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D L Oxender

Publications and source records attributed to D L Oxender.

At least 91 records · Page 5Linked to original sources
PubMed1977

Regulation of amino acid transport in Escherichia coli by transcription termination factor rho.

Amino acid transport rates and amino acid binding proteins were examined in a strain containing the rho-120 mutation (formerly SuA), which has been shown to lower the rho-dependent, ribonucleic acid-activated adenosine triphosphatase activity to 9% of the rho activity in the isogenic wild-type strain. Tryptophan and proline transport, which occur by membrane-bound systems, were not altered. On the other hand, arginine, histidine, leucine, isoleucine, and valine transport were variably increased by a factor of 1.4 to 5.0. Kinetics of leucine transport showed that the LIV (leucine, isoleucine, and valine)-I (binding protein-associated) transport system is increased 8.5-fold, whereas the LIV-II (membrane-bound) system is increased 1.5-fold in the rho mutant under leucine-limited growth conditions. The leucine binding protein is increased fourfold under the same growth conditions. The difference in leucine transport in these strains was greatest during leucine-limited growth; growth on complex media repressed both strains to the same transport activity. We propose that rho-dependent transcriptional termination is important for leucine-specific repression of branched-chain amino acid transport, although rho-independent regulation, presumably by a corepressor-aporepressor-type mechanism, must also occur.

Amino Acids↗
PubMed1976

Transport of amino acids in intact 3T3 and SV3T3 cells. Binding activity for leucine in membrane preparations of ehrlich ascites tumor cells.

Transport of amino acids into 3T3 and SV3T3 (SV40 virus-transformed 3T3) cells was measured on glass cover slips. The 3T3 and SV3T3 cells contain both A (alanine preferring) and L (leucine prefferring) systems for neutral amino acid transport. Initial rates of uptake of amino acids are about twofold higher in SV3T3 than in 3T3 cells. Other parameters measured, however, do not indicate marked differences in the transport of amino acids by the two cell types. L-system amino acids, such as leucine, are subject to trans-stimulation in both cell lines, whereas A-system amino acids, such as alanine and glycine, are not. Leucine was transported to higher levels in confluent cells than in nonconfluent cells. Glycine, however, shows distinctly less transport activity as the cells become confluent. Ehrlich ascites cell plasma membranes were prepared and assayed for amino acid-binding activity. Leucine-binding activity was detected by equilibrium dialysis in Triton X-100-treated membrane preparations.

Animals↗
PubMed1976

Mapping of two loci affecting the regulation of branched-chain amino acid transport in Escherichia coli K-12.

Two mutant loci resulting in derepression of, respectively, the L-leucine-specific transport system (lstR) and both the leucine-specific and the general branched-chain amino acid transport LIV-I systems (livR) were mapped by conjugation and transduction. Both livR and lstR were found to be closely linked to aroA at min 20 on the Escherichia coli genetic map. The merodiploid livR+/livR displayed wild-type regulation of L-leucine transport, indicating that the livR product is a diffusible, negative controlling element for high-affinity leucine transport systems. Isogenic strains carrying lstR, livR, and wild-type transport alleles were compared for leucine uptake kinetic parameters and leucine-binding protein levels. The higher levels of leucine transport in the mutant strains under repressing conditions were generally due to increased high-affinity systems, which were accompanied by striking increases in the level of leucine-binding proteins.

Biological Transport, Active↗
PubMed1976

Regulation of branched-chain amino acid transport in Escherichia coli.

The repression and derepression of leucine, isoleucine, and valine transport in Escherichia coli K-12 was examined by using strains auxotrophic for leucine, isoleucine, valine, and methionine. In experiments designed to limit each of these amino acids separately, we demonstrate that leucine limitation alone derepressed the leucine-binding protein, the high-affinity branched-chain amino acid transport system (LIV-I), and the membrane-bound, low-affinity system (LIV-II). This regulation did not seem to involve inactivation of transport components, but represented an increase in the differential rate of synthesis of transport components relative to total cellular proteins. The apparent regulation of transport by isoleucine, valine, and methionine reported elsewhere was shown to require an intact leucine, biosynthetic operon and to result from changes in the level of leucine biosynthetic enzymes. A functional leucyl-transfer ribonucleic acid synthetase was also required for repression of transport. Transport regulation was shown to be essentially independent of ilvA or its gene product, threonine deaminase. The central role of leucine or its derivatives in cellular metabolism in general is discussed.

Bacterial Proteins↗
PubMed1976

Regulation of transport in mammalian cell culture.

The regulation of amino acid transport has been investigated in Balb/3T3 cells. Transport for neutral amino acids in animal tissues is carried out by two distinct systems. The transport activity of the A system (alanine preferring) is inhibited by high internal of substrate amino acids, a process termed trans-inhibition. In contrast, the L system transport activity is greatly stimulated by high internal levels of substrate, a phenomenon termed trans-stimulation. Regulation of amino acid transport in animal cells occurs by a repression-derepression mechanism or by a feedback inhibition or feedback stimulation process. The level of endogenous amino acids is shown to be important in the regulation of transport activity. Cells which are slow growing or quiescent have increased levels of endogenous amino acids, which in turn effect the transport activity. The A system transport activity is decreased in quiescent cells, whereas L system transport activity increases. Conditions which effect endogenous amino acid levels have an effect on transport activity. Feedback regulation of transport activity through the amino acid pool levels is another way animal cells can regulate transport activity.

Alanine↗
PubMed1975

Binding proteins and membrane transport.

