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PubMed1975

Dendrites and neuroglia following hemisection of rat spinal cord: effects of puromycin.

Within 60 to 90 days of spinal cord hemisection or hemisection, Gelfoam, water implantation in the lesion site, ventral horn neurons 0 to 5 mm proximal to the site of lesion form varicosities along the dendrite. The varicosities form on peripheral dendrites at 10 days and progressively involve the entire dendritic shaft by 60 to 90 days. Following spinal cord hemisection and Gelfoam puromycin implantation the neurons in the ventral horn are resistant to varicosity formation. Sixty days after hemisection puromycin-treated neurons form a few varicosities on occasional peripheral dendrites with no further involvement of the dendrite by 90 days postlesion. The neuroglial reaction to hemisection is not altered by puromycin treatment. The correlation of inhibition of protein synthesis and resistance to dendritic varicosity formation is discussed.

Animals↗
PubMed1976

Termination of procollagen chain synthesis by puromycin. Evidence that assembly and secretion require a COOH-terminal extension.

Embryonic chick fibroblasts were incubated with [14C]proline and puromycin in the low concentrations of 1 to 3 mug/ml. The molecular weight of the synthesized procollagen chains, as measured by polyacrylamide gel electrophoresis in sodium dodecyl sulfate, was progressively reduced by increasing concentrations of puromycin in this range. For example, at 3 mug/ml the great majority of the [14C]proline was contained in procollagen chains having an average molecular weight of about 95,000 instead of the control value of 125,000. Associated with this decrease in molecular weight there was a marked decrease in the incorporation of cysteine although [14C]proline incorporation was relatively unaffedted. Disulfide bond formation was drastically inhibited as was triple helix formation as measured by resistance of the procollagen to pepsin digestion. Although the shortened procollagen chains were of normal hydroxyproline content, they nevertheless were secreted much more slowly than normal procollagen. Based upon these findings, we postulate that: (a) low concentrations of puromycin terminate procollagen chains before a COOH-terminal extension is completed, (b) these COOH-terminal extensions are required for normal assembly of the three individual procollagen chains and for triple helix formation, and (c) only assembled, triple helical procollagen molecules are selected for normal secretion.

Animals↗
PubMed1991

Translational inhibitors cycloheximide, emetine, and puromycin inhibit cellular autophagy in mouse liver parenchymal and pancreatic acinar cells in vivo.

The protein synthesis inhibitor cycloheximide is widely used (in vitro or in vivo) to inhibit the autophagic degradation of endogenous cellular proteins. Circumstantial evidence has been obtained largely from in vitro experiments for a similar effect of other translational inhibitors. In the present study, the in vivo effects of cycloheximide, emetine and puromycin on autophagy in murine exocrine pancreatic and liver cells were tested using electron microscopic morphometry. The experiments were based on the assumption that the autophagic compartment will regress if the formation of the vacuoles is blocked while degradation in the pre-existing vacuoles goes on. To make the measurements easier, autophagic compartment of the cells was greatly enlarged in both cell types by administering vinblastine (10 mg/kg b. wt.) for 2 h when the inhibitors were set on for an additional 30 min. During this half-an-hour, cycloheximide (0.2 mg/g b. wt.), emetine (0.12 mg/g b. wt.) and puromycin (0.2 mg/g b. wt.), respectively caused 58.5, 35.6, and 69.5% regression of the pancreatic and 46.7, 64.2, and 54.2% of the hepatocytic autophagic vacuole compartment. Thus, similarly to cycloheximide, both emetine and puromycin have proved to be inhibitors of autophagy in vivo. The results argue for a possible relationship between the synthesis and degradation of endogenous cellular proteins.

Animals↗
PubMed1989

Ultrastructural changes in rat-molar cementoblasts after administration of puromycin.

