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Journal of Biochemistry Mar 1981Puromycin-mediated in vitro release of nascent peptides from rat liver ribosomes was significantly stimulated by the presence of low concentrations of a detergent, and...
Puromycin-mediated in vitro release of nascent peptides from rat liver ribosomes was significantly stimulated by the presence of low concentrations of a detergent, and the stimulation was much more marked with bound ribosomes than with free ribosomes. The release of nascent peptides from ribosomes could be carried out in two steps, first with puromycin in the absence of a detergent and then with a detergent, to give two separate nascent peptide fractions S1 and S2, respectively. Although S1 and S2 fractions were not significantly different in hydrophobicity and in the size of the puromycin-conjugated peptides when examined by alkyl-Sepharose column chromatography and SDS-polyacrylamide gel electrophoresis, the fractionation of the released peptides by immunoprecipitation showed significant difference in the distribution of the nascent peptides of two specific proteins, serum albumin and NADPH-cytochrome c reductase, between these two fractions. The nascent peptides of serum albumin were found mainly in fraction S1 obtained from bound ribosomes. On the other hand, a larger portion of the nascent peptides of NADPH-cytochrome c reductase was detected in fraction S2 from free ribosomes than in other fractions. The presence of a detergent is indispensable for efficient in vitro release of nascent peptides from ribosomes by puromycin and this finding may be important in studying the synthesis of specific proteins in mammalian cells.
Topics: Animals; Detergents; Electrophoresis, Polyacrylamide Gel; Immunochemistry; In Vitro Techniques; Liver; Male; NADPH-Ferrihemoprotein Reductase; Peptides; Puromycin; Rats; Ribosomal Proteins; Serum Albumin; Surface-Active Agents
PubMed: 6793560
DOI: 10.1093/oxfordjournals.jbchem.a133259 -
Kidney International Feb 1986The cellular processes responsible for the proteinuria induced by the aminonucleoside of puromycin (PA) remain inadequately defined. Hypoxanthine is both a metabolic...
The cellular processes responsible for the proteinuria induced by the aminonucleoside of puromycin (PA) remain inadequately defined. Hypoxanthine is both a metabolic breakdown product of PA as well as a substrate for xanthine oxidase, which catalyzes its enzymatic conversion to xanthine and uric acid, yielding the superoxide anion in the process. We examined whether oxygen free radical production contributes to the development of proteinuria in this model. Seven groups of male Sprague-Dawley rats were studied. Proteinuria was quantitated and histology examined 7 days after rats were treated with PA intravenously over 5 min. PA-treated animals received either saline, dimethyl sulfoxide, superoxide dismutase, or catalase over 30 min prior to and 30 min following PA administration. Another group received allopurinol over 4 hr prior to PA. The superoxide dismutase and allopurinol treatment groups had a significant suppression of urinary protein excretion compared to the PA control group. There were also less severe glomerular morphologic changes in the superoxide dismutase group vs. the PA controls, which demonstrated a pathologic pattern that included epithelial cell blebbing, segmental mesangial cell proliferation and matrix expansion, loss of glomerular capillary lumina, and occasional adhesions between the glomerular tuft and Bowman's capsule. The allopurinol group exhibited normal glomerular morphology on light microscopy, with the exception of occasional epithelial cell blebs. All groups showed spreading of the epithelial cell cytoplasm along the glomerular basement membrane with loss of foot processes, focal areas of lifting of the epithelial cell from the glomerular basement membrane, cytoplasmic vacuolization, and protein reabsorption droplets; however, allopurinol-treated animals demonstrated these changes to a lesser extent.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Allopurinol; Animals; Catalase; Depression, Chemical; Dimethyl Sulfoxide; Disease Models, Animal; Free Radicals; Male; Nephrosis; Oxygen; Proteinuria; Puromycin; Puromycin Aminonucleoside; Rats; Rats, Inbred Strains; Superoxide Dismutase
PubMed: 3702206
DOI: 10.1038/ki.1986.24 -
The Journal of Cell Biology Feb 1968Bitemporal intracerebral injections of puromycin in mice suppress indefinitely expression of memory of avoidance-discrimination learning. Ultrastructural studies of the...
Bitemporal intracerebral injections of puromycin in mice suppress indefinitely expression of memory of avoidance-discrimination learning. Ultrastructural studies of the entorhinal cortex of puromycin-treated mice revealed the following: (a) Abnormalities were not observed in presynaptic terminals and synaptic clefts; many postsynaptic dendrites or somas contained swollen mitochondria. (b) Dispersion of polyribosomes into single units or condensation of ribosomes into irregular aggregates with loss of "distinctiveness" was noted in a few neurons 7-27 hr after puromycin treatment. (c) Cytoplasmic aggregates of granular or amorphous material were frequently noted within otherwise normal neuronal perikarya. (d) Mitochondria in many neuronal perikarya and dendrites were swollen. Mitochondria in axons, presynaptic terminals, and glial cells were unaltered. The relationships between these lesions and the effect of puromycin on protein synthesis and memory are examined. It is suggested that the disaggregation of polysomes is too limited to explain the effect of puromycin on memory. Special emphasis is given to the swelling of mitochondria. The possible mechanisms and the significance of this lesion are discussed.
