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Pharmacology, Biochemistry, and Behavior Jul 1976The ability of antibiotics to inhibit acetylcholinesterase was measured in homogenates of goldfish brain. Puromycin aminonucleoside was the most potent inhibitor... (Comparative Study)
Comparative Study
The ability of antibiotics to inhibit acetylcholinesterase was measured in homogenates of goldfish brain. Puromycin aminonucleoside was the most potent inhibitor followed by puromycin, cycloheximide and acetoxycycloheximide. Puromycin effectively impaired retention of active-avoidance learning in goldfish when injected either immediately before or after training, while puromycin aminonucleoside did not regardless of injection time. These results suggest that the known amnestic effects of puromycin, cycloheximide and acetoxycycloheximide are not a consequence of interference with acetylcholinesterase.
Topics: Acetylcholinesterase; Animals; Anti-Bacterial Agents; Avoidance Learning; Brain; Cholinesterase Inhibitors; Cycloheximide; Depression, Chemical; Goldfish; Memory; Puromycin; Puromycin Aminonucleoside; Time Factors
PubMed: 996038
DOI: 10.1016/0091-3057(76)90278-1 -
Proceedings of the National Academy of... Nov 1975A soluble protein factor was isolated, free of elongation factor (EF)-T and EF-G, based on its ability to stimulate the synthesis of peptide bonds using ribosomal bound...
A soluble protein factor was isolated, free of elongation factor (EF)-T and EF-G, based on its ability to stimulate the synthesis of peptide bonds using ribosomal bound 70S-AUG-N-formyl-[35S]methionyl-tRNA complex and added puromycin as substrates. Over 90% of this activity was found in the ribosome-free cytoplasm of Escherichia coli extracts. Otherfeatures such as molecular weight, purification properties, and catalytic activities distinguish this factor from ribosomal proteins and known activators of translation. The factor requires all components needed for peptide bond synthesis and is inhibited by antibiotics known to specifically block the peptidyl transferase activity of ribosomes. The factor increases the binding affinity of the ribosome for the aminoacyl-tRNA analog puromycin about 10-fold. We suggest that this extraribosomal factor modulates the intrinsic activity of ribosomes to catalyze peptide-bond synthesis, and regard it as a new factor required for peptide chain elongation, which we call EF-P.
Topics: Bacterial Proteins; Enzyme Activation; Escherichia coli; Kinetics; Peptide Chain Elongation, Translational; Peptidyl Transferases; Puromycin; RNA, Transfer; Ribosomes; Solubility
PubMed: 1105576
DOI: 10.1073/pnas.72.11.4257 -
The Biochemical Journal Feb 19671. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant...
1. An improved 11000g cell-free system for the incorporation of [(14)C]valine into gramicidin S has been obtained. The cell-free extract used was the supernatant obtained by treating Bacillus brevis with ultrasonics for 1min. followed by centrifugation at 11000g. The optimum pH for the incorporation was 8.2-8.4 and the optimum Mg(2+) concentration 0.05m. The presence of ammonium sulphate (0.1m) and K(+) (0.01m) increased the incorporation. 2. Cell-free extracts prepared from cells harvested in the early phase of growth (extinction value 0.1) incorporated negligible amounts of [(14)C]valine into gramicidin S compared with that incorporated by cell-free extracts prepared from cells harvested in the late phase of growth (extinction value 0.5). This was not due to the presence of inhibitors in the cell-free extracts prepared from cells harvested early, since there was no marked decrease in gramicidin S synthesis in a mixture of extracts prepared from cells harvested early and late in the growth phase. 3. The small incorporation of [(14)C]valine into protein, which took place in cell-free extracts from cells harvested in the late growth phase, was not inhibited by puromycin, chloramphenicol and ribonuclease. However, the substantial incorporation that took place in cell-free extracts prepared from cells harvested in the early phase of growth was completely inhibited by puromycin, chloramphenicol and ribonuclease. On mixing cell-free extracts prepared from cells harvested early and late in the growth phase, it appeared that the small incorporation that occurs in extracts from cells harvested in the late phase of growth was not due to cellular inhibitors.
