Medicine and the Division of Biology, Department of Molecular Biology, Albert Einstein College of Medicine, Bronx, New York 10461

The triphenylmethane dye, aurintricarboxylic acid (ATA), inhibits the synthesis of globin in reticulocyte lysates. The principal effect of low concentrations of ATA (0.01-0.1 mM) is on initiation of protein synthesis. This conclusion is based on the following observations: (a) a delay of 1-2 min occurs before inhibition of globin synthesis by ATA is observed; (b) complete breakdown of polyribosomes is induced by ATA; (c) peptide chains, previously identified as completed and chains of globin, are completed and released from the ribosome in the presence of the dye; and (d) an increase in the number of ribosomal subunits is observed in the presence of the dye.

ATA inhibits binding of polyuridylic and polycytidylic acids to reticulocyte ribosomes. This observation is in accordance with the view that a primary action of the dye is the inhibition of attachment of mRNA to ribosomes. Translocation, peptide bond formation, and rate of release of nascent globin peptides are unaffected by 0.1 mM concentrations of ATA. The activity of guanosine triphosphatase is inhibited, but only when the enzyme is assayed as an isolated preparation. If ATA is incubated with guanosine triphosphatase in the presence of all components required for amino acid polymerization, hydrolysis of GTP is not affected.

High concentrations of ATA (1 mM) prevent chain elongation. Under these conditions, breakdown of polyribosomes is incomplete and single ribosomes unfold, forming discrete particles sedimenting at 54-56 S. Such unfolding can be prevented by increasing the concentration of magnesium ions. ATA also induces a decrease in the sedimentation rate of polyribosomes, single ribosomes, and the smaller ribosomal subunit.

[³H]ATA binds to polyribosomes and 80 S and 40 S particles, but not to the 60 S subunit. Taken together, our results suggest that the dye binds to the 40 S ribosomal subunit, preventing the subsequent attachment of mRNA. As a consequence of this action, ATA inhibits initiation of globin synthesis in reticulocyte lysates.

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ACKNOWLEDGMENTS We are grateful to Dr.T. Hunt and M. L. Stewart-Blair for helpful discussions during the course of this work, and to Dr. R. Soffer for a critical reading of this manuscript.