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Science (New York, N.Y.) Nov 1969Intracerebral injections of puromycin produced memory deficits in naive quail trained to discriminate between red and green stimuli. Puromycin aminonucleoside,...
Intracerebral injections of puromycin produced memory deficits in naive quail trained to discriminate between red and green stimuli. Puromycin aminonucleoside, acetoxycycloheximide, and saline had no such effect. After a single reversal of the visual cues, naive quail treated with puromycin performed better than control birds. Also, puromycin had no effect on performance when injected into previously trained animals. High doses both of puromycin and acetoxycycloheximide inhibited ribonucleic acid and protein synthesis to a similar extent, while low doses of puromycin inhibited only protein synthesis. Since only puromycin inhibited memory, the basis for its effect appears more likely to be mediated by the action of peptidyl-puromycin rather than by the quantitative inhibition of macromolecular synthesis or by some nonspecific toxic action.
Topics: Animals; Birds; Brain; Cyclohexanes; Depression, Chemical; Discrimination Learning; Memory; Protein Biosynthesis; Puromycin; Pyridones; RNA
PubMed: 5348288
DOI: 10.1126/science.166.3909.1165 -
Biology of Reproduction Dec 1990Germinal vesicle breakdown (GVBD) in cumulus-enclosed and denuded cattle oocytes was sensitive to puromycin at concentrations at or above 50 micrograms/ml. Media...
Germinal vesicle breakdown (GVBD) in cumulus-enclosed and denuded cattle oocytes was sensitive to puromycin at concentrations at or above 50 micrograms/ml. Media supplemented with 5-25 micrograms/ml of puromycin did not significantly reduce either rate or sequence of GVBD after 8 h of culture (82-96% GVBD). In concentrations of 50, 75, and 100 micrograms/ml, GVBD occurred in 15, 4, and 2% of oocytes, respectively. However, 50 micrograms puromycin/ml did postpone the time sequence of GVBD, since all treated oocytes underwent GVBD after 20 h of culture. Oocytes arrested in the germinal vesicle (GV) stage possessed GV filled with highly condensed bivalents. The puromycin block (100 micrograms/ml) was fully reversible, and the time sequence of GVBD was two times faster than in control medium. Proteins important for GVBD were synthesized during the first 4 h of culture, and 81% of oocytes underwent GVBD when puromycin (100 micrograms/ml) was added after 4 h of preincubation in control medium. The first polar body (I PB) expulsion was more sensitive to inhibition of protein synthesis, as shown by the observation that 2.5 and 5 micrograms puromycin/ml significantly (69 and 61%) reduced the incidence of Metaphase II, and 10 micrograms/ml highly significantly (31%) reduced it. The I PB expulsion in concentrations of 25 and 37 micrograms puromycin/ml was less than 5%. The subsequent culture in puromycin (8 h) and 6-dimethylaminopurine (8 h) proved that nuclear membrane breakdown is less sensitive to inhibition of protein phosphorylation than the process of chromatin condensation.
Topics: Adenine; Animals; Cattle; Dose-Response Relationship, Drug; Female; In Vitro Techniques; Meiosis; Oocytes; Protein Biosynthesis; Puromycin; Time Factors
PubMed: 2291932
DOI: 10.1095/biolreprod43.6.994 -
Heterologous enzyme immunoassay for puromycin aminonucleoside using beta-D-galactosidase as a label.Journal of Immunological Methods Aug 1984A heterologous enzyme immunoassay (EIA) was developed to quantify puromycin aminonucleoside (PA). This double antibody assay was based on the use of anti-puromycin (PU)...
A heterologous enzyme immunoassay (EIA) was developed to quantify puromycin aminonucleoside (PA). This double antibody assay was based on the use of anti-puromycin (PU) antibody and used beta-D-galactosidase-labeled PA conjugate prepared via N-(m-maleimidobenzoyloxy)succinimide. The standard curve of the assay ranged from 1 ng to 30 ng, and the lower limit of detection was 22.7 nM (1 ng/tube). The EIA was found to be approximately 20 times more sensitive than the homologous EIA for PA with anti-PA antibody and PA-beta-D-galactosidase conjugate. The heterologous EIA was free from interference by any purine or pyrimidine analogs and drug levels were easily determined in rat tissue following i.v. administration at a dose of 15 mg/kg. The sensitivity and specificity of the EIA should provide a valuable new tool for use in pharmacokinetic and toxicity studies of PA.
Topics: Animals; Binding Sites, Antibody; Binding, Competitive; Dose-Response Relationship, Immunologic; Female; Galactosidases; Immunoenzyme Techniques; Nephrosis; Puromycin; Puromycin Aminonucleoside; Rabbits; Rats; Rats, Inbred Strains; Tissue Distribution; beta-Galactosidase
PubMed: 6431006
DOI: 10.1016/0022-1759(84)90438-1 -
Proceedings of the National Academy of... May 1970It has previously been shown that expression of memory of maze-learning in mice is blocked by puromycin injected intracerebrally one or more days after the training...
It has previously been shown that expression of memory of maze-learning in mice is blocked by puromycin injected intracerebrally one or more days after the training experience. Bilateral adrenalectomy before training has now been found to protect memory against the effects of puromycin. This protection is absent when adrenalectomy follows training. In view of control experiments, we conclude that adrenalectomy before training modifies factors necessary for the expression of memory and that this alteration makes puromycin ineffective in blocking memory.
Topics: Adrenal Glands; Adrenalectomy; Adrenocorticotropic Hormone; Animals; Behavior, Animal; Female; Male; Memory; Mice; Puromycin; Time Factors
PubMed: 4320463
DOI: 10.1073/pnas.66.1.48 -
Bioorganic & Medicinal Chemistry Dec 1995As part of our project aimed to introduce specifically glycosylated amino acids into proteins, new glycosylated puromycin analogues were chemically synthesized....