Ultrastructural changes in rat-molar cementoblasts after administration of puromycin were observed. Young Wistar male rats were injected with 200 mg/kg of puromycin dihydrochloride. At 10, 30, 60, and 180 minutes after the injection, the animals were fixed by perfusion with 2.5% glutaraldehyde-2.0% paraformaldehyde. After perfusion, the lower first molars were dissected out of the mandibles, and their cementoblast were observed in an electron microscope. Striking changes were seen in the Golgi apparatus and the rough endoplasmic reticulum after puromycin administration. The Golgi saccules were dilated and devoid of content. Golgi vacuoles and secretion granules were reduced in number. Numerous small, smooth-walled vesicles, 45-70 nm in diameter and a large number of coated vesicles were present in the Golgi area. The rough endoplasmic reticulum showed unusual shapes and arrangement. Ribosomes attached to the reticulum lost polysomal arrangement. These changes progressed following the time elapsed after experiment, and a great number of large vacuoles which contain fine flocculent material came to predominate the supranuclear areas of the cementoblasts.

Animals↗
PubMed1985

Dopamine-stimulated fucosylation of brain proteins in vitro is not inhibited by puromycin.

In slices of rat hippocampus, the influence of puromycin (0.5 mM) on dopamine (0.5 mM)-stimulated L-fucose incorporation into glycoproteins was studied. Puromycin (15 min, 2 h and 4 h pretreatment) inhibited protein synthesis by some -70% (estimated by the in vitro incorporation rate of L-leucine), whilst L-fucose incorporation into rat hippocampal glycoproteins was only inhibited about -20% (4 h pretreatment) compared to control values (100%). Under these experimental conditions, the dopamine-induced increase in both the L-fucose incorporation into glycoproteins and the activity of fucokinase was not affected by puromycin. These data suggest that these short-lasting dopamine actions do not depend on protein synthesis.

Animals↗
PubMed1987

Effect of cycloheximide or puromycin on induction of thermotolerance by heat in Chinese hamster ovary cells: dose fractionation at 45.5 degrees C1.

While studying the quantitative relationship between hyperthermia-induced heat shock proteins (HSPs) and thermotolerance (TT), we observed that heat induced a family of HSPs, particularly an HSP 70 family, that might be involved in the development of TT. When cells were heated for 10 min at 45.5 degrees C, they became thermotolerant to a second heat exposure at 45.5 degrees C, with a thermotolerance ratio of 5-6 at 10(-3) isosurvival at 12 h after heating. In parallel, during the 12-h interval, heat shock resulted in a 2-fold relative increase in the synthesis of three major HSP families (Mr = 110,000, 87,000, and 70,000). Rate of synthesis was expressed relative to total protein synthesis, as studied with one-dimensional polyacrylamide gels analyzed by counting radioactivity in selected protein bands. The increase of unique HSPs, if studied with two-dimensional gels, would probably be much greater. Furthermore, even though the development of TT was partially suppressed by treatment with cycloheximide (10 micrograms/ml) or puromycin (100 micrograms/ml) at concentrations that inhibited total protein synthesis by 96 or 99%, respectively, a family of HSP 70 was still preferentially synthesized. Nevertheless, when cells were labeled for 3 days, the total level of HSP families did not change either when TT developed after a triggering heat treatment or as the development of TT was partially inhibited by suppressing protein synthesis with cycloheximide or puromycin. Thus, TT could still occur when total levels of HSP families did not change and when synthesis of HSP families was less than in unheated control cells, which may imply that TT is unrelated to HSPs. However, the finding that the amount of TT increased with increased synthesis of both total protein and HSP families, as studied with different concentrations of cycloheximide or puromycin, suggests that heat-inducible proteins, in particular the observed preferential synthesis of the HSP 70 family, may be necessary for the development of TT.

Animals↗
PubMed1985

Localization of sites of photoaffinity labeling of the large subunit of Escherichia coli ribosomes by arylazide derivative of puromycin.

