Effect of protein synthesis inhibitors on xylose uptake and 125I-insulin binding by rat soleus muscle

Prolonged exposure (90–180 min) to cycloheximide (0.2 mg/ml), puromycin (0.2 mg/ml) or chloramphenicol (0.1 mg/ml) did not affect ¹²⁵I-insulin binding by rat soleus muscle. Chloramphenicol (2 mg/ml) depressed insulin binding and insulin-stimulated xylose uptake; these effects were attributed to the “toxic” effect of chloramphenicol on muscle ATP levels. Cycloheximide and puromycin inhibited insulin-stimulated xylose uptake without affecting ATP. Puromycin and chloramphenicol, but not cycloheximide, also inhibited basal sugar transport. This difference, and the rapid onset of all these inhibitory effects, suggest that they are not due to the inhibition of protein synthesis, but rather to some more direct effect on sugar transport itself.     

Synthesis of N-acetylglucosaminyl asparagine-substituted puromycin analogues

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 increases with hydrophobicity of the modifying protecting groups.     

Dual effect of adrenalin on sugar transport in rat diaphragm muscle

The effect of adrenalin on the membrane transport of the non-metabolized sugar, 3-methylglucose, was studied in isolated "intact" rat hemidiaphragms and related to simultaneously occurring changes in the internal levels of Na+, ATP, glucose-6-P, glycerol formation and 45Ca uptake and loss. Basal sugar transport was inhibited by low (10-8-10-5 M) concentrations of adrenalin; this was antagonized by propranolol and practolol. High concentrations (10-4-10-3 M) stimulated sugar transport, and this was blocked by propranolol and butoxamine and was dependent on external Ca2+. These results suggest interaction with two different classes of adrenergic receptors, possibly of beta 1 and beta 2 types. Both low and high concentrations increases Na+ and K+ gradients by a practolol-sensitive effect. Isoproterenol behaved identically but phenylephrine had only the two practolol-sensitive effects on sugar and ion transport. Insulin did not interfere with inhibition of sugar transport and decrease in internal Na+ but prevented stimulation of sugar transport. Under anoxia adrenalin had no effect on sugar transport but led to greater Na+ gain by tissue. Addition of 3.0 mM palmitate decreased inhibition of sugar transport without changing receptor specificity. ATP was decreased and lipolysis enchanged by high adrenalin but glucose-6-P was increased by the low concentration as well. Influx of 45 Ca was decreased by low and increased by high adrenalin; 45Ca efflux was also differentially affected. The results indicate that inhibition and stimulation of sugar transport depend on different receptors and that the latter response may override the former. The data are consistent with the earlier postulated regulatory role of sarcoplasmic Ca2+ on sugar transport in muscle, with adrenalin affecting Ca2+ fluxes and distribution both directly and indirectly.     

Interaction of arginine with the ribosomal peptidyl transferase centre.

Arginine inhibits the formation of acetylleucyl-puromycin from C(U)-A-C-C-A-LeuAc and puromycin ('fragment reaction'), catalized by Escherichia coli and yeast ribosomes. From 18 different L-amino acids assayed, arginine was the most effective in producing inhibition (50% inhibition at 20 mM, with 1 mM puromycin). L-Argininamide and D-arginine gave about the same inhibition as L-arginine. The inhibition by L-arginine is competitive with respect to puromycin. The plot of the slopes obtained in a Lineweaver and Burk representation versus [Arg]2, and the plot of 1/v versus [Arg]2 at a fixed concentration of puromycin, are linear, which seems to indicate that two arginine molecules must interact at the puromycin binding site to produce inhibition. In addition to the 'fragment reaction', arginine inhibits the non-enzymatic binding of AcPhe-tRNA, C(U)-A-C-C-A-Leu and C(U)-A-C-C-A-LeuAc to ribosomes. However, it does not inhibit poly(U)-directed polyphenylalanine synthesis or the reaction of puromycin with AcPhe-tRNA previously bound to the peptidyl site. The results agree with arginine binding to the acceptor site, and with a sequential mechanism for the 'fragment reaction', puromycin binding first.     

