Sodium-ion stimulated amino acid uptake in membrane vesicles of alkalophilic Bacillus no. 8-1

Membrane vesicles were isolated from alkalophilic Bacillus No. 8-1, and the active transport of amino acids was studied. The transport of amino acids was dependent upon substrate oxidation and the presence of Na+. Concentrative uptake of amino acids was stimulated by the addition of an artificial electron donor system, ascorbate-phenazine methosulfate (PMS), and to a lesser extent by NADH, while succinate, L-lactate, and alpha-glycerol-phosphate did not stimulate the uptake. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and cytochrome c were able to replace PMS, and reduced forms of these compounds were also very efficient electron donors. Amino acid transport was dependent on electron transfer, and inhibition of NADH oxidation by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), and sodium azide directly prohibited serine transport. The pH optima for serine transport lay between pH 8 and 9 for all energy sources. Sodium ion stimulated serine transport in the presence of NADH, NADH plus cytochrome c or succinate plus PMS, but had no stimulatory effect on the corresponding dehydrogenase activities. Sodium ion was also required for accumulation of serine in response to an artificial membrane potential where the respiratory chain was not operative. These results indicated that the stimulatory effect of Na+ on amino acid uptake was on the transport process itself.     

Inhibition of oxidative phosphorylation by organotin thiocarbamates

A series of triphenyl-, tricyclohexyl- and tribenzyltin compounds have been synthesized and examined as inhibitors of mitochondrial oxidative phosphorylation. All compounds tested inhibit oxidative phosphorylation linked to succinate oxidation by potato tuber mitochondria. All of the organotin compounds inhibit ADP-stimulated O2 uptake linked to succinate oxidation with concentrations for 50% inhibition in the range 2-50 microM. This inhibition is not due to inhibition of electron transport from succinate to O2 per se: none of the organotin compounds at 50 microM substantially inhibit the rate of succinate oxidation in the presence of 2,4-dinitrophenol. Representative organotin compounds at 0.5-50 microM do not act as uncouplers of succinate oxidation. It is concluded that the organotin compounds act as energy transfer inhibitors to inhibit oxidative phosphorylation in potato tuber mitochondria. A similar mode of action of representative organotin compounds was found with rat liver mitochondria. These organotin compounds inhibit a hydrophobic Ca2+-dependent plant protein kinase in the absence but not in the presence of thiols.     

Different mechanisms of energy coupling for transport of various amino acids in cells of Mycobacterium phlei.

Whole cells of Mycobacterium phlei were shown to actively accumulate proline, leucine, lysine, tryptophan, histidine, glutamine, and glutamic acid to different steady state levels. The transport of proline, in contrast to that of other amino acids, was found to be insensitive to various respiratory inhibitors, e.g. cyanide, arsenate, azide, and sulfhydryl reagents. However, oxygen was an obligatory requirement for the uptake of proline, as well as for the other amino acids. The results indicate that the energy requirements for proline uptake are different from those of other amino acids. In contrast to the system from Escherichia coli, the mode of energy transduction for the uptake of proline, glutamine, and glutamic acid is different even though these amino acids are shock resistant in the M. phlei system.     

Effects of phthalate esters on the respiration of rat liver mitochondria.

The effects of phthalate esters on the oxidation of succinate, glutamate, beta-hydroxybutyrate and NADH by rat liver mitochondria were examined and it was found that di-n-butyl phthalate (DBP) strongly inhibited the succinate oxidation by intact and sonicated rat mitochondria, but did not inhibit the State 4 respiration with NAD-linked substrates such as glutamate and beta-hydroxybutyrate. However, oxygen uptake accelerated by the presence of ADP and substrate (State 3) was inhibited and the rate of oxygen uptake decreased to that without ADP (State 4). It was concluded that phthalate esters were electron and energy transport inhibitors but not uncouplers. Phthalate esters also inhibited NADH oxidation by sonicated mitochondria. The degree of inhibition depended on the carbon number of alkyl groups of phthalate esters, and DBP was the most potent inhibitor of respiration. The activity of purified beef liver glutamate dehydrogenase [EC 1.4.1.3] was slightly inhibited by phthalate esters.     

