Energy Poisons Research Articles

Ureidosuccinic acid permeation in Saccharomyces cerevisiae

Some strains of Saccharomyces cerevisiae exhibit a specific transport system for ureidosuccinic acid, which is regulated by nitrogen metabolism. Ureidosuccinic acid uptake occurs with proline but with ammonium sulfate as nitrogen source it is inhibited. The V for transport is 20–25 μmol/ml cell water per min. The apparent K m is 3 · 10 -5. For the urep1 mutant (ureidosuccinic acid permease less) the internal concentration never exceeds the external one. In the permease plus strain ureidosuccinic acid can be concentrated up to 10 000 fold and the accumulated compound remains unchanged in the cells. Energy poisons such as dinitrophenol, carbonyl cyanide- m-chlorophenyl-drazone (CCCP) or NaN 3 inhibit the uptake. No significant efflux of the accumulated compound occurs even in the presence of these drugs. The specificity of the permease is very strict, only amino acids carrying an α- N-carbamyl group are strongly competitive inhibitors. The high concentration capacity of the cells and the lack of active exit of the accumulated compound support the hypothesis of a carrier mediated active transport system.

Mode of action of yeast toxins: energy requirement for Saccharomyces cerevisiae killer toxin

The role of the energy status of the yeast cell in the sensitivity of cultures to two yeast toxins was examined by using 12K release from cells as a measure of toxin action. The Saccharomyces cerevisiae killer toxin bound to sensitive cells in the presence of drugs that interfered with the generation or use of energy, but it was unable to efflux 12K from the cells under these conditions. In direct contrast, the Torulopsis glabrata pool efflux-stimulating toxin induced efflux of the yeast 42K pool was insensitive to the presence of energy poisons in cultures. The results indicate that an energized state, maintained at the expense of adenosine 5'-triphosphate from either glycolytic or mitochondrial reactions, is required for the action of the killer toxin on the yeast cell.

Siderophore protection against colicins M, B, V, and Ia in Escherichia coli.

A variety of natural and synthetic siderophores capable of supporting the growth of Escherichia coli K-12 on iron-limited media also protect strain RW193+ (tonA+ ent-) from the killing action of colicins B, V, and Ia. Protective activity falls into two categories. The first, characteristic of enterobactin protection against colicin B and ferrichrome protection against colicin M, has properties of a specific receptor competition between the siderophore and the colicin. Thus, enterobactin specifically protects against colicin B in fes- mutants (able to accumulate but unable to utilize enterobactin) as predicted by our proposal that the colicin B receptor functions in the specific binding for uptake of enterobactin (Wayne and Neilands, 1975). Similarly ferrichrome specifically protects against colicin M in SidA mutants (defective in hydroxamate siderophore utilization). The second category of protective response, characteristic of the more general siderophore inhibition of colicins B, V, and Ia, requires the availability or metabolism of siderophore iron. Thus, enterobactin protects against colicins V and Ia, but only when the colicin indicator strain is fes+, and hydroxamate siderophores inhibit colicins B, V, and Ia, but only when the colicin indicator strain is SidA+. Moreover, ferrichrome inhibits colicins B, V, and Ia, yet chromium (III) deferriferrichrome is inactive, and ferrichrome itself does not prevent adsorption of colicin Ia receptor material in vitro. Although the nonspecific protection against colicins B, V, and Ia requires iron, the availability of siderophore iron for cell growth is not sufficient to bring about protection. None of the siderophores tested protect cells against the killing action of colicin E1 or K, or against the energy poisons azide, 2, 4-dinitrophenol, and carbonylcyanide m-chlorophenylhydrazone. We suggest that nonspecific siderophore protection against colicins B, V, and Ia may be due either to an induction of membrane alterations in response to siderophore iron metabolism or to a direct interference by siderophore iron with some unknown step in colicin action subsequent to adsorption.

Transport of maltose by Pseudomonas fluorescens W.