Kinetics of inhibition of ribonuclease P activity by peptidyltransferase inhibitors – Effect of antibiotics on RNase P

A cell-free system derived from Dictyostelium discoideum has been used to study the kinetics of inhibition of RNase P by puromycin, amicetin and blasticidin S. Detailed kinetic analysis showed that the type of inhibition of RNase P activity by puromycin is simple competitive, whereas the type of inhibition by amicetin and blasticidin S is simple non-competitive. On the basis of K_i values amicetin is stronger inhibitor than puromycin and blasticidin S.     

The influence of uncouplers on proton-sugar symport in Saccharomyces fragilis

Uncouplers of oxidative phosphorylation inhibit proton-sugar symport in Saccharomyces fragilis. However, they do not induce efflux of accumulated sugar. It is shown that the effect cannot be explained by uncoupler-induced alterations in the transmembrane potential or transmembrane pH difference. It is also indicated that a decrease in intracellular pH is not involved in inhibition of sugar transport. It is argued that inhibition of transport by uncouplers is most likely caused by a direct interaction with the translocator.     

Density-dependent resistance to puromycin in cell cultures

Pig kidney cells exhibit resistance to puromycin when cultured at high cell densities, growing progressively in otherwise lethal concentrations of drug. Comparison of population growth of cultures inoculated at different densities indicates that survival in puromycin is a function of cell density. Uptake of puromycin from culture media is less at higher cell densities resulting in decreased inhibition of protein synthesis by puromycin. Enhanced survival in puromycin at high population densities is attributed to reduced accumulation of puromycin from the culture media.     

Inhibitors of protein synthesis cause increased hexose transport in cultured human fibroblasts by a mechanism other than transporter translocation.

We have investigated the effect of various inhibitors of protein synthesis on hexose transport in human skin fibroblasts using 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose (3-OMG) to measure hexose transport. Exposure of glucose-fed, serum-free cultures to cycloheximide (CHX) (50 micrograms/ml) for 6 h resulted in increased 2-DG transport (3.81 +/- .53 vs. 6.62 +/- .88 nmoles/mg protein/2 min; n = 9) and 3-OMG transport (1.36 +/- .66 vs. 3.18 +/- .83 nmoles/mg protein/30 sec; n = 4) in the CHX exposed group. Under these conditions inhibition of protein synthesis was greater than 90%. This CHX induced transport increase was time dependent (approaching maximum within 1 h of exposure to CHX) and related to an increase in the Vmax of hexose transport in the CHX exposed group (18.4 +/- 2.4 vs. 4.8 +/- 1.1 nmoles 2-DG/mg protein/min) with no difference in the transport Km (1.55 +/- .63 vs. 2.92 +/- .59 mM). Further, the CHX induced increase in hexose transport was reversible. Exposure of human fibroblasts to inhibitors of protein synthesis with different mechanisms of action (e.g., puromycin, pactamycin, or CHX) all generated hexose transport increases in a concentration-dependent fashion correlating with their increasing inhibitory effects on protein synthesis. Nucleotidase enriched (i.e., plasma membrane) fractions of control and CHX-exposed cells showed no differences in D-glucose inhibitable cytochalasin B binding activity. Further, quantitative Western analysis of nucleotidase enriched fractions indicated CHX exposure resulted in no significant increase in glucose transporter mass compared with control plasma membrane fractions. Glucose deprived cells, however, which exhibited increased sugar transport comparable to the CHX-exposed group, did show increased glucose transporter mass in the plasma membrane fraction. The data indicate that inhibitors of protein synthesis can cause a significant elevation in hexose transport and that the hexose transporter mass in the isolated plasma membrane fractions did not reflect the whole cell transport change. It is suggested that a mechanism other than glucose transporter translocation to the plasma membrane may be involved in causing this sugar transport increase.     