Topics: Animals; Avoidance Learning; Axons; Behavior, Animal; Cell Aggregation; Cerebral Cortex; Cytoplasm; Dendrites; Discrimination Learning; Injections; Memory; Mice; Microscopy, Electron; Mitochondria; Puromycin; Ribosomes; Synapses
PubMed: 5638887
DOI: 10.1083/jcb.36.2.379 -
PloS One 2012Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a...
Nearly thirty percent of all newly synthesized polypeptides are targeted for rapid proteasome-mediated degradation. These rapidly degraded polypeptides (RDPs) are a source of antigenic substrates for the MHC class I presentation pathway, allowing for immunosurveillance of newly synthesized proteins by cytotoxic T lymphocytes. Despite the recognized role of RDPs in MHC I presentation, it remains unclear what molecular characteristics distinguish RDPs from their more stable counterparts. It has been proposed that premature translational termination products may constitute a form of RDP; indeed, in prokaryotes translational drop-off products are normal by-products of protein synthesis and are subsequently rapidly degraded. To study the cellular fate of premature termination products, we used the antibiotic puromycin as a means to experimentally manipulate prematurely terminated polypeptide production in human cells. At low concentrations, puromycin enhanced flux into rapidly degraded polypeptide pools, with small polypeptides being markedly more labile then high molecular weight puromycin adducts. Immunoprecipitation experiments using anti-puromycin antisera demonstrated that the majority of peptidyl-puromycins are rapidly degraded in a proteasome-dependent manner. Low concentrations of puromycin increased the recovery of cell surface MHC I-peptide complexes, indicating that prematurely terminated polypeptides can be processed for presentation via the MHC I pathway. In the continued presence of puromycin, however, MHC I export to the cell surface was inhibited, coincident with the accumulation of polyubiquitinated proteins. The time- and dose-dependent effects of puromycin suggest that the pool of peptidyl-puromycin adducts differ in their targeting to various proteolytic pathways that, in turn, differ in the efficiency with which they access the MHC I presentation machinery. These studies highlight the diversity of cellular proteolytic pathways necessary for the metabolism and immunosurveillance of prematurely terminated polypeptides that are, by their nature, highly heterogeneous.
Topics: Antigen Presentation; Cell Line; HEK293 Cells; Histocompatibility Antigens Class I; Humans; Monitoring, Immunologic; Peptide Chain Termination, Translational; Peptides; Proteasome Endopeptidase Complex; Proteolysis; Puromycin
PubMed: 23251665
DOI: 10.1371/journal.pone.0051968 -
European Journal of Biochemistry Sep 1986In a model system derived from Escherichia coli, Ac[3H]Phe-puromycin is produced in a pseudo-first-order reaction between the preformed Ac[3H]Phe-tRNA-poly(U)-ribosome...
In a model system derived from Escherichia coli, Ac[3H]Phe-puromycin is produced in a pseudo-first-order reaction between the preformed Ac[3H]Phe-tRNA-poly(U)-ribosome complex (complex C) and excess puromycin [Kalpaxis et al. Eur. J. Biochem. 154, 267, 1986]. Amicetin and gougerotin inhibit this reaction to various degrees depending on whether or not complex C is allowed to interact with the inhibitor (I) prior to the addition of puromycin (S). The kinetic analysis shows a phase where competitive inhibition can be observed provided that S and I are added simultaneously. After preincubating C with I, the inhibition becomes of the mixed non-competitive type. The Ki (the dissociation constant of the CI complex), calculated from the competitive plot, is 20.0 microM for amicetin and 15.0 microM for gougerotin. This inhibition constant (Ki) cannot distinguish amicetin from gougerotin. Its acceptance as a criterion of potency does not explain why after preincubation amicetin proves to be a stronger inhibitor than gougerotin. The determination of the apparent catalytic rate constants of peptidyltransferase at various inhibitor concentrations and the appropriate replotting of these rate constants distinguish amicetin from gougerotin. A new approach for evaluating the potency of these inhibitors is proposed. The familiar Ki is supplemented with an apparent kinetic constant obtained from a replot in which the intercepts of the double-reciprocal plots (1/kobs versus 1/[S]) are plotted versus the inhibitor concentration.
Topics: Depression, Chemical; Guanidines; Kinetics; Models, Chemical; Nucleosides; Protein Biosynthesis; Puromycin; Pyrimidine Nucleosides
PubMed: 3758072
DOI: 10.1111/j.1432-1033.1986.tb09911.x -
Kidney International Jul 1991Two puromycin aminonucleoside (PAN) excretion products were purified by HPLC from urine of PAN-treated rats and characterized by nuclear magnetic resonance as...