The system for uptake of maltose in Pseudomonas fluorescens W was inducible. Using a mutant strain unable to hydrolyze maltose, it was shown that maltose was taken up unaltered against a concentration gradient. Uptake of 14C maltose was only significantly inhibited by nonradioactive maltose or maltotriose. These were the only sugars that could displace accumulated radioactive maltose in the strain unable to hydrolyze maltose. Uptake exhibited saturation kinetics and was inhibited by energy poisons, indicating that this system was one of active transport. Sulfhydryl-binding reagents reversibly inhibited maltose uptake. No transport ability was lost when cells were subjected to osmotic shock. Using the protein-binding dye 7-diazonium-1, 3-naphthalene disulfonate a protein or proteins located in or external to the cell membrane was implicated in maltose transport. The hydrolysis of p-nitrophenyl-alpha-D-glucoside (PNPG) was used as an indirect measure of transport ability since penetration of PNPG, not its hydrolysis, was the rate-limiting step.

Transport of pyrimidine nucleosides in cells of Escherichia coli K 12.

1. The transport of pyrimidine mucleosides into cells of Escherichis coli has been investigated in mutant strains which cannot metabolize these nucleosides. Such cells transport and concentrate purimidine mucleosides several hindredfold. 2. The transport is inhibited by energy poisons and by sulfhydryl reagents. 3. Pyrimidine mucleosides compete mutually for transport. Adenosine is also a strong competitor while guanosine and inosine are weak competitors. 4. The rate of pyrimidine mucleoside transport is shown to be under control of the cytR and deoR gene products, which are also known to regulate the synthesis of nucleoside-catabolizing enzymes. The transport system is repressed by growth on glucose, as is the synthesis of the enzymes.

Transport of Sugars and Amino Acids in Bacteria<subtitle>XV. Comparative Studies on the Effects of Various Energy Poisons on the Oxidative and Phosphorylating Activities, and Energy Coupling Reactions for the Active Transport Systems for Amino Acids in <italic>E. coli</italic><xref ref-type="fn" rid="fn1"><sup>1</sup></xref></subtitle>

The effects of various energy poisons on oxidation of respiratory substrate, synthesis of cellular ATP, and energy transformation reaction in intact Escherichia coli cells were studied systematically. Various mutants were, therefore, used in which specific functions in the energy-transducing reactions are defective or altered. The energy poisons examined were: sodium azide, DPPA and azidebenzenes which are inhibitors of respiratory-chain phosphorylation, SF6847, and CCCP which are known to be uncouplers, zinc sulfate which is an inhibitor for certain dehydrogenases, and sodium arsenate and sodium fluoride which are inhibitors of glycolytic synthesis of ATP. The preferential inhibitions occurred in the oxidation reactions with certain respiratory substrates by energy poisons used. DPPA inhibited glycerol oxidation much more strongly than succinate oxidation. However, DPPA could inhibit the oxidation of both glycerol 3-phosphate and succinate by membrane fraction strongly while the oxidation of NADH and D-lactate slightly. It inhibited glycerol 3-phosphate dehydrogenase [EC 1. 1. 2. 1] strongly as well as succinate dehydrogenase [EC 1. 3. 99. 1], but not D-lactate dehydrogenase of membrane fraction. MAB and other azidebenzene derivatives inhibited succinate oxidation preferentially. SF6847 and CCCP inhibited succinate oxidation strongly, while sodium azide inhibited it weakly and these three poisons were less inhibitory for glycerol oxidation. DPPA, sodium azide, SF6847, and CCCP inhibited the synthesis of ATP coupled with respiration but not with glycolysis. Zinc sulfate inhibited the cellular ATP synthesis coupled with either respiration or glycolysis. The effects of these energy poisons on the active transport reactions for isoleucine and proline were examined in detail. SF6847, CCCP, and sodium azide inhibited the active transport reaction for proline preferentially to that for isoleucine. These effects were distinct particularly in a mutant which lacks coupling factor of the oxidative phosphorylation. DPPA was an inhibitor which preferentially block active transport reaction for isoleucine. Zinc sulfate inhibited strongly the active transport reaction for isoleucine but was without effect on that for proline, coupled with endogenous sources of energy. These effects were consistent with its effect on cellular synthesis of ATP. Zinc sulfate extensively enhanced the steady level of proline accumulation which was coupled to D-lactate, without changing the Kt value for entry of proline. This was explained by assuming that the exchange-exit reaction for proline was greatly affected by zinc sulfate.

