Puromycin dihydrochloride

Puromycin dihydrochloride [1] is an analog of aminoacyl-tRNA. It inhibits the incorporation of aminoacyl-tRNA into the C-terminal on a synthesizing polypeptide, resulting in the premature termination of the polypeptide chain.

Puromycin is toxic to the growth of various eukaryote cells including mammalian cells. Concentrations of puromycin sufficient to inhibit the cell growth of mammalian cells range from 0.5-10 μg/ml. IC90s for puromycin to inhibit the growth of Plasmodium falciparum and Giardia lamblia are 60 ng/ml and 54 μg/ml, respectively [2]. Aminoacyl-tRNA is used as the material for elongation of a synthesizing polypeptide on a ribosome [2].

When treated with puromycin dihydrochloride at different concentrations, the growth rates of T. thermophila changed. In the first 24 h, puromycin dihydrochloride at a concentration of 50 µg/ml reduced the growth rate by 80%, but did not completely block the cell growth; until 72 h, there was a gradual cell number increase. At 100 μg/ml, puromycin dihydrochloride completely blocked the cell growth; in the first 48 h under this condition, almost all of the cells died, surviving cells grew rapidly after 48 h. Puromycin dihydrochloride at 150 μg/ml completely inhibited the cell growth for 72 h. By 72 h, the majority of cells died, and then surviving cells grew. Puromycin dihydrochloride at 200 μg/ml made almost all the cells die by 48 h, and hence no survivors appeared [2].

In animals of 25 days old, 180 or 120 min of previous exposure to puromycin dihydrochloride inhibited subsequent amino acid transport. In animals of 50 days old, however, puromycin dihydrochloride failed to inhibit α-aminoisobutyric acid uptake [3].

References:
[1].  Mi. Sundaralingami and S. K. Arora. Stereochemistry of nucleic acids and their constituents. IX. The conformation of the antibiotic puromycin dihydrochloride pentahydrate. Proc. N. A. S., 1969, 64(3):1021-6.
[2].  Masaaki Iwamoto, ChieMori, Yasushi Hiraoka, et al. Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena. Gene, 2014, 534:249–255.
[3].  Elsas L.J. II, MacDonell R.C. Jr. and Rosenberg L.E. Influence of age on insulin stimulation of amino acid uptake in rat diaphragm. J. Biol. Chem., 1971, 246(21):6452-6459.

Stereochemistry of nucleic acids and their constituents. IX. The conformation of the antibiotic puromycin dihydrochloride pentahydrate.[Pubmed:5264134]

Proc Natl Acad Sci U S A. 1969 Nov;64(3):1021-6.

The elucidation of puromycin structure represents the first case where both a nucleic acid and a protein fragment are found together in a crystal. The molecule displays an elongated conformation with the nucleoside and the p-methoxy-L-phenylalanine in the preferred conformation. The purine and the tyrosine rings form alternating stacks with interplanar spacings of 3.4 A. The inhibitory effect of this antiobiotic may be due, in part, to its unique conformation and the ability of the amide proton on N(3') to hydrogen bond to the active site of the ribosomal enzyme, peptide synthetase.

Puromycin inhibits protein import into mitochondria by interfering with an intramitochondrial ATP-dependent reaction.[Pubmed:8334141]

Biochim Biophys Acta. 1993 Jul 25;1150(1):89-97.

We have performed experiments which demonstrate that puromycin inhibits the import of proteins into mitochondria in in vitro reactions containing mitochondria isolated from the yeast Saccharomyces cerevisiae and precursor proteins synthesized in a nuclease-treated rabbit reticulocyte lysate. Puromycin inhibited the import of several precursor proteins including; a fusion protein consisting of the first 22 N-terminal residues of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase, both a destabilized and truncated form of this same fusion protein, the beta-subunit of the yeast mitochondrial F1-ATPase and yeast alcohol dehydrogenase III. The insertion of the yeast outer mitochondrial protein porin was not inhibited by puromycin. Puromycin-induced import inhibition could be overcome by adding additional ATP to the import reactions. However, if access of ATP to the mitochondrial matrix was prevented by blocking the adenine nucleotide translocase with carboxyatractyloside, ATP addition was unable to overcome the inhibitory effect of puromycin on protein import. Collectively, these results demonstrate that puromycin inhibits protein import into mitochondria by interfering with an ATP-dependent step in the import process and that the ATP-dependent component in the reaction is located inside the inner mitochondrial membrane. In addition to supporting the view that ATP is required in the matrix for efficient protein import, these results may provide a useful tool for identifying the ATP-binding components of the import apparatus.