Effect of dicyclohexylcarbodiimide on growth and membrane-mediated processes in wild type and heptose-deficient mutants of Escherichia coli K-12

The effects of N,N'-dicyclohexylcarbodiimide (DCCD) on the growth of Streptococcus faecalis, and on the growth, beta-galactosidase synthesis, and various membrane-mediated processes, were studied in wild-type Escherichia coli JE1011 and its lipopolysaccharide-defective mutant NS1. DCCD (0.1 mM) completely inhibited the growth of S. faecalis and E. coli NS1 but had little effect on strain JE1011. The same amount of DCCD with E. coli NS1, but not with E. coli JE1011, inhibited the induction of beta-galactosidase, increased the permeability of the cells to o-nitrophenyl-beta-d-galactoside without causing extensive cell lysis or release of ultraviolet-absorbing materials, and inhibited the oxidation of certain intermediates of the tricarboxylic acid cycle. Inhibition of the oxidation of malate, fumarate, and alpha-ketoglutarate by DCCD appeared to be at the level of the transport system for these compounds. Inhibition of the membrane-bound adenosine triphosphatase by DCCD was not entirely responsible for these effects, since oxidation of these substances, and transport of [(14)C]succinate and [(14)C]fumarate, was inhibited by DCCD in a mutant, N(144), which lacked adenosine triphosphatase activity. It is concluded that lipopolysaccharide forms a barrier to DCCD in wild-type E. coli, and that DCCD can inhibit several processes in the cell.     

Relationship between phospholipid compositions and transport activities of amino acids in Escherichia coli membrane vesicles.

Cells of Escherichia coli were incubated in broth medium in the presence of 5 mM of hydroxylamine which completely inhibited growth but did not affect viabilities. Hydroxylamine is known to inhibit phosphatidylserine decarboxylase. A large amount of phosphatidylserine (up to 20% of total phospholipids), which did not occur in normal cells, accumulated accompanied with a decrease in phosphatidylethanolamine. Higher uptake activities of serine and glutamate were observed with the hydroxylamine-treated cells than control cells. When membrane vesicles from hydroxylamine-treated cells were prepared, they also displayed higher uptake activities of serine, proline, glutamate, and threonine than those of normal membranes. When hydroxylamine-treated cells were incubated with chloramphenicol, at concentrations which almost completely inhibited protein synthesis, the composition of phosphatidylserine decreased with a concomitant increase in that of phosphatidylethanolamine. The phospholipid composition of these cells incubated for 5 h with chloramphenicol became almost normal. Membranes vesicles prepared from such cells displayed reduced uptake activities, which were close to those of normal vesicles. These results were interpreted as indicating the altered transport activities due to the altered phospholipid composition.     

Uptake of amino acids by actidione-treated yeast cells

The active uptake ofl-aspartic acid, glycine andl-lysine by actidione-treated cells ofSaccharomyces cerevisiae was found to be inhibited by anaerobic conditions in the absence of a source of energy, only facilitated diffusion persisting. Similarly, metabolic inhibitors (iodoacetamide, sodium fluoride and potassium sorbate) inhibited the uptake very substantially. 2,4-Dinitrophenol and sodium azide appeared to inhibit the movement of the transport carrier itself, while uranyl ions showed a complex interaction pattern, ranging from inhibition at concentrations of 10^?6–10^?4 m, to stimulation at concentrations of 3×10^?4–10^?3 m, to pronounced inhibition at higher concentrations. The uptake was pH-dependent with optima forl-aspartic acid near pH 4, for glycine near pH 5, forl-lysine near pH 6.5.     

Uptake of the neutral amino acids glutamine, leucine, and serine by Pneumocystis carinii.

Experiments to elucidate the mechanism by which Pneumocystis carinii transports glutamine, leucine, and serine were performed in this study. Uptake of all three radiolabeled amino acids exhibited first-order, saturation kinetics as extracellular substrate concentrations were increased, thus ruling out simple diffusion and indicating carrier-mediated transport. Kinetic analyses of amino acid uptake and the results of competitive inhibition experiments suggested that leucine, serine, and glutamine were taken up via a common transporter system. The uptake of serine was examined in greater detail to characterize the nature of the carrier. Serine uptake was not affected by N, N'-dicyclohexylcarbodiimide, carbonyl cyanide m-chlorophenyl hydrazone, ouabain, gramicidin, valinomycin, sodium azide, salicylhydroxamine acid (SHAM), iodoacetate, iodoacetate plus SHAM, KCN, and azide. Thus serine uptake did not require sodium or energy from ATP, an electrochemical proton gradient or a membrane potential across the cell surface (i.e., proton-motive force). Serine uptake was dependent on glucose in the extracellular compartment. In the presence of glucose, serine uptake was inhibited by chloramphenicol but not cycloheximide. The results from these experiments are most consistent with facilitated diffusion as the mechanism. After 30 min of incubation, most of the radioactivity was in the cellular soluble fraction. In most cases, incorporation into the extractable total lipids and the remaining particulate cellular components were detectable after this incubation period.     