Leucine transport in Escherichia coli. The resolution of multiple transport systems and their coupling to metabolic energy.

The multiple active transport systems mediating L-leucine accumulation in Escherichia coli strain 7 (K12) and ML 308-225 have been examined. In addition to the previously characterized osmotic shock-sensitive LS (L-leucine-specific) and LIV-I (L-leucine; L-isoleucine-, and L-valine-specific) activities, a third system (designated LIV-II) has been detected, confirming a report by Rahmanian et al. (RAHMANIAN, M., CLAUS, D.R., and OXENDER, D. L. (1973) J. Bacteriol. 116, 1258-1266). This third system transports L-leucine, L-isoleucine, and L-valine with a relatively low affinity (apparent transport Km equals 4 muM for L-leucine) and it is resistant to repression by cell growth on L-leucine. Exploitation of these properties and of the differential sensitivity of the three transport activities to inhibition by L-leucine analogues permits estimation of the contribution by each system to the total transport activity under varying conditions. Such experiments show that, unlike systems LS and LIV-I, system LIV-II is resistant to osmotic shock. The L-leucine, L-iosleucine, and L-valine transport activity in membrane vesicles from strain ML 308-225 has the properties of system LIV-II. Although the L-leucine transport activities in strains 7 and ML 308-225 are in all other respects similar, membrane vesicles from strain 7 do not transport L-leucine, L-isoleucine, or L-valine. L-leucine transport under various conditions of energy supply has been measured in strain ML 308-225 and the corresponding Mg-2+-ATP-ASE-DEFICIENT STRAIN, DL-54. These measurements support the view that the osmotic shock-sensitive LS and LIV-I activities depend on the synthesis of ATP, while the osmotic shock-resistant LIV-II activity depends on the energized membrane state generated by electron flow but not on ATP synthesis, per se. This conclusion is not supported by the inhibitory effects of the energy poisons arsenate and 2,4-dinitrophenol, but these compounds may have secondary chemical effects on the transport systems.

Unimportance of counterflux in the energetics of "downhill" transport.

Adam Kepes suggested that the cellular transport and hydrolysis of orthonitrophenyl-beta-d-galactopyranoside is powered by the counterflux of the d-galactose resulting from beta-galactosidase action within the cell. His explanation would rationalize the unique insensitivity of this galactoside transport to energy poisons such as azide. But contrary to the predictions of this hypothesis, (i) there is no initial large inhibition that progressively lessens as galactose is produced. This was shown with a double wavelength stopped-flow spectrophotometer developed to eliminate interference from turbidity transients. (ii) The azide sensitivity does not increase with an external concentration of galactose sufficient to reverse the thermodynamic gradient. (iii) Mutation in galactose utilization or growth on highly catabolite-repressing regimens did not increase the azide sensitivity, and induction of galactose transport and metabolism did not decrease azide sensitivity. It was found that Kepes measurements must have contained two artifacts. One is that the control rate of hydrolysis decreases with time as the dense cell suspension becomes anaerobic. The other is that azide causes turbidity changes for some time after its introduction. If the former is avoided by magnetic stirring and the latter by double wavelength spectrophotometry or controls without substrate, the inhibition is constant from the earliest time that can be measured. It is therefore concluded that energy-unstarved cells, exposed to azide, still have adequate energy reserves to couple to the downhill transport, although their potential is not adequate to drive accumulation against a concentration gradient.

