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At least 19 recordsLinked to original sources
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↗
PubMed1990

Renal purine efflux and xanthine oxidase activity during experimental nephrosis in rats: difference between puromycin aminonucleoside and adriamycin nephrosis.

1. The hypothesis was tested that the renal xanthine oxidase system provides a source of oxygen free radicals in puromycin aminonucleoside and adriamycin experimental nephrosis by generating uric acid from hypoxanthine and xanthine. 2. The concentrations in renal tissue of the putative intermediary products of puromycin aminonucleoside metabolism, hypoxanthine and xanthine, and of their precursors, adenosine and inosine, were lower in rats treated with puromycin aminonucleoside than in normal controls, whereas concentrations of the metabolites were normal after adriamycin intoxication. Their daily urinary excretion was lower in the 24 h after puromycin aminonucleoside administration compared with the baseline values and returned to near normal levels within 5 days. After adriamycin the 24 h urinary excretion of xanthine and uric acid was double the baseline levels (P less than 0.001). 3. When equimolar amounts of hypoxanthine were injected instead of puromycin aminonucleoside, the concentration of all bases increased slightly in renal tissue and their urinary efflux was double the baseline level: allantoin, uric acid, the unmodified nucleotide and xanthine were the most represented compounds in urine. 4. The enzymatic activities relative to xanthine oxidase (EC 1.1.3.22) and xanthine dehydrogenase (EC 1.1.1.204) in renal tissues were unchanged 1 day after puromycin aminonucleoside or hypoxanthine intoxication and only moderately increased in both groups at 13 days (the time of appearance of heavy proteinuria in the puromycin aminonucleoside-treated group). In contrast, xanthine oxidase and xanthine dehydrogenase activities were higher in adriamycin-treated rats at 1 and 15 days after the treatment (P less than 0.001). 5. Feeding rats with normoprotein diets containing tungsten induced a marked and constant decrease of renal xanthine oxidase and xanthine dehydrogenase activities to 20% of the baseline values in both puromycin aminonucleoside- and adriamycin-treated rats. Inhibition of renal xanthine oxidase and xanthine dehydrogenase activities by tungsten was associated with a marked reduction (P less than 0.001) of proteinuria in adriamycin-treated rats and the same occurred with allopurinol, a specific inhibitor of xanthine oxidase activity. In contrast, tungsten treatment did not reduce the proteinuria associated with puromycin aminonucleoside, which reached a maximum 13 days after puromycin aminonucleoside intoxication. Hypoxanthine-treated rats were normoproteinuric after 2 months of observation. 6. These data demonstrate an activation of renal xanthine oxidase and xanthine dehydrogenase after adriamycin intoxication which is relevant to the induction of proteinuria. They also argue against the involvement of the renal xanthine oxidase system as a source of free radicals in puromycin aminonucleoside nephrosis and suggest that the nucleotide cycle is not a normal route for puromycin aminonucleoside degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Allantoin↗
PubMed1987

The effect of dilazep on puromycin-induced rat renal mitochondrial dysfunction.

The effect of tetrahydro-1 H-1,4 (5H)-dipropanol bis(3,4,5-trimethoxybenzoate)hydrochloride monohydrate (dilazep, Comelian) on puromycin-induced rat renal damage was investigated. In vivo study: Rats were divided into 3 groups, the control group; untreated, the puromycin group; puromycin (150 mg/kg) was injected intraperitoneally once, the dilazep + puromycin group; puromycin (150 mg/kg) was injected 1 h after intraperitoneal dilazep injection (2 mg/kg), and dilazep (2 mg/kg) was injected every 12 h until the end of the experiment. In each group, 84 h after puromycin injection, kidneys were isolated and renal mitochondria were prepared. The endogenous phospholipase activity in kidney homogenate was determined by high performance liquid chromatography. The activities of three segments (NADH-cytochrome c reductase, succinate-cytochrome c reductase and cytochrome c oxidase) of the electron-transport chain in mitochondria were measured enzymatically. In the puromycin group, phospholipase activity was increased and activities of all of three segments of the electron-transport chain were decreased. In the dilazep + puromycin group, premedication with dilazep prevented activation of phospholipase and maintained mitochondrial electron-transport activity. In vitro study: Mitochondria prepared from intact rat kidney were incubated with phospholipase C. Activities of the mitochondrial electron-transport chain were deteriorated by phospholipase C. These results indicated that activation of endogenous phospholipase, which digests membrane phospholipids, essential components in maintaining mitochondrial electron-transport activity, is responsible for the puromycin-induced renal damage. Premedication with dilazep prevented the damage by inhibition of the activation of phospholipase.