Anaerobic transport of amino acids coupled to the glycerol-3-phosphate-fumarate oxidoreductase system in a cytochrome-deficient mutant of Escherichia coli.

The uptake of proline and glutamine by cytochrome-deficient cells of Escherichia coli SASX76 grown aerobically on glucose or anaerobically on pyruvate was stimulated by these two substrates. Pyruvate could not stimulate transport in the glucose-grown cells. Uptake of these amino acids energized by glucose was inhibited by inhibitors of the Ca2+, Mg2+-stimulated ATPase such as DCCD, pyrophosphate, and azide, and by the uncouplers CCCP and 2,4-dinitrophenol. Glycerol (or glycerol 3-phosphate) in the presence of fumarate stimulated the transport of proline and glutamine under anaerobic conditions in cytochrome-deficient cells but not in membrane vesicles prepared from these cells although glycerol 3-phosphate-fumarate oxidoreductase activity could be demonstrated in the vesicle preparation. In contrast, in vesicles prepared from cytochrome-containing cells of E. coli SASX76 amino acid transport was energized under anaerobic conditions by this system. Inhibitors of the Ca2+, Mg2+-activated ATPase and uncoupling agents inhibited the uptake of proline and glutamine in cytochrome-deficient cells dependent on the glycerol-fumarate oxidoreductase system. Ferricyanide could replace fumarate as an electron acceptor to permit transport of phenylalanine in cytochrome-deficient or cytochrome-containing cells under anaerobic conditions. It is concluded that in cytochrome-deficient cells using glucose, pyruvate, or glycerol in the presence of fumarate, transport of both proline and glutamine under under anaerobic conditions is energized by ATP through the Ca2+, Mg2+-activated ATPase. In cytochrome-containing cells under anaerobic conditions electron transfer between glycerol and fumarate can also drive transport of these amino acids.     

Energy coupling in the active transport of amino acids by bacteriohodopsin-containing cells of Halobacterium holobium.

Growth of Halobacterium halobium under illumination with limiting aeration induces bacteriorhodopsin formation and renders the cells capable of photophosphorylation. Cells depleted of endogenous reserves by a starvation treatment were used to investigate the means by which energy is coupled to the active transport of [14C]proline, -leucine, and -histidine. Proline was readily accumulated by irradiated cells under anaerobiosis even when the photophosphorylation was abolished by the adenosine triphosphatase inhibitor N,N'-dicyclohexylcarbodimiide (DCCD). The uptake of proline in the dark was limited except when the cells were allowed to accumulate adenosine 5'-triphosphate (ATP) by prior light exposure or by the oxidation of glycerol. DCCD inhibited this dark uptake. These findings essentially support Mitchell's chemiosmotic theory of active transport. The driving force is apparently the proton-motive force developed when protons are extruded from irradiated bacteriorhodopsin or by the dydrolysis of ATP by membrane adenosine triphosphatase. Carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton permeant known to abolish membrane potential, was a strong inhibitor of proline uptake. Leucine transport was also apparently driven by proton-motive force, although its kinetic properties differed from the proline system. Histidine transport is apparently not a chemiosmotic system. Dark- or light-exposed cells show comparable initial rats of histidine uptake, and these processes were only partially inhibited by DCCD or CCCP. The histidine system apparently does not utilize ATP per se since comparable rates of uptake were exhibited by cells of differing intracellular ATP levels. Irradiated cells did effect a greater total accumulation of histidine than dark-exposed cells. These findings suggest that ATP is needed for sustained transport.     

Effects of trialkyllead compounds on growth, respiration and ion transport in Escherichia coli K12