Direct Measurement of the Binding of Labeled Sugars to the Lactose Permease M Protein

Abstract Previous studies of the interaction of sugars with the membrane protein (M protein) component of the lactose permease system have employed an indirect procedure based on the protection of a reactive cysteine in the M protein against attack by labeled N-ethylmaleimide when certain sugars are bound. A method has now been devised for measuring the binding of labeled sugars to M protein directly, avoiding the use of N-ethylmaleimide. The particulate, membrane-containing fraction is equilibrated with 50 µm β-[3H]galactosyl-1-thio-d-β-galactoside (thiodigalactoside), in phosphate buffer containing 32P equal to the total count of 3H. The membrane fragments are then sedimented by high speed centrifugation, and, after careful removal of the supernatant (but without washing), the pellet is resuspended in a detergent solution and counted. The absorption of thiodigalactoside to the membrane fragments is revealed by an increment in the ratio of 3H to 32P. Control experiments show that 32P equilibrates with at least 95% of the total aqueous compartment of the pellet. The increment in the ratio of 3H: 32P is, therefore, not the result of the preferential filling of cell-free vesicles by the radioactive sugar. This binding reaction is not affected by energy poisons such as azide. Membranes from cells that have not been induced for the lac system and thus contain no M protein have no specific binding sites for thiodigalactoside as defined by this assay. The specific binding of thiodigalactoside involves a limited number of saturable binding sites. The value for half-saturation with thiodigalactoside is about 67 µm, in excellent agreement with previous studies of the affinity of the M protein for thiodigalactoside obtained with the indirect N-ethylmaleimide procedure. The site for binding thiodigalactoside (Site II) has also been found to have a surprising affinity for certain α-galactosides, such as p-nitrophenyl-α-galactoside, for which the Kd is about 7 µm. Other properties of the system are described. A procedure is described for the preparation and purification of tritiated thiodigalactoside.

Effect of inhibition of macromolecule synthesis on the association of bacteriophage T4 DNA with membrane.

The "Mg(2+)-Sarkosyl crystals" (M band) technique distinguishes between membrane-bound and free intracellular DNA. This procedure was employed to investigate the nature of the reactions necessary to convert input T4 DNA to a rapidly sedimenting form. Energy poisoning inhibits this attachment reaction. Neither protein nor DNA synthesis appears to be required, but experiments with rifampin and extensively irradiated T4 suggest that RNA synthesis is involved. These results were confirmed by a second procedure for the determination of rapidly sedimenting DNA.

Glutamate transport in Escherichia coli K-12: nonidentity of carriers mediating entry and exit.

The exit of glutamate from Escherichia coli K-12 cells preloaded with the radioactive amino acid and its relation to the reaction of entry were studied. Experiments with cells preloaded to different intracellular concentrations of radioactive glutamate confirmed our earlier conclusion that glutamate exit was a first-order reaction. l-Glutamate, competitive inhibitors of glutamate uptake (d-glutamate and l-glutamate-gamma-methyl ester), noncompetitive inhibitors of glutamate uptake (l-serine and l-alanine), and the energy poison NaN(3) all accelerated glutamate exit 2.8-fold. No additive effect was observed in the presence of NaN(3) together with l-glutamate. Preloading with cold l-glutamate did not increase the rate of uptake of radioactive glutamate. It is concluded that the acceleration of glutamate exit in the presence of l-glutamate in the medium is not due to exchange diffusion and that l-glutamate and azide affect exit indirectly by preventing recapture. Sucrose, 25%, slowed down glutamate exit by a factor of about 4.7 and increased the steady-state level of glutamate accumulation to about the same extent. Increasing the intracellular K(+) concentration enhanced glutamate uptake but did not affect the half-time of exit. It is concluded that separate carriers are most probably involved in mediating the entry and exit reactions.

A new spin-labeled substrate for β-galactosidase and β-galactoside permease

A spin-labeled β-galactoside has been synthesized. It is a substrate for β-galactosidase and is accumulated by E. coli ML 308-225 and K12-N2244. The accumulation appears to be active transport via the lactose permease since it is inhibited with lactose and the energy poison, amytal. Induction of the K12 strain with isopropylthiogalactoside results in a 100-fold stimulation of uptake of the spin-labeled β-galactoside. Binding of the spin-labeled sugar to membranes derived from the ML strain is inhibited by lactose.