Effect of osmotic pressure on membrane energy-linked functions in Escherichia coli

Osmotic upshock of E. coli cells in NaCl or sucrose medium resulted in a large decrease in the cytoplasmic volume and the inhibition of growth, of the electron transfer chain and of four different types of sugar transport system: the lactose proton symport, the glucose phosphotransferase system, the binding-protein dependent maltose transport system and the glycerol facilitator. In contrast to NaCl and sucrose, the permeant solute glycerol had no marked effect. These inhibitions could be partially relieved by glycine betaine. Despite these inhibitions, the internal pH, the protonmotive force and the ATP pool were maintained. It is concluded that inhibition of electron transfer and of sugar transport is the consequence of conformational changes caused by the deformation of the membrane. It is also concluded that the arrest of growth observed upon osmotic upshock is not due to energy limitations and that it cannot be explained by the inhibition of carbohydrate transport.     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Direct interaction of phenylarsine oxide with hexose transporters in isolated rat adipocytes

It has previously been shown that phenylarsine oxide (PhAsO), an inhibitor of protein internalization, also inhibits stereospecific uptake of D-glucose and 2-deoxyglucose in both basal and insulin-stimulated rat adipocytes. This inhibition of hexose uptake was found to be dose-dependent. PhAsO rapidly inhibited sugar transport into insulin-stimulated adipocytes, but at low concentrations inhibition was transient. Low doses of PhAsO (1 microM) transiently inhibit stereospecific hexose uptake and near total (approx. 90%) recovery of transport activity occurs within 20 min. Interestingly, once recovered, the adipocytes can again undergo rapid inhibition and recovery of transport activity upon further treatment with PhAsO (1 microM). In addition, PhAsO is shown to inhibit cytochalasin B binding to plasma membranes from insulin-stimulated adipocytes in a concentration-dependent manner which parallels the dose-response inhibition of hexose transport by PhAsO. The data presented suggest a direct interaction between the D-glucose transporter and PhAsO, resulting in inhibition of transport. The results are consistent with the current recruitment hypothesis of insulin activation of sugar transport and indicate that a considerable reserve of intracellular glucose carriers exists within fat cells.     

Effect of the Inhibition of Protein Synthesis on the Establishment of Transformation by Polyoma Virus

Puromycin was used to study the effect of the inhibition of protein synthesis on transformation of hamster cells (BHK21) by polyoma virus. The drug was used at a concentration (10(-4)m) which caused in these cells a drastic but fully reversible inhibition of protein synthesis. A two- to threefold enhancement of transformation rate was obtained when the cells were exposed to puromycin for a period of 5 hr that started at the end of the virus adsorption period. No further enhancement was produced by prolonging puromycin treatment up to 13 hr after infection. The possibility that the observed effect on transformation rate could be mainly attributed to cell selection by puromycin was excluded. In addition, the relevance of a number of possible secondary effects of puromycin (inhibition of cell division, inhibition of deoxyribonucleic acid synthesis, etc.) was also ruled out. The effect of puromycin on transformation appeared to be dependent on the time (relative to infection) of addition of the drug. In fact, no transformation enhancement was observed when the cells were exposed to puromycin prior to infection or beyond the 10th hr after infection. Since another drug known to affect protein synthesis (p-fluorophenylalanine) was also shown to produce similar effects, it is suggested that transformation enhancement results from the inhibition of protein synthesis during a sensitive period closely following adsorption of the virus.     