Triethyllead and tripropyllead cations affected growth, energy metabolism and ion transport in Escherichia coli K12. The tripropyllead compound was more liposoluble than the triethyl analogue and was also more effective in inhibiting cell growth and the oxygen uptake of both intact cells and membrane particles. Triethyllead acetate (5 microM) inhibited growth on non-fermentable carbon sources, such as glycerol and succinate, more markedly than on glucose. At higher concentrations, triethyllead caused significant inhibition of respiration rates of intact cells; the concentration giving 50% inhibition was 60 microM for glycerol-grown cells and 150 microM for glucose-grown cells. Oxidation of succinate by membrane particles was less sensitive to inhibition by the tripropyl- or triethyllead compounds than were the oxidations of DL-lactate or NADH. Triethyllead acetate [1.9 mumol (mg membrane protein)-1] inhibited the reduction by NADH of cytochromes; evidence for more than one site of inhibition in the respiratory chain was obtained. Membrane-bound ATPase activity was strongly inhibited by triethyllead acetate in the absence or presence of Cl-. The concentration of inhibitor giving 50% inhibition [0.02 mumol (mg membrane protein)-1] was about two orders of magnitude lower than that required for 50% inhibition of substrate oxidation rates in membranes. Triethyllead acetate (1 microM) induced swelling of spheroplasts in iso-osmotic solutions of either NH4Cl or NH4Br, presumably as a result of the mediation by the organolead compound of Cl-/OH- and Br-/OH- antiports across the cytoplasmic membrane. Similar exchanges of OH- for F-, NO3- or SO4(2)- or the uniport of H+ could not be demonstrated. Comparisons are drawn between the effects of trialkyllead compounds and those of the more widely studied trialkyltin compounds.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Fatty acid oxidation

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pentanoic acid, pent-2-enoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on the oxidation of saturated fatty acids by rat liver mitochondria were determined. 2. The formation of (14)CO(2) from [1-(14)C]palmitate was strongly inhibited by 0.01mm-pent-4-enoic acid. 3. The inhibition of oxygen uptake was less than that of (14)CO(2) formation, presumably because fumarate was used as a sparker. 4. The oxidation of [1-(14)C]-butyrate, -octanoate or -laurate was not strongly inhibited by 0.01mm-pent-4-enoic acid. 5. The other four non-hypoglycaemic compounds did not inhibit the oxidation of any saturated fatty acid when tested at 0.01mm concentration, though they all inhibited strongly at 10mm. 6. The oxidation of [1-(14)C]-myristate and -stearate, but not of [1-(14)C]decanoate, was strongly inhibited by 0.01mm-pent-4-enoic acid. 7. The oxidation of [1-(14)C]palmitate was about 50% carnitine-dependent under the experimental conditions used. 8. The percentage inhibition of [1-(14)C]palmitate oxidation by pent-4-enoic acid was the same whether carnitine was present or not. 9. Acetoacetate formation from saturated fatty acids was inhibited by 0.1mm-cyclopropanecarboxylic acid to a greater extent than their oxidation. 10. The other compounds tested inhibited acetoacetate formation from saturated fatty acids proportionately to the inhibition of oxidation. 11. Possible mechanisms for the inhibition of long-chain fatty acid oxidation by pent-4-enoic acid are discussed. 12. There was a correlation between the ability to inhibit long-chain fatty acid oxidation and hypoglycaemic activity in this series of compounds.     

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Oxidative phosphorylation and mitochondrial oxidation of pyruvate, 3-hydroxybutyrate and tricarboxylic acid-cycle intermediates

1. The effects of the hypoglycaemic compound pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pent-2-enoic acid, pentanoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on several reactions in rat liver mitochondria were determined. 2. The use of manometric techniques for measurements of oxidations and of phosphorylation is critically discussed. 3. Pent-4-enoic acid and pentanoic acid uncoupled oxidative phosphorylation at low concentrations, but usually by not more than about 50%. 4. All the compounds, except cyclobutanecarboxylic acid, strongly inhibited the oxidation of pyruvate and 2-oxoglutarate, but the oxidations of succinate, citrate and 3-hydroxybutyrate were not strongly inhibited. 5. All the compounds, except cyclobutanecarboxylic acid, inhibited decarboxylation of [1-(14)C]pyruvate with ferricyanide as electron acceptor. 6. All the compounds, except pent-2-enoic acid, caused mitochondrial swelling after a time-lag.     

Stimulatory effect of lithium ion on proline transport by whole cells of Escherichia coli.