Transport of Succinate in Escherichia coli: II. CHARACTERISTICS OF UPTAKE AND ENERGY COUPLING WITH TRANSPORT IN MEMBRANE PREPARATIONS

Abstract Membrane vesicles prepared from a strain of Escherichia coli which lacks membrane-bound succinate dehydrogenase and fumarate reductase activity are capable of accumulating succinate in the presence of d(-)lactate or reduced phenazine methosulfate as electron donors. The steady state, intramembranal concentration of succinate reaches 30- to 50-fold of the external concentration. Both the initial and the steady state rates are higher with ascorbate-phenazine methosulfate than with d(-)lactate. ATP, ADP, and various other metabolic intermediates do not stimulate succinate transport. Magnesium ions are required for maximum uptake. Monovalent cations, such as Na+, K+, or Li+ also stimulate transport to varying degrees. With ascorbate-phenazine methosulfate as electron donor the Michaelis constant for succinate is about 10 µm, and the inhibition constants for fumarate and l-malate are 12 µm and 22 µm, respectively. Both the ascorbate-phenazine methosulfate-dependent and d(-)lactate-dependent uptake of succinate are inhibited by energy poisons, uncouplers, and various other respiratory chain inhibitors. Compounds known to interfere with phosphorolytic and transphosphorylation reactions are without significant effect on transport. Amongst the thiol reagents, N-ethylmaleimide and showdomycin do not inhibit succinate uptake even at very high concentrations, but p-chloromercuribenzoate, Hg++ and Ag++ are very potent inhibitors. Both N-ethylmaleimide and p-chloromercuribenzoate, however, strongly inhibit d-lactate oxidation. From these observations, and from oxamate inhibition data, it is concluded that electrons are coupled to succinate transport directly at the initial step of lactate dehydrogenation. It is proposed that succinate uptake is mediated by alternate reduction and oxidation of a succinate carrier in membranes. Membranes prepared from strains carrying genes dct A or dct B are defective in the uptake of succinate. It thus appears that at least two genetic loci are involved in the formation of the succinate transport system in the membranes.

Transport of Succinate in Escherichia coli: I. BIOCHEMICAL AND GENETIC STUDIES OF TRANSPORT IN WHOLE CELLS

Abstract A mutant of Escherichia coli unable to metabolize succinate has been used to study the nature of succinate transport in whole cells. It is demonstrated that cells accumulate succinate against a concentration gradient. The uptake of succinate is competitively inhibited by fumarate and malate. The Michaelis constants for the dicarboxylic acids are all in the range of 15 to 30 µm. The accumulation of succinate is prevented by various energy poisons and sulfhydryl reagents. Inhibitors of mitochondrial dicarboxylate transport are without any effect on the succinate uptake system of E. coli. The influx and efflux of succinate in whole cells is temperature dependent. The influx increases rapidly between 15 and 36° and efflux between 36–50°. The transport system is induced by succinate and repressed by glucose. Mutants lacking adenyl cyclase and some lacking enzyme I of phosphotransferase system acquire succinate transport activity when grown in the presence of succinate and cyclic 3',5'-AMP. Mutants of E. coli unable to transport succinate fall into two broad categories which are referred to as dct (dicarboxylate transport) and ct (carboxylate transport). The dct mutants fail to grow on dicarboxylic acids (fumarate, malate, and succinate) but grow normally on the monocarboxylic acid, lactate. The ct mutants do not grow on either the dicarboxylic acids or on lactate. Both classes of mutants grow well on acetate. The dct class of mutants can be genotypically separated into two categories. The dct A mutants map at around 69 min, and the dct B mutant maps at about 17 min (linked to galactose locus) on the genetic map. The ct mutants are also linked to galactose. It is inferred from these experiments that there are at least two components involved in the transport of succinate in E. coli. It is suggested that the ct gene possibly specifies a carboxylate binding protein. This suggestion is based on the evidence that osmotically shocked cells of E.coli lose the capacity for uptake of succinate.