Previous work (Nicholson, A. W., Hall, C. C., Strycharz, W. A., and Cooperman, B. S. (1982) Biochemistry 21, 3797-3808) showed that [3H]p-azidopuromycin photoaffinity labeled 70 S Escherichia coli ribosomes and that photoincorporation into 50 S subunit proteins was in the order L23 greater than L18/22 greater than L15. In the present work we report on immunoelectron microscopic studies of the complexes formed by p-azidopuromycin-modified 50 S subunits with antibodies to the N6,N6-dimethyladenosine moiety of the antibiotic. The p-azidopuromycin-modified 50 S subunits appear to be identical to unmodified control subunits in electron micrographs. Complexes of modified subunits with antibodies to the N6,N6-dimethyladenosine moiety of p-azidopuromycin were visualized in micrographs. Individual subunits with a single bound antibody (monomeric complexes) and pairs of subunits cross-linked by a single antibody (dimeric complexes) were separately evaluated and showed similar results. Two regions of p-azidopuromycin photoincorporation were identified. The primary site, seen in about 75% of the complexes, is between the central protuberance and small projection, on the side away from the L7/L12 arm, in a region thought to contain the peptidyltransferase center. The secondary site, of unknown significance, is at the base of the subunit maximally distant from the arm. These placements are essentially identical to those we observed in analyses of puromycin photoincorporation (Olson, H. M., Grant, P. G., Cooperman, B. S., and Glitz, D. G. (1982) J. Biol. Chem. 257, 2649-2656) and quantitatively similar to evaluations of monomeric puromycin-50 S subunit complexes. The data support the placement of proteins L23, L18/22, and L15 at or near the peptidyltransferase center at the primary site and suggest, in addition, that the secondary site includes a genuine area of puromycin affinity.

Affinity Labels↗
PubMed1984

Changes in the expression of SRBC receptors on human lymphocytes caused by azathioprine and puromycine.

The expression of human peripheral T lymphocyte receptors for sheep red blood cells in vitro was investigated. Changes in the expression of SRBC receptors were analysed in successive culture days depending on the stimulation of lymphocytes by PHA or Con A and the action of immunosuppressive agents, such as azathioprine and puromycine. The SRBC receptor variability of expression in cultures of whole peripheral blood lymphocytes was compared with that of T lymphocytes subpopulation isolated by the rosetting method. The experiments demonstrated: a) the appearance of different morphological E rosette types in cultures stimulated by plant mitogens, b) the inhibition of SRBC receptor expression by azathioprine and puromycine on lymphocytes from stimulated and non-stimulated cultures, c) higher sensitivity of SRBC receptors to immunosuppressive agents on cultured isolated T lymphocytes, d) the difference of azathioprine and puromycine action on the ability to form all types of E rosettes in stimulated and nonstimulated cultures.

Azathioprine↗
PubMedDate not supplied

[Puromycin interacts with the donor (P) site of Escherichia coli ribosomes].

Puromycin inhibits the interaction of peptidyl-tRNA analogs AcPhe-tRNA Phe ox-red, AcPhe-tRNA Phe and FMet-tRNA f Met with the donor (P) site of Escherichia coli ribosomes. It affects both template-free and poly(U)-dependent systems. The inhibition is apparently due to direct competition for the P-site. On isolated 30S ribosomal subunits it was shown that the puromycin binding site is situated far from the peptidyl transferase center. Quantitative measurements of the inhibition revealed that the affinity constant of puromycin for the P-site is not less than its affinity for the A-moiety of the peptidyl transferase center [1.1 divided by 3.8) X 10(3) M-1).

Binding Sites↗
PubMed1980

[Puromycin action on intestinal lipid absorption in male and female rats].

The effect of puromycin on absorption balance and mucosal lipid distribution was studied during intestinal absorption of 14C oleic acid and 3H palmitic acid, using the isolated intestinal loop of male and female rats in situ. In puromycin-treated animals, mucosal lipid uptake was reduced and mucosal lipid content increased, particularly with oleic acid and in male rats. The phospholipids were significantly reduced. As revealed by mucosal accumulation of free fatty acids, esterification was particularly inhibited with oleic acid and in female rats. These results might explain why portal blood radioactivity was higher in treated rats than in the controls. Puromycin effect occurred at different stages of lipid absorption and varied with sex.

Animals↗
PubMed1994

[Role of tubular Na-K-ATPase in nephrotic syndrome induced by puromycin in the rat].

Na-K-ATPase is an ubiquitous enzyme involved in the tubular reabsorption process. Na-K-ATPase is specifically controlled in each nephron segment. Sodium retention is one of the main features of the nephrotic syndrome. Hypervolemia is found in most of the nephrotic syndromes in adults, suggesting a primary renal origin. In the puromycin-induced nephrotic syndrome in rats, the collecting duct is the site of sodium retention. We have shown that Na-K-ATPase activity is specifically enhanced in collecting ducts from rats with puromycin-induced nephrotic syndrome. The stimulation of Na-K-ATPase activity was independent of aldosterone and endogenous inhibitors of the Na-K-ATPase, suggesting a primary paracrin or cellular mechanism. We have demonstrated that two different isoforms of the Na-K-ATPase are coexpressed all along the rat nephron. In the puromycin-induced nephrotic syndrome, the activity of one isoform is specifically enhanced. These results demonstrate that the different isoforms of the Na-K-ATPase can be individually controlled.