The effect of monovalent cations on proline transport in whole cells of Escherichia coli K-12 has been examined. Lithium ion added to the uptake medium stimulated proline transport severalfold and K+ and Na+ were slightly effective, whereas Rb+, Cs+, and NH4+ were completely without effect. The stimulatory effect of Li+ on proline transport was not due to an increase in osmolarity of the uptake medium, and d 5 mM p-chloromercuribenzene sulfonic acid completely blocked this effect of Li+ without having any effect on the basal rate of proline transport. The Arrhenius plots for Li+-stimulated transport showed a clear transition point at 35 degrees C in addition to 20 degrees C which was also detectable in the basal transport. Lithium ion stimulated proline transport synergistically in the presence of glucose and succinate as a carbon source. The addition of 2.5 mM KCN or 0.5 mM arsenate did not inhibit this synergistic effect, although the presence of these inhibitors inhibited completely the stimulation of proline transport induced by the addition of carbon source. Carbonylcyanide m-chlorophenylhydrazone and 2,4-dinitrophenol blocked both the basal and Li+-stimulated proline transport. When membrane potential of E. coli cells was measured by the dibenzyldimethylammonium uptake method, the incubation of Li+ with the cells did not affect the preexisting membrane potential. These results suggest that Li+ stimulates proline transport by intact cells of E. coli in a manner somewhat affecting membrane component(s) different from the transport carrier of proline. It is uncertain whether the effect of Li+ is directly involved in the mechanisms of energy coupling of proline transport.     

Permeability of Isolated Infected Cells from Soybean Nodules

Infected cells from the central zone of mature soybean noduleswere isolated by incubating nodule slices with hydrolytic enzymes,and their permeability to various organic compounds measured.The cells were effectively impermeable to sucrose, fructoseand glucose, readily permeable to malate and succinate and partiallypermeable to glutamate and glutamine. Malate uptake showed saturationkinetics and was competitively inhibited by succinate. Malateuptake was also inhibited by a protonophore and the respiratorypoison antimycin, as well as by butylmalonate, cyanocinnamicacid and other substrate analogues, but not by phthalonate.We conclude that there is a dicarboxylate carrier on the plasmamembraneof soybean infected cells, which catalyses the uptake of malateand which depends on mitochondrial ATP for maximum rates.     

Aromatic amino acid transport in Yersinia pestis.

The uptake and concentration of aromatic amino acids by Yersinia pestis TJW was investigated using endogenously metabolizing cells. Transport activity did not depend on either protein synthesis or exogenously added energy sources such as glucose. Aromatic amino acids remained as the free, unaltered amino acid in the pool fraction. Phenylalanine and tryptophan transport obeyed Michaelis-Menten-like kinetics with apparent Km values of 6 x 10(-7) to 7.5 x 10(-7) and 2 x 10(-6) M, respectively. Tyrosine transport showed biphasic concentration-dependent kinetics that indicated a diffusion-like process above external tyrosine concentrations of 2 x 10(-6) M. Transport of each aromatic amino acid showed different pH and temperature optima. The pH (7.5 TO8) and temperature (27 C) optima for phenylalanine transport were similar to those for growth. Transport of each aromatic amino acid was characterized by Q10 values of approximately 2. Cross inhibition and exchange experiments between the aromatic amino acids and selected aromatic amino acid analogues revealed the existence of three transport systems: (i) tryptophan specific, (ii) phenylalanine specific with limited transport activity for tyrosine and tryptophan, and (iii) general aromatic system with some specificity for tyrosine. Analogue studies also showed that the minimal stereo and structural features for phenylalanine recognition were: (i) the L isomer, (ii) intact alpha amino and carboxy group, and (iii) unsubstituted aromatic ring. Aromatic amino acid transport was differentially inhibited by various sulfhydryl blocking reagents and energy inhibitors. Phenylalanine and tyrosine transport was inhibited by 2,4-dinitrophenol, potassium cyanide, and sodium azide. Phenylalanine transport showed greater sensitivity to inhibition by sulfhydryl blocking reagents, particularly N-ethylmaleimide, than did tyrosine transport. Tryptophan transport was not inhibited by either sulfhydryl reagents or sodium azide. The results on the selective inhibition of aromatic amino acid transport provide additional evidence for multiple transport systems . These results further suggest both specific mechanisms for carrier-mediated active transport and coupling to metabolic energy.     

The oxidation of fatty acids combined with albumin by isolated rat liver mitochondria