Effects of colicins E1 and K on permeability to magnesium and cobaltous ions.

The energy-dependent exchange of intracellular Mg(2+) with extracellular Mg(2+) or Co(2+) is inhibited by colicin E1 and, less strongly, by colicin K. Treatment with either colicin causes a net loss of intracellular Mg(2+). This loss begins immediately in cells treated with colicin E1, but in colicin K-treated cells the onset of Mg(2+) loss is delayed 1 to 10 min, depending upon the temperature and the multiplicity of colicin K. Both colicins differ from chemical inhibitors of energy-yielding metabolism; energy poisons block transport of Mg(2+) and Co(2+), but both colicins increase passive permeability to Mg(2+) and Co(2+). Inhibitors of energy-yielding metabolism (and of Mg(2+) exchange) block the initiation of Mg(2+) loss by either colicin, but do not stop colicin-promoted efflux once it has begun. Colicin E1 added before colicin K prevents the more rapid Mg(2+) efflux characteristic of colicin K-treated cells. Quantitative comparisons of the effects of colicins E1 and K upon permeability to Mg(2+) and Co(2+) lead us to conclude that the two colicins are not identical in their mode of action.

Energy expenditure is obligatory for the downhill transport of galactosides

Galactoside permease-containing cells of Escherichia coli have been depleted of energy reserves to such a low level that uptake of α-methyl-glucoside and cellular hydrolysis of o-nitrophenyl-β- d-galactopyranoside (ONPG) no longer take place. In all previous studies these transport processes had been found to be resistant to energy poisons and starvation. The cells' energy supplies can be sufficiently depleted by inducing the glucose permease and then making the cells pump α-methyl-glucoside to exhaustion in the absence of a utilizable energy source and in the presence of azide. Such cells recover their measured transport capabilities when a utilizable energy source is added back. The recovery is sufficiently fast and takes place in the presence of protein synthesis inhibitors which eliminates the possibility that protein synthesis is needed for recovery. Since the cellular hydrolysis assay of ONPG is dependent on permease, but constitutes a transport process from the outside region of high concentration to the inside where the concentration is very low, transport even down a thermodynamic electrochemical potential requires energy expenditure. I must therefore retract the concepts previously proposed (Koch, 1964); I now conclude that permease-mediated transport per se is dependent on energy and that energy coupling to transport, even down a gradient, is obligatory.

Metabolism of T4 bacteriophage ghost-infected cells: effect of bacteriophage and ghosts on the uptake of carbohydrates in Escherichia coli B.

Ghosts of T4 bacteriophage inhibit the uptake of thiomethyl-beta-galactoside (TMG), alpha-methylglucoside, glucose-6-phosphate, and glycerol in Escherichia coli B. The transport of orthonitrophenyl-beta-galactoside (ONPG) is also inhibited to a lesser degree and without alteration of the apparent K(m) of transport. These effects of ghosts parallel those of energy poisons on these systems. However, no one energy poison can produce such pronounced inhibitory effects in all these systems. The effect of the intact phage in these systems was either absent or very slight relative to the ghost. The effect of ghosts on the uptake of TMG was not immediate; at 10 C, no effect of the ghosts was apparent for at least 2 min. This suggests that a step, more temperature dependent than the attachment of the ghost, is necessary for the inhibitory action. The intracellular level of adenosine triphosphate (ATP) in the ghost-infected cells fell to less than 25% of the control value, and the ATP lost from the cell appeared in extracellular medium. Phage, on the other hand, caused no decrease in the intracellular ATP level. This loss of ATP from the cells after ghost infection suggests an alteration of the barrier properties of the membrane so that ATP can leave the cell; however, the accessibility of extracellular ONPG to intracellular beta-galactosidase does not increase. The dissimilarity of the actions of phage and ghosts on all properties examined does not support the model that the initial events in their infections are identical but that the intact phage, unlike the ghost, can provide information for the repair of its effects.