Topics: Bacillus; Bacterial Proteins; Chloramphenicol; Chromatography; Puromycin; Ribonucleases; Tyrothricin; Valine
PubMed: 6029614
DOI: 10.1042/bj1020586 -
The Journal of Organic Chemistry Dec 2008Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the...
Conformationally locked North and South versions of puromycin analogues built on a bicyclo[3.1.0]hexane pseudosugar template were synthesized. The final assembly of the products was accomplished by the Staudinger-Vilarrasa coupling of the corresponding North (2 and 3) and South (6 and 7) 3'-azidopurine carbanucleosides with the Fmoc-protected 1-hydroxybenzotriazole ester of 4-methoxy-L-tyrosine. North azides 2 and 3 were reported earlier. The 3'-azido intermediates 6 and 7 that are necessary for the synthesis of the South puromycin analogues are described herein for the first time.
Topics: Antimetabolites, Antineoplastic; Chemistry, Organic; Chemistry, Pharmaceutical; Drug Design; Models, Chemical; Molecular Conformation; Nucleosides; Peptides; Puromycin; RNA, Transfer; Ribosomes
PubMed: 18991379
DOI: 10.1021/jo8016132 -
Pulsating electromagnetic field stimulation prevents cell death of puromycin treated U937 cell line.Journal of Physiology and Pharmacology... Apr 2010Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities...
Aim of study was to verify whether pulsating electromagnetic field (PEMF) can affect cancer cells proliferation and death. U937 human lymphoid cell line at densities starting from 1 x 10(6) cells/ml to 0.0625 x 10(6) cells/ml, were exposed to a pulsating magnetic field 50 Hz, 45+/-5 mT three times for 3 h per each stimulation with 24 h intervals. Proliferation has been studied by counting number of cells stimulated and non-stimulated by PEMF during four days of cultivation. Viability of cells was analyzed by APC labeled Annexin V and 7-AAD (7-amino-actinomycin D) dye binding and flow cytometry. Growing densities of cells increase cell death in cultures of U937 cells. PEMF exposition decreased amount of cells only in higher densities. Measurement of Annexin V binding and 7-AAD dye incorporation has shown that density-induced cell death corresponds with decrease of proliferation activity. PEMF potentiated density-induced death both apoptosis and necrosis. The strongest influence of PEMF has been found for 1 x 10(6)cells/ml and 0.5 x 10(6) cells/ml density. To eliminate density effect on cell death, for further studies density 0.25 x 10(6) cells/ml was chosen. Puromycin, a telomerase inhibitor, was used as a cell death inducer at concentration 100 microg/ml. Combined interaction of three doses of puromycin and three fold PEMF interaction resulted in a reduced of apoptosis by 24,7% and necrosis by 13%. PEMF protects U937 cells against puromycin- induced cell death. PEMF effects on the human lymphoid cell line depends upon cell density. Increased density induced cells death and on the other hand prevented cells death induced by puromycin.
Topics: Antimetabolites, Antineoplastic; Apoptosis; Cell Proliferation; Cell Survival; Electromagnetic Fields; Humans; Lymphoma, Large B-Cell, Diffuse; Necrosis; Puromycin; U937 Cells
PubMed: 20436221
DOI: No ID Found -
The American Journal of Pathology Sep 1975The metabolic characteristics of the mitochondria of Agaricus bisporus are altered in the zygote by specific inhibitors that permit them to retain structural integrity...