Properties of the human erythrocyte glucose transport protein are determined by cellular context

Human erythrocyte hexose transfer is mediated by the glucose transport protein GLUT1 and is characterized by a complexity that is unexplained by available hypotheses for carrier-mediated sugar transport [Cloherty, E. K., Heard, K. S., and Carruthers, A. (1996) Biochemistry 35, 10411-10421]. The study presented here examines the possibility that the operational properties of GLUT1 are determined by host cell environment. A glucose transport-null strain of Saccharomyces cerevisiae (RE700A) was transfected with the p426 GPD yeast expression vector containing DNA encoding the wild-type human glucose transport protein (GLUT1), mutant GLUT1 (GLUT1(338)(-)(A3)), or carboxy-terminal hemagglutinin-polyHis-tagged GLUT1 (GLUT1-HA-H6). GLUT1 and GLUT1-HA-H6 are expressed at the yeast cell membrane and restore 2-deoxy-d-glucose, 3-O-methylglucose, and d-glucose transport capacity to RE700A. GLUT1-HA-H6 confers GLUT1-specific sugar transport characteristics to transfected RE700A, including inhibition by cytochalasin B and high-affinity transport of the nonmetabolized sugar 3-O-methylglucose. GLUT1(338)(-)(A3), a catalytically inactive GLUT1 mutant, is expressed but fails to restore RE700A sugar uptake capacity or growth on glucose. In contrast to transport in human red cells, K(m(app)) for 2-deoxy-d-glucose uptake equals K(i(app)) for 2-deoxy-d-glucose inhibition of 3-O-methylglucose uptake. Unlike transport in human red cells or transport in human embryonic kidney cells transfected with GLUT1-HA-H6, unidirectional sugar uptake in RE700A-GLUT1-HA-H6 is not inhibited by reductant and is not stimulated by intracellular sugar. Net uptake of subsaturating 3-O-methylglucose by RE700A-GLUT1-HA-H6 is a simple, first-order process. These findings support the hypothesis that red cell sugar transport complexity is host cell-specific.     

Uptake of Nitrite by Neurospora crassa

Like the nitrate transport system, the nitrite uptake system in Neurospora crassa is induced by either nitrate or nitrite. This induction is prevented by cycloheximide, puromycin, or 6-methyl purine. The K(m) for nitrite of the induced nitrite uptake system is 86 muM, and the V(max) is 100 mumol of nitrite per g (wet weight) per h. Nitrite uptake is inhibited by metabolic poisons such as arsenate, dinitrophenol, cyanide, and antimycin A. No repression or inhibition of the nitrite transport system by ammonia, nitrate, or Casamino Acids was observed.     

Characterization of the D-allose-mediated regulation of sugar transport in Chinese hamster fibroblasts

Exposure to D-allose has been demonstrated to lead to decreased 2-deoxy-D-glucose (2-DG) and 3-0-methyl-D-glucose transport in the V79 Chinese hamster lung fibroblast cell line. The effect of D-allose 1) was maximal after 4 hours exposure to the cells; 2) was optimal between 2.77 and 5.55 mM D-allose; and 3) led to a decreased Vmax for 2-DG transport with no change in the transport Km value. The decrease in 2-DG transport induced by D-allose was reversible and the reversal was differentially affected by cycloheximide, being blocked by a low concentration of cycloheximide (0.05 micrograms/ml) but not a high concentration of the inhibitor (5 micrograms/ml). D-allose did not competitively inhibit the transport of 2-DG while D-glucose under similar conditions yielded a Kl for 2-DG transport inhibition of 1.7 mM. Additionally, D-allose did not affect the phosphorylation of 2-DG by hexokinase in cell-free cytosol. The data indicate that D-allose has significant lowering effects on sugar transport activity. Additionally, while the sugar itself may be the active component in sugar transport regulation, the effect is not blocked by inhibition of protein synthesis but the synthesis of a regulatory protein(s) may be involved in the return of sugar transport following D-allose removal.     

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.     