Animals↗
PubMed1989

Stimulation by insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine-dependent inhibition of phosphofructokinase 2 activation.

Activation of glycolysis by insulin in cultured rat hepatocytes is preceded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructose 2,6-bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated. 1. 50 microM ATP completely prevented the 3-5-fold insulin-dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content. 50 microM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin cells with initial high Fru(2,6)P2 content. 2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 microM ATP or 15 microM adenosine 5'[beta, gamma-methylene]triphosphate. ADP and adenosine-5-[gamma-thio]triphosphate were as effective as ATP, whereas 100 microM adenosine 5'[alpha, beta-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis. 3. Extracellular ATP (100 microM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3-phosphate levels stayed constant. Puromycin did not inhibit PFK 2. 4. Both puromycin and ATP prevented the insulin-dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase. 5. 100 microM ATP elevated the cAMP-dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16-fold within 5 min, whereas puromycin was without effect on either level. It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.

Adenosine Triphosphate↗
PubMed1978

Testing with puromycin and amino acyl tRNAs that limit the rate of peptide chain extension.

With puromycin one can recognize when the synthesis of a given protein is dependent on amino acyl tRNA that is present in rate limiting amount. We demonstrate this use of puromycin by its interaction with another inhibitor, L-o-methylthreonine. L-o-methylthreonine lowers the Ile-tRNA concentration in the cell, thereby inhibiting synthesis of proteins containing isoleucine. In certain rabbits, the alpha hemoglobin chain has three isoleucyl residues and the beta chain none. L-o-methylthreonine thus inhibits alpha globin synthesis in intact reticulocytes from these rabbits. When puromycin and L-o-methylthreonine are used together, the two inhibitors synergize in inhibiting alpha globin synthesis. Hence, puromycin is a more effective inhibitor when the Ile-tRNA concentration is lowered. Cycloheximide and sodium fluoride have different modes of action from puromycin. Neither synergizes with L-o-methylthreonine; instead, the interaction is less than additive. We have found that beta chain synthesis in rabbit reticulocytes is more sensitive than alpha to inhibition by puromycin. This difference could reflect either differences in amino acid sequence or tRNA dependent limitations of beta chain elongation. The switch from fetal to adult hemoglobin in humans does not involve changes in limiting amino acyl tRNA because, for cord blood from infants of different developmental ages, the puromycin sensitivity of incorporation into gamma and beta chains remains constant.

Animals↗
PubMed1992

Parthenogenetic activation of human oocytes by puromycin.

OBJECTIVE: Our objective was to obtain parthenogenetic activation of unfertilized human oocytes by puromycin and to try to use this procedure for cytogenetic purposes. SETTING: The setting was our IVF laboratory. METHODS: Eighty-two spare oocytes from 34 IVF patients were used. In the first series of experiments 39 unfertilized oocytes were cultured in medium containing 100, 50, or 10 micrograms/ml puromycin for 6 to 24 hr. After the appearance of pronuclei they were transferred to plain medium, further cultured, and cytogenetically analyzed. In the second series of experiments 43 oocytes were cultured for 5 to 10 hr in 10 micrograms/ml puromycin, transferred to plain medium, and fixed for cytogenetic analysis 2 hr after nuclear envelope breakdown. RESULTS: Ninety-one percent of the oocytes in the first experiment showed the presence of one or more nuclei. From the pronucleate oocytes additionally cultured in puromycin-free medium, 46% developed further to the metaphase of the first mitotic division or the two-cell stage and 54% remained arrested at the pronuclear stage. In the second experiment 88% of the treated oocytes showed pronuclei or had cleaved, and after withdrawal from puromycin 96% of the pronucleate oocytes entered mitosis. CONCLUSION: Puromycin induces haploid as well as diploid parthenogenesis in aged human oocytes. A 5- to 10-hr treatment of oocytes with 10 micrograms/ml puromycin yields the highest percentage of activation, and almost all parthenogenetically activated oocytes enter or develop beyond the first cleavage mitosis. Analysis of mitotic metaphase chromosomes from parthenogenetically activated human oocytes may be a promising new approach to preimplantation cytogenetics.

Cell Division↗
PubMed1988

Species-specific differences in the toxicity of puromycin towards cultured human and Chinese hamster cells.

The toxicity of the protein synthesis inhibitor puromycin towards a number of human and Chinese hamster cell lines has been examined. In comparison to cells of human origin, Chinese hamster cells exhibited about 25-fold higher resistance towards puromycin. These differences appeared to be species related as all the cell lines from any one species showed similar sensitivity towards puromycin. The incorporation of [3H]leucine in the hamster cell lines was accordingly found to be more resistant to the inhibitory effects of puromycin as compared to human cells. Studies on the cellular uptake of [3H]puromycin showed that in comparison to human cells, the drug uptake/binding in the hamster cell lines was greatly reduced. However, protein synthesis in the extracts of hamster and human cells showed no significant differences in sensitivity towards puromycin. These results show that the observed species related differences in cellular toxicity to puromycin are due to differences in the cellular uptake/binding of the drug.