The metabolic characteristics of the mitochondria of Agaricus bisporus are altered in the zygote by specific inhibitors that permit them to retain structural integrity in the dormant spore and enable them to initiate energy production, with apparent protein synthesis and replication during the initial phase of germination. The insensitivity of the earliest events of germination to selective cytoplasmic and nuclear inhibitors characterizes this as a transient period of unusual mitochondrial autonomy. To define the intrinsic metabolic potentials of the organelle and its role in cryptobiosis, mitochondria were fractionated aseptically from presporulating zygotes and were placed in dialysis chambers surrounded by nutrient media at 15 C. For periods through 48 hours, the isolated mitochondria manifested the capacity to incorporate labeled amino acids linearly into proteins and retained stable electrophoretic protein profiles for more than 5 days. They maintained fine structural integrity for at least 10 days, some developed septational membranes, and they increased numerically. These metabolic activities were dependent upon a nutrient substrate.
Topics: Amino Acids; Basidiomycota; Fungal Proteins; Mitochondria; Molecular Weight; Puromycin; Spores, Fungal
PubMed: 1163641
DOI: No ID Found -
European Journal of Biochemistry Jun 1991Erythromycin binds to the large subunit of Escherichia coli ribosomes at a specific site that is very close to the amino acid of aminoacyl-tRNA bound into the...
Erythromycin binds to the large subunit of Escherichia coli ribosomes at a specific site that is very close to the amino acid of aminoacyl-tRNA bound into the peptidyltransferase center, and to the site to which puromycin is bound, the P and A sites, respectively, of the classical two-site model of ribosome function. Both erythromycin and puromycin affect fluorescence from fluorescent derivatives of aminoacyl-tRNAs, while both puromycin and aminoacyl-tRNAs affect fluorescence of fluorescent derivatives of erythromycylamine. The results demonstrate unequivocally that erythromycin, deacylated tRNA, a peptidyl-tRNA analogue and puromycin can be bound simultaneously to the same ribosome. Nascent peptides of more than a few amino acids in length block binding of erythromycin to the ribosomes but, unlike most other peptides, long polyphenylalanine chains can be synthesized on ribosomes to which erythromycin is bound. It is suggested that this refractory synthesis in the presence of erythromycin reflects the atypical physical and structural properties of polyphenylalanine.
Topics: Binding Sites; Erythromycin; Escherichia coli; Fluorescein-5-isothiocyanate; Fluoresceins; Fluorescent Dyes; Kinetics; Peptides; Poly U; Puromycin; RNA, Transfer, Phe; Ribosomes; Spectrometry, Fluorescence; Thiocyanates
PubMed: 1904819
DOI: 10.1111/j.1432-1033.1991.tb16071.x -
Mutation Research Mar 2012DNA double-strand breaks (DSBs) are most often repaired by two pathways in mammalian cells, homologous recombination or non-homologous end joining. Biochemical and...
DNA double-strand breaks (DSBs) are most often repaired by two pathways in mammalian cells, homologous recombination or non-homologous end joining. Biochemical and genetic studies showed that DSBs can also be joined via microhomology-mediated end joining (MHEJ), which is always mutagenic and may result in diseases, such as cancer. In this study we established a human cell-based reporter system to determine the prevalence of MHEJ events and factors that modulate MHEJ. A nonfunctional puromycin acetyltransferase (Pac) gene, disrupted by an insertion flanked by two microhomologous repeats, was integrated into chromosomes of human HT1080 cells. Repair of DSBs via MHEJ using the repeats resulted in deletion of the insertion and restoration of the Pac gene function, thus rendering the cells puromycin resistant. Our results showed that MHEJ spontaneously occurs at the reporter locus (loci), manifested by formation of puromycin resistant (puro(r)) colonies after culturing reporter cells in medium containing puromycin. The frequency of puro(r) cells can be greatly increased by site-directed DSB inside the insertion. Our results also demonstrated that the frequency of puro(r) cells is affected by the length of the repeat and by the size of the intervening sequence. Thus, this cell-based assay provides a platform for evaluating factors modulating in vivo MHEJ.
Topics: Cell Line, Tumor; DNA Breaks, Double-Stranded; DNA End-Joining Repair; Drug Resistance, Neoplasm; Genetic Techniques; Humans; Puromycin
PubMed: 22197482
DOI: 10.1016/j.mrfmmm.2011.12.003 -
European Journal of Biochemistry Jan 1985The requirements for peptide-bond synthesis and transesterification reactions of Escherichia coli 70S ribosomes, 50S native or reconstructed 50S subunits were examined...