Long chain fatty acids at concentrations inhibiting mitochondrial respiration were, in the presence of serum albumin, found to produce almost as high a rate of oxygen uptake as alpha-ketoglutarate, succinate, or acetate. This oxidation was characterized in terms of its coupling to phosphorylation, need for cofactors, and production of different metabolites during the reactions. Fatty acids were oxidized to carbon dioxide, acetoacetate, beta-hydroxybutyrate, and other water-soluble metabolites, tentatively identified as intermediates of the citric acid cycle. An agent to spark the citric acid cycle and adenosine tri- or monophosphate were necessary for optimal oxidation rate, as described for other fatty acid oxidation systems. Balance experiments with different amounts of malate were performed with incubations lasting as long as oxygen uptake took place. In the presence of 1 mumole of malate, practically all added palmitic acid was used up and found to be converted primarily to carbon dioxide, acetoacetate, and other water-soluble metabolites of which the major part was tentatively identified as succinate. A significant portion was found in mitochondrial phospholipids. With 10 mumoles of malate some palmitic acid remained in the system, while a comparatively small amount was converted to carbon dioxide, and a major part was found as succinate. Here also incorporation into phospholipids occurred. With no malate added, fatty acid oxidation was much smaller than with malate, although significant conversion to carbon dioxide took place. Only a little succinate and phospholipid were found. Oxygen uptake was greater than a theoretical value calculated from radioactive balance experiments. It was concluded that albumin contains oxidizable material even after extraction and dialysis. Albumin at high concentrations inhibited both fatty acid and alpha-ketoglutarate oxidation. The oxidation of long chain fatty acids in high concentrations in the form of albumin-fatty acid complex was coupled to phosphorylation. Thus P:O ratios above 2 were found as well as evidence for respiratory control. It was concluded that oxidation of long chain fatty acids by isolated mitochondria occurs from their albumin complex. This process can also be studied at high concentrations of fatty acids, where high rates of oxygen uptake are obtained from oxidation which is coupled to phosphorylation.     

Transport of amino acids and ammonium in mycelium of Agaricus bisporus.

Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis-Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH(+)(4), with a K(i) of 3.7 microM for the high-velocity system. The K(m) values for glutamate, glutamine and arginine transport were 124, 151 and 32 microM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH(+)(4) concentration. Moreover, increase of the intracellular NH(+)(4) level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH(+)(4) concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH(+)(4) did not inhibit glutamine uptake, suggesting that NH(+)(4) and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed.     

Oxidative phosphorylation. Halide-dependent and halide-independent effects of triorganotin and trioganolead compounds on mitochondrial functions.

1. Each of five triorganotin and five triorganolead compounds was shown to perturb mithochondrial functions in three different ways. One is dependent and two are independent of Cl- in the medium. 2. Structure-activity relationships for the three interactions are described, and compounds suitable as tools for the separate study of each process are defined. 3. In a Cl- -containing medium trimethyltin, triethyltin, trimethyl-lead, triethyl-lead and tri-n-propyl-lead all produce the same maximum rate of ATP hydrolysis and O2 uptake; this rate is much less than that produced by uncoupling agents such as 2,4-dinitrophenol. 4. Increase in ATP hydrolysis and O2 uptake are measures on energy ultilization when triogranotin and triorganolead compounds bring about an exchange of external C1- for intramitochondrial OH- ions. Possible rate-limiting steps in this process are discussed. 5. In a C1- -containing medium ATP synthesis linked to the oxidation of beta-hydroxybutyrate or reduced cytochrone c is less inhibited by triethyltin or triethyl-lead than is ATP synthesis linked to the oxidation of succinate, pyruvate or L-glutamate. 6. The inhibition of ATP synthesis linked to the oxidation of both beta-hydroxybutyrate and reduced cytochrome c consists of two processes: one is a limited uncoupling and is C1- -dependent and the other is a C1- -independent inhibition of the energy-conservation system. 7. The different sensitivities to inhibition by triethyltin of mitochondrial functions involving the oxidation of beta-hydroxybutyrate and succinate are compared and discussed.     

Genetic and biochemical studies of transport systems for branched-chain amino acids in Escherichia coli.

Mutants of Escherichia coli K-12 requiring high concentrations of branched-chain amino acids for growth were isolated. One of the mutants was shown to be defective in transport activity for branched-chain amino acids. The locus of the mutation (hrbA) was mapped at 8.9 min on the E. coli genetic map by conjugational and transductional crosses. The gene order of this region is proC-hrbA-tsx. The hrbA system was responsible for the uptake activity of cytoplasmic membrane vesicles. It was not repressed by leucine. The substrate specificities and kinetics of the uptake activities were studied using cytoplasmic membrane vesicles and intact cells of the mutants grown in the presence or absence of leucine. Results showed that there are three transport systems for branched-chain amino acids, LIV-1, -2, and -3. The LIV-2 and -3 transport systems are low-affinity systems, the activities of which are detectable in cytoplasmic membrane vesicles. The systems are inhibited by norleucine but not by threonine. The LIV-2 system is also repressed by leucine. The LIV-1 transport system is a high-affinity system that is sensitive to osmotic shock. When the leucine-isoleucine-valine-threonine-binding protein is derepressed, the high-affinity system can be inhibited by threonine.