As part of our project aimed to introduce specifically glycosylated amino acids into proteins, new glycosylated puromycin analogues were chemically synthesized. Introduction of a free N-acetylglucosaminyl asparaginyl side chain abolished the activity of puromycin completely, but when the sugar OH groups were rendered increasingly hydrophobic by acetylation or benzylation, up to 8% of the activity was recovered. The results of our preliminary inhibition tests suggest that the interaction of puromycin analogues and therefore also of glycosylated aminoacyl tRNA, with the ribosomal A site increase with hydrophobicity of the modifying protecting groups.
Topics: Acetylglucosamine; Anti-Bacterial Agents; Escherichia coli; Glycosylation; Magnetic Resonance Spectroscopy; Molecular Structure; Protein Synthesis Inhibitors; Puromycin; RNA, Transfer, Amino Acyl; Ribosomes; Structure-Activity Relationship
PubMed: 8770387
DOI: 10.1016/0968-0896(95)00148-4 -
Biochemistry Apr 1990In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall,...
In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & Stöffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].
Topics: Chromatography, High Pressure Liquid; Escherichia coli; Escherichia coli Proteins; Kinetics; Protein Binding; Puromycin; RNA, Transfer, Phe; Ribosomal Proteins; Ribosomes
PubMed: 2191716
DOI: 10.1021/bi00466a006 -
Journal of the American Chemical Society Jul 2003We synthesized a series of puromycin analogues to probe the chemical specificity of the ribosome in an intact eukaryotic translation system. These studies reveal that...
We synthesized a series of puromycin analogues to probe the chemical specificity of the ribosome in an intact eukaryotic translation system. These studies reveal that both d-enantiomers and beta-amino acid analogues can be incorporated into protein, and provide a quantitative means to rank natural and unnatural residues. Modeling of a d-amino acid analogue into the 50S ribosomal subunit indicates that steric clash may provide part of the chiral discrimination. The data presented provide one metric of the chiral and regiospecificity of mammalian ribosomes.
Topics: Amino Acids; Animals; Globins; Protein Biosynthesis; Protein Synthesis Inhibitors; Proteins; Puromycin; RNA, Messenger; Rabbits; Ribosomes; Stereoisomerism; Structure-Activity Relationship
PubMed: 12837064
DOI: 10.1021/ja034817e -
Life Sciences Aug 1983We have established optimal conditions for the in vitro formation of peptidyl-[3H] puromycin by mammalian ribosomes. The growth conditions of cultured Ehrlich ascites...
Radioactive puromycin formation as an assay for the proportion of active ribosomes in mammalian cells: correlation with polysome profiles under different physiological conditions.
We have established optimal conditions for the in vitro formation of peptidyl-[3H] puromycin by mammalian ribosomes. The growth conditions of cultured Ehrlich ascites tumor cells were manipulated to produce changes in the polysome profiles. The correlation between polysome content and peptidyl-[3H] puromycin formation was linear and excellent when different cell densities were compared. The percentage of ribosomes actively engaged in protein synthesis, calculated from the number of 3H-peptide bonds formed, was similar in rapidly growing Ehrlich cells (47%) and in young rat gastrocnemius muscle (44%). Starvation resulted in a 50% reduction in the number of puromycin-reactive ribosomes in rat gastrocnemius.
Topics: Animals; Carcinoma, Ehrlich Tumor; Cell Fractionation; Centrifugation, Density Gradient; Kinetics; Mice; Polyribosomes; Protein Biosynthesis; Puromycin; Ribosomes; Tritium
PubMed: 6888182
DOI: 10.1016/0024-3205(83)90126-1 -
Biochemical and Biophysical Research... Jun 1983Streptomyces alboniger produces the antibiotic puromycin and expresses an enzymic activity which acetylates the drug using acetyl CoA. The N-acetyl-puromycin formed is...
Streptomyces alboniger produces the antibiotic puromycin and expresses an enzymic activity which acetylates the drug using acetyl CoA. The N-acetyl-puromycin formed is biologically inactive against protein synthesis in Bacillus subtilis (as assayed in vivo).
Topics: Acetylation; Inactivation, Metabolic; Puromycin; Streptomyces
PubMed: 6870889
DOI: 10.1016/0006-291x(83)91066-5 -
Proceedings of the National Academy of... Jan 2012Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation,...
Synthesis of many proteins is tightly controlled at the level of translation, and plays an essential role in fundamental processes such as cell growth and proliferation, signaling, differentiation, or death. Methods that allow imaging and identification of nascent proteins are critical for dissecting regulation of translation, both spatially and temporally, particularly in whole organisms. We introduce a simple and robust chemical method to image and affinity-purify nascent proteins in cells and in animals, based on an alkyne analog of puromycin, O-propargyl-puromycin (OP-puro). OP-puro forms covalent conjugates with nascent polypeptide chains, which are rapidly turned over by the proteasome and can be visualized or captured by copper(I)-catalyzed azide-alkyne cycloaddition. Unlike methionine analogs, OP-puro does not require methionine-free conditions and, uniquely, can be used to label and assay nascent proteins in whole organisms. This strategy should have broad applicability for imaging protein synthesis and for identifying proteins synthesized under various physiological and pathological conditions in vivo.
Topics: Alkynes; Azides; Copper; Diagnostic Imaging; Magnetic Resonance Spectroscopy; Microscopy, Fluorescence; Molecular Structure; Protein Biosynthesis; Puromycin
PubMed: 22160674
DOI: 10.1073/pnas.1111561108