Animals↗
PubMed1990

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences.

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].

Chromatography, High Pressure Liquid↗
PubMed1986

Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined.

The standard technique for determination of the ribosomal site location of bound tRNA, viz. the puromycin reaction, has been analyzed with regard to its applicability under tRNA saturation conditions. The criteria derived have been used to re-examine the exclusion principle for peptidyl-tRNA binding, which states that only one peptidyl-tRNA (AcPhe-tRNA) can be bound per ribosome although in principle two sites (A and P site) are available. The following results were obtained. The puromycin reaction is only appropriate for a site determination if the reaction conditions prevent one ribosome from performing more than one puromycin reaction. With an excess of AcPhe-tRNA over ribosomes, and in the absence of EF-G, this criterion is fulfilled at 0 degree C, where the P-site-bound material reacts with puromycin (quantitative reaction after 50 h), while the A-site-bound material does not. In contrast, at 37 degrees C the extent of the puromycin reaction can exceed the binding values by 2-4-fold ('repetitive reaction'). In the presence of EF-G a repetitive puromycin reaction is seen even at 0 degree C, i.e. EF-G can already promote a translocation reaction at 0 degree C. However, the extent of translocation becomes negligibly low for short incubation times (up to 60 min) at 0 degree C, if only catalytic amounts of EF-G are used. Using the criteria outlined above, the validity of the exclusion principle for Escherichia coli ribosomes was confirmed pursuing two different experimental strategies. Ribosomes were saturated with AcPhe-tRNA at one molecule per 70S ribosome, and a quantitative puromycin reaction demonstrated the exclusive P-site location of the AcPhe-tRNA. The same result was also found in the presence of viomycin, which blocks the translocation reaction. These findings also indicate that here nearly 100% of the ribosomes participate in AcPhe-tRNA binding to the P site. Precharging the P sites of 70S ribosomes with one Ac[14C]Phe-tRNA molecule per ribosome prevented additional Ac[3H]Phe-tRNA binding. In contrast, 70S particles carrying one molecule of [14C]tRNAPhe per ribosome were able to bind up to a further 0.64 molecule Ac[3H]Phe-tRNA per ribosome.

Binding Sites↗
PubMed1978

Effects of puromycin and actinomycin D on fertilized rabbit eggs cultured in vitro.

Rabbit zygotes were cultured in media containing varying concentrations of puromycin and actinomycin D in order to determine what effect these agents have on events of fertilization and early development. Concentrations of puromycin greater than 10(-5) M inhibited mitosis, cytokinesis of fertilized eggs and leucine incorporation. In approximately 50% of the zygotes treated with puromycin (10(-5) M or greater) the female pronucleus remained at its site of formation, the cortex, and did not migrate centrad. In these instances the male pronucleus moved from its site of development to the female pronucleus. Concentrations of actinomycin D greater than 10(-7) M also inhibited mitosis and cytokinesis of fertilized eggs. Breakdown of the nuclear envelopes and association of the paternally- and maternally-derived chromatin did not occur in either puromycin- or actinomycin D-treated embryos. Actinomycin D at all of the concentrations employed (10(-9) to 10(-4) M) had little effect upon the structure of nucleolus-like bodies within male and female pronuclei. Male and female pronuclei of puromycin (10(-4) M) and actinomycin D (10(-5) M) treated zygotes, incubated with tritiated thymidine and prepared for scintillation counting and autoradiography, synthesized DNA at a reduced level when compared to control specimens. Although "side effects" of puromycin and actinomycin D treatment are not excluded, these results suggest that nascent proteins may be required for events of fertilization.

Animals↗
PubMed1976

Puromycin-resistant biosynthesis of a specific outer-membrane lipoprotein of Escherichia coli.

The reported puromycin resistance of the in vivo biosynthesis of a specific outer-membrane lipoprotein of Escherichia coli was further investigated. The biosynthetic machinery making the lipoprotein was made more accessible to puromycin by disruption of the cell structure using ethylenediaminetetracetate or toluene, and finally in an in vitro protein biosynthesis system using polyribosomes. Puromycin sensitivity of overall protein synthesis increased by about 10-fold for each method of disruption of the cell structure; 50% inhibitions were obtained at 330, 35, 2.7, and 0.22 mug of puromycin per ml for intact cells, ethylenediaminetetraacetate-treated cells, toluene-treated cells, and the polyribosome system, respectively. However, the lipoprotein biosynthesis remained more resistant to puromycin than the biosynthesis of other proteins in all systems tested. These results strongly suggest that puromycin resistance of the lipoprotein biosynthesis is due to an intrinsic property of the lipoprotein biosynthetic machinery.

