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At least 19 recordsLinked to original sources

PubMed1985

The mechanism of resistance to puromycin and to the puromycin-precursor O-demethyl-puromycin in Streptomyces alboniger.

Ribosomes from Streptomyces alboniger are sensitive in vitro to puromycin and, to a lesser extent, to the puromycin-precursor O-demethyl-puromycin. The puromycin-inactivating enzyme (puromycin N-acetyltransferase) from S. alboniger also N-acetylates O-demethyl-puromycin. This finding indicates that in certain antibiotic-producing organisms the antibiotic-inactivating enzymes may play a role in self-defence against toxic precursor molecules.

Acetylation↗

PubMed1973

Effect of puromycin analogues and other agents on peptidyl-puromycin synthesis on polyribosomes.

The incorporation of [(3)H]puromycin into nascent polypeptide chains of polyribosomes has proved to be a sensitive method of evaluating effects of inhibitors on peptide bond synthesis. Several analogues of puromycin were found to react with polyribosomes from both bacteria and rat liver. The K(m) for puromycin is 4 muM with bacterial polyribosomes; under the same conditions, the K(i) for psi-hydroxy-puromycin (6-dimethylamino-9-[3-(l-beta-phenyllactylamino)-3-deoxy-beta- d-ribofuranosyl] purine) is 240 muM and for a carbocyclic analogue of puromycin (6-dimethylamino-9- {R- [2R-hydroxy-3R- (p-methoxyphenyl-l-alanylamino)]-cyclopentyl}purine) is 1 muM. Both were found to be competitive inhibitors of puromycin. The K(m) for C-A-C-C-A(Phe) is 250 muM. In addition, the dissociation constant for C-A-C-C-A(Phe) binding to washed ribosomes was found to be 1 and 0.03 muM in the absence and presence, respectively, of 20% (vol/vol) ethanol. The results with these analogues lead to the following conclusions. Substitution of a hydroxyl group for the alpha-amino group of puromycin results in an active analogue with about one-sixtieth the affinity of puromycin in the reaction. Omission of the 5'-hydroxymethyl group or substitution of the furanosyl ring oxygen by a carbon atom in the carbocyclic analogue reduces its activity compared with puromycin only slightly. Additionally, the relatively high K(m) for C-A-C-C-A(Phe) as an acceptor compared with puromycin suggests the existence of a protective mechanism on polyribosomes, which prevents aminoacyl-transfer ribonucleic acid (tRNA) free in solution from stripping nascent chains from polyribosomes so that only aminoacyl-tRNA bound to ribosomes through the appropriate coding mechanism can form a peptide bond.

Animals↗

PubMed1983

Puromycin binding to the small subunit of Escherichia coli ribosomes. Localization of the antibiotic in subunits reconstituted with puromycin-modified components.

Small (30 S) ribosomal subunits from Escherichia coli strain TPR 201 were photoaffinity-labeled with [3H]puromycin in the presence of chloramphenicol under conditions in which more than 1 mol of antibiotic was incorporated per mol of ribosomes. The subunits were than washed with 3 M NH4Cl to yield core particles and a split protein fraction; the split proteins were further fractionated with ammonium sulfate. Subunits were then reconstituted using one fraction (core, split proteins, or ammonium sulfate supernatant) from photoaffinity-modified subunits and other components from unmodified (control) subunits. The distribution of [3H]puromycin in ribosomal proteins was monitored by one-dimensional polyacrylamide gel electrophoresis, and the sites of puromycin binding were visualized by immunoelectron microscopy. Two areas of puromycin binding were identified. A high affinity puromycin site, found on the upper third of the subunit and distant from the platform, is identical to the primary site previously identified (Olson, H. M., Grant, P. G., Glitz, D. G., and Cooperman, B. S. (1980) Proc. Natl. Acad. Sci. U. S. A. 77, 890-894). Binding at this site is maximal in subunits reconstituted with high levels of puromycin-modified protein S14, and is decreased when unmodified S14 is incorporated. Because the percentage of antibody binding at the primary site always exceeds the percentage of puromycin label in protein S14, the primary site must include components other than S14. A secondary puromycin site of lower affinity is found on the subunit platform. This site is enriched in subunits reconstituted from puromycin-modified core particles and may include protein S7. Our results demonstrate the feasibility of localizing specifically modified components in reconstituted ribosomal subunits.

Electrophoresis, Polyacrylamide Gel↗

PubMed1985

Biosynthesis of puromycin by Streptomyces alboniger: characterization of puromycin N-acetyltransferase.