The requirements for peptide-bond synthesis and transesterification reactions of Escherichia coli 70S ribosomes, 50S native or reconstructed 50S subunits were examined using fMet-tRNA as donor substrate and puromycin or alpha-hydroxypuromycin as acceptors. We report that the soluble protein EF-P, purified to apparent homogeneity, stimulates the synthesis of N-formylmethionylpuromycin or N-formylmethionylhydroxypuromycin by 70S ribosomes or reassociated 30S and 50S subunits. In the presence of EF-P, 70S ribosomes are significantly more efficient than 50S particles in catalysing either peptide-bond synthesis or transesterification. The involvement of 50S subunit proteins in EF-P-stimulated peptide-bond formation and transesterification was studied. 50S subunits were dissociated by 2.0 M LiCl into core particles and 'split' proteins, several of which were purified to homogeneity. When added to 30S X A-U-G X f[35S]Met-tRNA, 50S cores or 50S cores reconstituted with L6 or L11 promoted peptide-bond synthesis or transesterification poorly. EF-P stimulated peptide-bond synthesis by both these types of core particles to approximately the same extent. On the other hand, EF-P stimulated a low level of transesterification by cores reconstituted with L6 and L11. In contrast, core particles reconstituted with L16 exhibited both peptide-bond-forming and transesterification activities and EF-P stimulated both reactions twentyfold and fortyfold respectively. Thus different proteins differentially stimulate the intrinsic or EF-P-stimulated peptide-bond and transesterification reactions of the peptidyl transferase. Ethoxyformylation of either 50S subunits or purified L16 used to reconstitute core particles, resulted in loss of peptide-bond formation and transesterification. Similarly ethoxyformylation of EF-P resulted in a 25-50% loss of its ability to stimulate both reactions. 30S subunits were resistant to treatment by this reagent. These results suggest the involvement of histidine residues in peptidyltransferase activities. The role of EF-P in the catalytic mechanism of peptidyltransferase is discussed.
Topics: Acyltransferases; Catalysis; Escherichia coli; Kinetics; Peptide Biosynthesis; Peptide Elongation Factors; Peptidyl Transferases; Puromycin; Ribosomal Proteins; Solubility; Substrate Specificity
PubMed: 3881259
DOI: 10.1111/j.1432-1033.1985.tb08651.x -
European Journal of Biochemistry Aug 1984The puromycin-induced stimulation of [3H]dihydrorosaramicin binding is due to a twofold increase in affinity of the macrolide antibiotic, with no change in the number of...
The puromycin-induced stimulation of [3H]dihydrorosaramicin binding is due to a twofold increase in affinity of the macrolide antibiotic, with no change in the number of binding sites. Conversely, the binding of [3H]puromycin (A site) is stimulated by rosaramicin. The synergistic effect observed between the two antibiotics can be explained by a conformational change with positive effect, which occurs at the level of their binding sites. Various effectors of [3H]dihydrorosaramicin binding have been tested. Adenosine and dimethyladenosine stimulate the binding; phenylalanine, uridine and gougerotin (A site) have no effect whereas AMP, ADP, ATP, GTP, puromycin 5'-phosphate and lincomycin (P site) are inhibitors. These results point to the importance of the purine moiety in the stimulatory effect and of the phosphate function in reversing this effect. It is concluded that rosaramicin binds to the ribosomal P site and that the synergism observed between rosaramicin and puromycin may be related to interactions between the A and P sites.
Topics: Adenosine Triphosphate; Binding Sites; Escherichia coli; Guanosine Triphosphate; Kinetics; Leucomycins; Lincomycin; Phenylalanine; Puromycin; Ribosomes
PubMed: 6381054
DOI: 10.1111/j.1432-1033.1984.tb08333.x