Bacterial Proteins↗
PubMed1992

Cloning of the complete biosynthetic gene cluster for an aminonucleoside antibiotic, puromycin, and its regulated expression in heterologous hosts.

Puromycin, produced by Streptomyces alboniger, is a member of the large group of aminonucleoside antibiotics. The genes pac and dmpM, encoding a puromycin N-acetyl transferase and an O-demethyl puromycin O-methyltransferase, respectively, are tightly linked in the DNA of S. alboniger. The entire set of genes encoding the puromycin biosynthesis pathway was cloned by screening a gene library from S. alboniger, raised in the low copy number cosmid pKC505, with a DNA fragment containing pac and dmpM. Puromycin was identified by biochemical and physicochemical methods, including 1H NMR, in the producing transformants. This pathway was located in a single DNA fragment of 15 kb which included the resistance, structural and regulatory genes and was expressed when introduced into two heterologous hosts Streptomyces lividans and Streptomyces griseofuscus. In addition to pac and dmpM, two other genes have been identified in the pur cluster: pacHY, which determines an N-acetylpuromycin hydrolase and prg1, whose deduced amino acid sequence is significantly similar to that of degT, a Bacillus stearothermophilus pleiotropic regulatory gene.

Acetyltransferases↗
PubMed1978

Effect of puromycin treatment on the regeneration of hemisected and transected rat spinal cord.

The effect of puromycin on spinal cord regeneration was studied following implantation into the site of spinal cord hemi- or transection of Gel-foam saturated with puromycin (1 mM) in a saline carrier, implantation of Gel-foam sponge saturated with saline (carrier control), or lesion alone (lesion control). The spinal cords of 107 rats were studied with light and electron microscopy 7, 14, 30, 60 and 90 days postoperative (DPO). Spinal cord hemisected animals developed a dense cicatrix at the site of lesion replete with connective tissue, blood vessles, and myelinated and unmyelinated nerve fibres which could be traced to peripheral sources. Rostrally at the C.N.S.--cicatrix interface, there were reactive neuroglial cells, occasional nerve fibres and finger-like projections of spinal cord (due to cavitation lesions) which contained neuroglia, axons and dendrites. Implantation of saline in Gel-foam resulted in the same morphology as in hemisected animals except for increased lesion size due to mechanical factors and decreased cicatrix density during the first 30 DPO. Puromycin treatment resulted in a cicatrix with initial decreased cell density but which contained a new class of nerve fibres at 30 DPO. These nerve fibres were oriented in a rostro-caudal direction, were unmyelinated, 0.1-0.2 micron in diameter and had expanded smooth endoplasmic reticulum. Some of these nerve fibres were degenerating at 30 DPO and all were absent by 60 DPO. The puromycin-treated spinal cord within 200 micron rostral to the basal lamina contained nerve terminal conglomerates, which resembled boutons, in fascicles from 30-90 DPO (duration of experiment). Hemisection of the spinal cord by crushing 1-1 1/2 segments rostral to the site of puromycin implantation at 30 DPO resulted in degeneration of these nerve fibres in the cicatrix as well as the degeneration of nerve terminal conglomerates just rostral to the basal lamina. The regenerative capacity of the spinal cord is discussed in relationship to these findings.

Animals↗
PubMed1978

Peptidyl transferase substrate specificity with nonaromatic aminoacyl analogues of puromycin.

A series of puromycin analogues, 3'-N-(S-substituted L-cysteinyl) puromycin aminonuleosides, has been prepared and examined as substrates for ribosomal peptidyl transferase. S-Substituted N-tert-butyloxycarbonyl-L-cysteines were coupled with puromycin aminonucleoside using dicyclohexylcarbodiimide and N-hydroxysuccinimide. Removal of the t-Boc blocking group with anhydrous trifluoroacetic acid gave the desired puromycin analogues. Kinetic studies indicate that the nonaromatic aminoacyl analogues of puromycin are effective substrates for the peptidyl transferase reaction. In addition, the discovery of the existence of hydrophilic character beyond the region normally occupied by hydrophobic amino acid R groups of the aminoacyladenyl termini of tRNA molecules, and the proper exploitation of this information, has provided the first active purmoycin analogue possessing a hydrophilic amino acid.

Acyltransferases↗
PubMed1990

Two sensitivity levels of cattle oocytes to puromycin.

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.

Adenine↗