Puromycin N-acetyltransferase from Streptomyces alboniger inactivates puromycin by acetylating the amino position of its tyrosinyl moiety. This enzyme has been partially purified by column chromatography through DEAE-cellulose and Affigel Blue and characterized. It has an Mr of 23 000, as determined by gel filtration. In addition to puromycin, the enzyme N-acetylates O-demethylpuromycin, a toxic precursor of the antibiotic, and chryscandin, a puromycin analogue antibiotic. The Km values for puromycin and O-demethylpuromycin are 1.7 and 4.6 microM, respectively. The O-demethylpuromycin O-methyltransferase from S. alboniger, which apparently catalyzes the last step in the biosynthesis of puromycin [Rao, M. M., Rebello, P. F., & Pogell, B. M. (1969) J. Biol. Chem. 244, 112-118], also O-methylates N-acetyl-O-demethylpuromycin. The Km values of the methylating enzyme for O-demethylpuromycin and N-acetyl-O-demethylpuromycin are 260 and 2.3 microM, respectively. These findings suggest that O-demethylpuromycin, if present in S. alboniger, would be N-acetylated and then O-methylated to be converted into N-acetylpuromycin. It might even be possible that N-acetylation of the puromycin backbone takes place at an earlier precursor.

Acetylation↗

PubMed1985

Isolation and properties of a puromycin acetyltransferase from puromycin-producing Streptomyces alboniger.

Puromycin 2"-N-acetyltransferase was isolated from cell extracts of puromycin-producing Streptomyces alboniger KCC S-0309 by ammonium sulfate fractionation, heat treatment to eliminate contaminant proteins and chromatography on DEAE-Toyopearl 650S. After PAGE (polyacrylamide gel electrophoresis) of the final fraction, a single protein band corresponding to puromycin 2"-N-acetyltransferase was detected. The molecular weight of the enzyme determined by SDS-PAGE and Sephadex G-150 chromatography was about 21,000 and 85,000, respectively, suggesting that the enzyme consisted of four subunits. The isoelectric point and the optimum pH for reaction were 6.2 and 7.7, respectively. The Km values for puromycin and acetyl coenzyme A were 40 microM and 67 microM, respectively. The enzyme was thermostable up to 70 degrees C for 12 minutes. It was shown, by using an in vitro protein synthesizing system from a puromycin-susceptible organism S. flavotricini subsp. pseudochromogenes V-13-1, that the isolated puromycin 2"-N-acetyltransferase could protect polyphenylalanine synthesis from inhibition by puromycin.

Acetyltransferases↗

PubMed1975

Photoincorporation of puromycin and N-(ethyl-2-diazomalonyl)puromycin into Escherichia coli ribosomes.

[3H]Puromycin and N-(ethyl-2-diazomalonyl)[3H]puromycin are incorporated into E. coli ribosomes on irradiation at 253.7 nm. Both compounds incorporate into both protein and nucleic acid. Two-dimensional gel electrophoresis of ribosomal protein shows that L23 is the major protein labeled by puromycin. Although incorporation is clearly a complex process, evidence is presented that L23 is labeled via an affinity labeling process, thus placing L23 at the aminoacyl-tRNA receptor (A) site. N-(ethyl-2-diazomalonyl)puromycin is a ribosomal ligand, as shown by its inhibition of two ribosomal assays, but it is not a good puromycin analog, and it is unclear whether its incorporation, which proceeds via both carbene-dependent and carbene-independent processes, results from affinity labeling.

Azo Compounds↗

PubMed1981

Inhibition of aminopeptidase and acetylcholinesterase by puromycin and puromycin analogs.

Puromycin analogs in which the o-methyl-L-tyrosine moiety was substituted by a number of amino acids were examined as inhibitors of the puromycin-sensitive rat brain aminopeptidase and bovine erythrocyte acetylcholinesterase. In the case of the aminopeptidase, the structure and stereochemistry of the amino acid substituent were important factors in determining inhibitor effectiveness. In the case of the acetylcholinesterase reaction, the aminonucleoside of puromycin was nearly as effective an inhibitor as puromycin itself, with little effect dependent of the nature or stereochemistry of the amino acid.

Aminopeptidases↗

PubMed1971

The rate-limiting step of protein synthesis in vivo and in vitro and the distribution of growing peptides between the puromycin-labile and puromycin-non-labile sites on polyribosomes.

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0 degrees C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.

Alloxan↗

PubMed1981

Puromycin analogues. Effect of aryl-substituted puromycin analogues on the ribosomal peptidyltransferase reaction.

A series of ortho- and para-substituted L-phenylalanylpuromycin analogues were synthesized and evaluated as substrates for the peptidyltransferase reaction of Escherichia coli ribosomes. Kinetic results reveal that substitution of the p-methoxy group of the puromycin molecule alters the peptidyltransferase activity of the molecule with the following decreasing order of substrate efficiencies: p-NH2 greater than p-NHCOCH3 greater than p-NO2 = p-NHCO(CH2)2CH3 greater than p-NHCOCH2Br. However, the inability of the ribosome to tolerate a nitro group at the ortho position of the phenylalanine ring precluded the use of the photosensitive puromycin analogue, 2-nitro-4-azidophenylalanylpuromycin aminonucleoside (7a), as a photoaffinity label for the peptidyltransferase site.

Acyltransferases↗

PubMed1975