Two puromycin aminonucleoside (PAN) excretion products were purified by HPLC from urine of PAN-treated rats and characterized by nuclear magnetic resonance as N6-dimethyl-3'amino-3'deoxyadenosine (DA-Ado) and N6-methyl-3'amino-3'deoxyadenosine (MA-Ado), respectively, the former corresponding to unmodified PAN. DA-Ado was not a substrate for adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP) or xanthine oxidase (XO), while MA-Ado was consecutively converted into hypoxanthine by a mixture of ADA and PNP. A different rate of transformation of DA-Ado and MA-Ado into hypoxanthine by isolated glomeruli was observed and was higher for the monomethylated analogue by a factor of 3 (79% vs. 21%); this was ascribed to the rate-limiting level of a demethylase activity acting on DA-Ado. Furthermore, DA-Ado was not transformed by glomerular epithelial cells in culture, while a little amount of MA-Ado was converted into hypoxanthine after six hours of incubation. In spite of this different metabolic behavior, the same order of cytotoxicity on glomerular epithelial cells in culture was observed for MA-Ado, DA-Ado and commercial PAN. All these molecules induced a dose response inhibition of [3H]thymidine incorporation into DNA after exposure for two hours and a marked alteration of cell viability which was not inhibited by free radical scavengers and deferoxamine. This study provides the first evidence for a glomerular metabolism of PAN and its urinary metabolite MA-Ado involving their transformation via the purine cycle enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)
Topics: Animals; Cell Survival; Cells, Cultured; Epithelial Cells; Epithelium; In Vitro Techniques; Kidney Glomerulus; Male; Purines; Puromycin Aminonucleoside; Rats; Rats, Inbred Strains
PubMed: 1921153
DOI: 10.1038/ki.1991.176 -
Antimicrobial Agents and Chemotherapy Oct 2009Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture)....
Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin-N-acetyltransferase from Streptomyces alboniger. Puromycin is an antibiotic that mimics the 3'-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation--an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli, which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.
Topics: Acetyltransferases; Anti-Bacterial Agents; Genome, Bacterial; Models, Genetic; Mycoplasma; Puromycin
PubMed: 19687239
DOI: 10.1128/AAC.00388-09 -
Proceedings of the National Academy of... Jun 1970Puromycin was injected bitemporally in mice one day after training in a Y-maze. Eight days later various psychotropic drugs were injected intraperitoneally or...
Puromycin was injected bitemporally in mice one day after training in a Y-maze. Eight days later various psychotropic drugs were injected intraperitoneally or subcutaneously at maximum tolerable doses. Ten days after the drug injection the mice were tested for their memory of the maze-learning. Memory was lost in control animals injected with saline but restored in most of the animals injected with imipramine, tranylcypromine, or D-amphetamine. Some indication of restoration was observed after injection of reserpine or L-dopa. These results suggest that the blockage caused by puromycin is due to adsorption of peptidyl-puromycin to adrenergic sites and that these sites may be involved in the memory trace.
Topics: Animals; Behavior, Animal; Dextroamphetamine; Dihydroxyphenylalanine; Imipramine; Memory; Mice; Puromycin; Reserpine; Sensory Receptor Cells; Temporal Lobe; Tranylcypromine
PubMed: 5271166
DOI: 10.1073/pnas.66.2.310 -
Nature Communications 2013Eradication of cancer cells while minimizing damage to healthy cells is a primary goal of cancer therapy. Highly selective drugs are urgently needed. Here we demonstrate...
Eradication of cancer cells while minimizing damage to healthy cells is a primary goal of cancer therapy. Highly selective drugs are urgently needed. Here we demonstrate a new prodrug strategy for selective cancer therapy that utilizes increased histone deacetylase (HDAC) and tumour-associated protease activities produced in malignant cancer cells. By coupling an acetylated lysine group to puromycin, a masked cytotoxic agent is created, which is serially activated by HDAC and an endogenous protease cathepsin L (CTSL) that remove the acetyl group first and then the unacetylated lysine group liberating puromycin. The agent selectively kills human cancer cell lines with high HDAC and CTSL activities. In vivo studies confirm tumour growth inhibition in prodrug-treated mice bearing human cancer xenografts. This cancer-selective cleavage of the masking group is a promising strategy for the next generation of anticancer drug development that could be applied to many other cytotoxic agents.
Topics: Animals; Antineoplastic Agents; Cathepsin L; Cell Line, Tumor; Cell Survival; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Histone Deacetylases; Humans; Mice; Neoplasms, Experimental; Prodrugs; Puromycin
PubMed: 24193185
DOI: 10.1038/ncomms3735 -
Journal of Virology Dec 1967Cultures of chick cells exposed to interferon continued to decrease in virus-producing ability during incubation after the interferon was removed. The rate of...
Cultures of chick cells exposed to interferon continued to decrease in virus-producing ability during incubation after the interferon was removed. The rate of development of the additional interference and the degree of viral interference finally manifested were dependent on the concentration of interferon to which the cultures were exposed and the time of exposure. Additional interference occurred also in infected cells. Additional interference was inhibited by actinomycin D and puromycin. The best explanation of additional interference is that it results from interferon that is fixed to the cells during their initial period of contact.
Topics: Animals; Antiviral Agents; Chick Embryo; Culture Techniques; Dactinomycin; Encephalitis Viruses; Horses; Interferons; Kinetics; Puromycin; RNA, Messenger; Viral Interference; Virus Replication
PubMed: 5621487
DOI: 10.1128/JVI.1.6.1158-1163.1967