Melanin counter act puromycin-induced inhibition of collagen and DNA biosynthesis in human skin fibroblasts

Puromycin is an experimental anti-tumor antibiotic acting through inhibition of protein synthesis. Because of its untoward side effects (as inner ear and renal lesions) the antibiotic was not approved for clinical trials. The mechanism underlying the organ specificity of the side effect is not understood. In view of the fact that a number of drugs form with melanin complexes that affect their pharmacological activity, we determined whether puromycin interacts with melanin and how this process affects biosynthesis of collagen in cultured human skin fibroblasts. Our results indicate that puromycin forms complexes with melanin. The amount of puromycin bound to melanin increases with increase of initial drug concentration. The Scatchard plot analysis of the drug binding to melanin has shown that at least two classes of independent binding sites are implicated in the puromycin-melanin complex formation: one class of strong binding sites with the association constant K1 = 1.84 x 10(6) M(-1), and the second class of weak binding sites with the association constant K2 = 5.26 x 10(3) M(-1). The number of total binding sites were n1 = 0.1260 and n2 = 0.2861 mumol puromycin per 1 mg melanin. We found that puromycin induced inhibition of collagen and DNA biosynthesis (IC50 approximately 2 microM). Melanin at 100 microg/ml produced about 20% inhibition of DNA synthesis, but it had no effect on collagen biosynthesis in cultured fibroblasts. However, the addition of melanin (100 microg/ml) to puromycin - treated cells (2 microM) abolished the inhibitory action of puromycin on collagen and DNA biosynthesis. We have suggested that IGF-I receptor expression, involved in collagen metabolism, may be one of the targets for puromycin - induced inhibition of collagen biosynthesis. It was found that melanin abolished puromycin induced decrease in the expression of IGF-I receptor as well MAP kinases expression: ERK1 and ERK2 as shown by Western immunoblot analysis. These data suggest that tissue specific pharmacological activity of puromycin may depend on the melanin abundance in tissues.     

ANISOMYCIN,ACETOXYCYCLOHEXIMIDE, CYCLOHEXIMIDE,AND PUROMYCIN AS INHIBITORS OF RAT BRAIN ACETYLCHOLINESTERASE IN VITRO1

A kinetic analysis of the interaction of anisomycin, acetoxycycloheximide, cycloheximide, and puromycin with acetylcholinesterase (acetylcholine acetyl-hydrolase, EC 3.1.1.7) in rat brain homogenate shows that all of these protein synthesis inhibitors are also inhibitors or this enzyme. Puromycitl aminonucleoside, a puromycin analog without antibiotic activity, was also found to be an inhibitor of acetylcholinesterase activity much like puromycin. Anisomycin appeared to be a competitive inhibitor whereas all of the other compounds showed mixed inhibition. The apparent 10.5 values for inhibition of rat brain acetylcholinesterase at 50 μM substrate were: anisomycin, 3 mM; acetoxycycloheximide, 1 mM; cycloheximide, 2.2 mM; puromycin, 0.5 mM and puromycin aminonucleoside, 0.6 mM.     

Uranyl action on sugar transport across rat jejunum

The effect of uranyl on sugar transport across rat jejunum has been studied in vitro and in vivo. D-glucose and D-galactose accumulation in jejunum rings at pH 6.0 is inhibited about 40-65% by 1 mM uranyl nitrate. This inhibition is lower than that produced by 0.5 mM phlorizin. The effect was very similar when the incubation of the rings with the sugar was made in the absence of uranyl, after preincubation with the inhibitor. Washing with 10 mM EDTA reverted uranyl inhibition only slightly. D-fructose entry was weakly inhibited by uranyl. Glucose absorption in vivo along perfusion periods of 1 min was not affected by the presence of uranyl (0.001 to 1 mM) in the sugar solution, but the exposure of the mucosa to 0.1 mM uranyl at pH 6.5 for 10 min inhibited sugar absorption at the same pH in the subsequent periods of perfusion. Results suggest that uranyl impairs sugar transport by binding to protein chemical groups at the surface or in deeper sites of enterocyte membranes, a process that requires some minutes to be accomplished.