Aldosterone↗
PubMed1975

Toxic effect of puromycin on erythrocyte membranes which is unrelated to inhibition of protein synthesis.

Exposure of rabbit or human erythrocytes to concentrations of puromycin as low as 7 x 10(-4)M for 2 hr causes damage to the cell membrane, as evidenced by increased susceptibility of the cells to hyposmotic lysis, increased cell rigidity, and ultrastructural changes consistent with severe membrane damage. Puromycin causes a concentration-dependent internalization of the erythrocyte membrane, resulting in vacuolization of the cells, at concentrations between 7 x 10(-4) M and 10(-2) M. Since the erythrocyte does not synthesize protein, the data indicate that puromycin has a direct toxic effect on erythroid cell membranes which is unrelated to its action in inhibiting the synthesis of protein.

Animals↗
PubMed1977

Puromycin inhibition of cholesterol absorption in the rat.

The effect of puromycin on the intestinal absorption of cholesterol has been studied in rats with indwelling catheters in the left thoracic lymphatic duct. Puromycin administration to female rats produced a marked depression of cholesterol absorption under conditions where the absorption of simultaneously administered fatty acid was also dramatically inhibited. The same treatment of male rats also produced a significant depression in cholesterol absorption, but was without effect on absorption of the fatty acid. Despite the depressions of lipid absorption in puromycin-treated animals, there was no accumlation of either cholesterol or fatty acid in the intestinal mucosa of either sex. Actinomycin D treatment of fasting male and female rats, receiving constant infusions of saline, had no effect on the rate of lymph production. This suggest that altered lymph production was not responsible for the depressed lipid absorption observed in fed animals treated with protein synthesis inhibitors. The selective inhibition of cholesterol absorption in male rats also precludes the possibility that the major effect of the inhibitor is on delayed gastric emptying.

Animals↗
PubMed1995

Degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M.

The degradation of dynorphin-related peptides by the puromycin-sensitive aminopeptidase and aminopeptidase M was examined using these peptides as alternate substrate inhibitors. Ki determinations showed that both aminopeptidases exhibit a higher affinity for longer dynorphin-related peptides, i.e., Ki for dynorphin A-17 = 23-30 nM with the Ki increasing to 25-50 microM for the enkephalin pentapeptides. Binding appears dependent not only on peptide length, but also on its sequence. With aminopeptidase M, as the peptide size increases from five to 10 amino acids, kcat remains relatively constant; however, as the peptide size increases beyond a decapeptide, kcat decreases significantly. With the puromycin-sensitive aminopeptidase, similar results were obtained except that kcat was greatest for the pentapeptide. Thus, if one considers kcat/Km as the relevant kinetic constant for estimating in vivo peptide hydrolysis, these results are consistent with the involvement of aminopeptidase M and the puromycin-sensitive aminopeptidase in the degradation of extended dynorphin-related peptides.

Amino Acid Sequence↗
PubMed1971

Puromycin-peptide bond formation with reticulocyte initiation factors M1 and M2.

The ability to form a "peptide" bond between various forms of Met-tRNA or Phe-tRNA and puromycin has been studied in the reticulocyte cell-free system. When Met-tRNA(F), fMet-tRNA(F), or N-acetylPhe-tRNA are used as substrate at low Mg(++) concentration (3 mM), reticulocyte initiation factors M(1) and M(2) (M(2A) + M(2B)) are required for puromycin-peptide synthesis. In contrast to bacterial systems, this reaction is also stimulated by the elongation factor T(1). When Met-tRNA(M) or Phe-tRNA is used as substrate, there is no M-factor requirement for the puromycin reaction; T(1) is absolutely required, and the reaction is stimulated by T(2). These studies indicate that reticulocyte factors M(1) and M(2) may function in part by placing the initiator tRNA into the P site. The detailed mechanism for mammalian initiation, however, may be more complex than that for bacterial systems.