Nucleoside transport by Novikoff rat hepatoma cells growing in suspension culture: Specificity and mechanism of transport reactions and relationship to nucleoside incorporation into nucleic acids

The transport of adenosine, guanosine, inosine, uridine and cytidine by Novikoff rat hepatoma cells (subline N1S1-67) is competitively inhibited by each of the other nucleosides, thymidine, persantin and phenethyl alcohol. Comparisons of the transport kinetics of the various nucleosides ( K m and v max), of the K m / K i ratios for the inhibitions and of the effect of heat shock (47.5°, 5 min) on nucleoside transport suggest that guanosine and inosine are transported by a single system, whereas different specific systems transport adenosine and the pyrimidine nucleosides. Uridine and cytidine also seem to be transported by a single system. All systems are inactivated to about the same extent by treatment of the cells with p-chloromercuribenzoate. Incubation of the cells in media containing 2% trypsin or chymotrypsin, 1% neuraminidase or 0.025% phospholipase C for 15 min has no significant effect on uridine transport, and energy poisons have an effect only at relatively high concentrations. The incorporation of each of the nucleosides into nucleic acids by the cells also follows simple Michaelis-Menten kinetics and the apparent K m for each nucleoside is similar to that of its transport into the cell. Competitive inhibition of nucleoside transport by heterologous nucleosides, persantin or phenethyl alcohol results in an apparent competitive inhibition of nucleoside incorporation into nucleic acids and the apparent K i values for the inhibitions are similar for both processes. The results suggest that the rate-limiting step in the incorporation of each of the nucleosides into nucleic acids is its transport into the cell.

Nucleoside transport by Novikoff rat hepatoma cells growing in suspension culture: Specificity and mechanism of transport reactions and relationship to nucleoside incorporation into nucleic acids

The transport of adenosine, guanosine, inosine, uridine and cytidine by Novikoff rat hepatoma cells (subline N1S1-67) is competitively inhibited by each of the other nucleosides, thymidine, persantin and phenethyl alcohol. Comparisons of the transport kinetics of the various nucleosides ( K m and v max), of the K m K i ratios for the inhibitions and of the effect of heat shock (47.5°, 5 min) on nucleoside transport suggest that guanosine and inosine are transported by a single system, whereas different specific systems transport adenosine and the pyrimidine nucleosides. Uridine and cytidine also seem to be transported by a single system. All systems are inactivated to about the same extent by treatment of the cells with p-chloromercuribenzoate. Incubation of the cells in media containing 2% trypsin or chymotrypsin, 1% neuraminidase or 0.025% phospholipase C for 15 min has no significant effect on uridine transport, and energy poisons have an effect only at relatively high concentrations. The incorporation of each of the nucleosides into nucleic acids by the cells also follows simple Michaelis-Menten kinetics and the apparent K m for each nucleoside is similar to that of its transport into the cell. Competitive inhibition of nucleoside transport by heterologous nucleosides, persantin or phenethyl alcohol results in an apparent competitive inhibition of nucleoside incorporation into nucleic acids and the apparent K i values for the inhibitions are similar for both processes. The results suggest that the rate-limiting step in the incorporation of each of the nucleosides into nucleic acids is its transport into the cell.

An Additional Step in the Transport of Iron Defined by the tonB Locus of Escherichia coli

Abstract The uptake of iron by cells of Escherichia coli involves at least three components. One component is chelators of iron which are excreted into the medium by iron-deficient cells. One function of these dihydroxybenzoylserine-containing compounds is to increase the availability of transportable ferric ion to the cells by solubilization of hydroxypolymers of iron. Another component is a tonB-associated step which is characterized by a high affinity of the cells for ferric ion. Point and deletion mutants of the tonB locus are resistant to phage T1, unable to grow normally on iron-deficient medium, and transport ferric ion with a Km 10-fold higher than for wild type cells. Also involved in the uptake of iron is an energy-dependent component which can be demonstrated by treatment of wild type or tonB- cells with azide (30 mm) and iodoacetamide (1 mm). The energy poisons decrease the Vmax of iron accumulation by approximately 10-fold, but they do not alter the apparent Km.