Cell-Free System↗
PubMed2003

The Caenorhabditis elegans orthologue of mammalian puromycin-sensitive aminopeptidase has roles in embryogenesis and reproduction.

Mammals possess membrane-associated and cytosolic forms of the puromycin-sensitive aminopeptidase (PSA; EC 3.4.11.14). Increasing evidence suggests the membrane PSA is involved in neuromodulation within the central nervous system and in reproductive biology. The functional roles of the cytosolic PSA are less clear. The genome of the nematode Caenorhabditis elegans encodes an aminopeptidase, F49E8.3 (PAM-1), that is orthologous to PSA, and sequence analysis predicts it to be cytosolic. We have determined the spatio/temporal gene expression pattern of pam-1 by using the promoter region of F49E8.3 to control expression in the nematode of a second exon translational fusion of the aminopeptidase to green fluorescent protein. Cytosolic fluorescence was observed throughout development in the intestine and nerve cells of the head. Neuronal expression was also observed in the tail of adult males. Recombinant PAM-1, expressed and purified from Escherichia coli, hydrolyzed the N-terminal amino acid from peptide substrates. Favored substrates had positively charged or small neutral amino acids in the N-terminal position. Peptide hydrolysis was inhibited by the metal-chelating agent 1,10-phenanthroline and by the aminopeptidase inhibitors actinonin, amastatin, and leuhistin. However, the enzyme was approximately 100-fold less sensitive toward puromycin (IC50, 135 mum) than other PSA homologues. Following inactivation of the enzyme, aminopeptidase activity was recovered with Zn2+, Co2+, and Ni2+. Silencing expression of pam-1 by RNA interference resulted in 30% embryonic lethality. Surviving adult hermaphrodites deposited large numbers of oocytes throughout the self-fertile period. The overall brood size was, however, unaffected. We conclude that pam-1 encodes an aminopeptidase that clusters phylogenetically with the PSAs, despite attenuated sensitivity toward puromycin, and that it functions in embryo development and reproduction of the nematode.

Amino Acid Sequence↗
PubMed1981

Bound Ribosomes of Pea Chloroplast Thylakoid Membranes: Location and Release in Vitro by High Salt, Puromycin, and RNase.

The mode of attachment of 70S ribosomes to thylakoid membranes from pea leaves was studied by determining the proportion of the bound RNA which was released by various incubation conditions. The results supported a model in which several classes of bound ribosomes could be distinguished: (a) very tightly bound, not released by any conditions yet tested (20% of the total); (b) monomeric ribosomes attached by electrostatic interaction with the membranes (30 to 40% of the total) and released by high salt; and (c) polysomes, with some of the ribosomes attached by a combination of electrostatic interactions and insertion of the nascent polypeptide chain into the membrane. These required a combination of puromycin and high salt for release. Other ("hanging") ribosomes of the polysomes were inferred to be attached through mRNA but not actually attached to the membranes directly; they could be released by RNase under low salt conditions, as well as by puromycin plus high salt.To obtain these results, chloroplasts had to be prepared in media containing 0.2 molar Tris at pH 8.5. Using Tricine buffers at pH 7.5 yielded thylakoid membranes whose ribosomes were removed almost completely by high salt alone; these showed no response to puromycin. However, pH 7.5 had to be used in all cases for ribosome dissociation in high salt media, as the ribosome structure appeared to be degraded by high salt at pH 8.5, and release then occurred without the need for puromycin.The kinetics of ribosome release by high salt showed a rapid initial phase with a half-life of 20 seconds. The extent of release by high salt was very dependent on the temperature of the incubation. Plotting the data according to the Arrhenius interpretation shows a significant break at about 15 C, with apparent activation energy of 20 kilocalories per mole below that temperature and 5 kilocalories per mole above that temperature. This result suggests that membrane fluidity might be an important factor permitting release of ribosomes under high salt conditions.Electron microscope pictures of the washed thylakoids showed polysomes closely associated with the outer membranes of grana stacks, and with the stroma lamellae. Following digitonin treatment of the membranes and centrifugation, fractions enriched in Photosystem I and presumed stroma lamellae were also enriched in bound RNA.

Journal Article↗
PubMed1984

Heterologous enzyme immunoassay for puromycin aminonucleoside using beta-D-galactosidase as a label.

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.

Animals↗