The recent studies have clearly established two types of active transport systems. One type is membrane-bound and can be observed in membrane vesicles and the other type is osmotic-shock-sensitive and requires binding proteins to produce active transport. It appears that the membrane-bound systems derive cellular energy from the energy-rich membrane state which can be formed from respiration or ATP-hydrolysis, while the binding protein systems are more directly coupled to phosphate bond energy derived from glycolysis or oxidative phosphorylation. The following conclusions concerning the role of the binding proteins are offered: 1. The binding proteins are present in relatively large amounts (approximately 10(-6) or 10%-5) M) and appear to reside in the periplasmic space. 2. They do not appear to be involved in solute translocation steps, although they cantain a second binding site that could interact with membrane components. 3. The binding proteins appear to increase the affinity of the transport system for the solute by interacting with a membrane component. This may substrate for the membrane transport system.

Biological Transport↗
PubMed1975

Role of leucyl-tRNA synthetase in regulation of branched-chain amino-acid transport.

The regulation of the transport of leucine, isoleucine, and valine in Escherichia coli B/r was studied in a mutant with a complete deletion of the leucine biosynthetic operon and a temperature-sensitive leucyl-tRNA synthetase [L-leucine:tRNALeu ligase (AMP-forming), EC 6.1.1.4]. Under conditions of excess leucine and a functional leucyl-tRNA synthetase transport activity was repressed. Shifting the culture to a temperature at which the activation of leucine to an appropriate tRNA species became growth-rate-limiting led to a large increase in the high-affinity transport of leucine, isoleucine, and valine (system LIV-I) while the uptake of histidine and proline was unchanged. A similar increase was observed for branched-chain amino-acid binding protein activity. The temperature change did not alter the transport activity for any of these substrates or the level of the binding proteins in an isogenic strain with a normal leucyl-tRNA synthetase. The increase in transport activity observed in the mutant was prevented by inhibitors of protein and RNA synthesis and probably represents an increase in the differential rate of synthesis of the protein(s) required for transport. These experiments demonstrate that the repression of branched-chain amino-acid transport involves the interaction of leucine with its aminoacyl-tRNA synthetase and its cognate leucyl-tRNA species.

Amino Acids↗
PubMed1975

Transport properties of merodiploids covering the dagA locus in Escherichia coli K-12.

A membrane componenet of the dag transport system which serves for glycine, D-alanine, and D-serine is coded for by the dagA gene at minute 83 of the Escherichia coli chromosome. Merodiploid strains (dagA+/dagA+) show two to three times the transport activity for only those amino acids that are substrates of the dag transport system. The increased transport activity is a result of a two-to threefold increase in Vmax for amino acid uptake with little or no change in the Km value. The two- to threefold gene dose effect of the merodiploid strains is maintained even during carbon starvation, eliminating the possibility that a greater energy supply for transport activity may account for the effect. Since merodiploids which carry more than one copy of the dagA allele show a gene dose response for transport activity, we conclude that the membrane componenet of the dag transport system which is coded for by the dagA allele is present in limiting amounts.

Alanine↗
PubMed1975

Transport of L-4-azaleucine in Escherichia coli.

The uptake of L-4-azaleucine was examined in Escherichia coli K-12 strains to determine the systems that serve for its accumulation. L-4=Azaleucine in radio-labeled form was synthesized and resolved by the action of hog kidney N-acylamino-acid amidohydrolase (EC 3.5.1.B) on the racemic alpha-N-acetyl derivative of DL-[dimethyl-14C]4-azaleucine. L-4-Azaleucine is taken up in E. coli by energy-dependent processes that are sensitive to changes in the pH and to inhibition by leucine and the aromatic amino acids. Although a single set of kinetic parameters was obtained by kinetic experiments, other evidence indicates that transport systems for both the aromatic and the branched-chain amino acids serve for azaleucine. Azaleucine uptake in strain EO317, with a mutation leading to derepression and constitutive expression of branched-chain amino acid (LIV) transport and binding proteins, was not repressed by growth with leucine as it was in parental strain EO300. Lesions in the aromatic amino acid transport system, aroP, also led to changes in the regulation of azaleucine uptake activity when cells were grown on phenylalanine. Experiments on the specificity of azaleucine uptake and exchange experiments with leucine and phenylalanine support the hypothesis that both LIV and aroP systems transport azaleucine. The ability of external azaleucine to exchange rapidly with intracellular leucine may be an important contributor to azaleucine toxicity. We conclude from these and other studies that at least four other process may affect azaleucine sensitivity: the level of branched-chain amino acid biosynthetic enzymes; the level of leucine, isoleucine, and valine transport systems; the level of the aromatic amino acid, aroP, uptake system; and, possibly, the ability of the cell to racemize D and L amino acids. The relative importance of these processes in azaleucine sensitivity under various conditions is not known precisely.

Biological Transport, Active↗
PubMed1975

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.

Acetolactate Synthase↗
PubMed1973

Transport systems for alanine, serine, and glycine in Escherichia coli K-12.

At least two transport systems serve for the entry of alanine, glycine, and serine into Escherichia coli. One of these systems serves mainly for glycine, d-alanine, and d-serine and to some extent for l-alanine, whereas the second serves for l-alanine and perhaps l-serine. These two transport systems have been characterized by kinetic studies and by inhibition analysis. Reciprocal plots for l-alanine entry are distinctly biphasic, giving rise to K(m) values of about 2 and 27 muM. The major route of glycine entry can be described by a single K(m) value of about 4 muM. A higher K(m) value for glycine of around 70 to 100 muM shows that other routes of entry may serve at high concentrations of amino acid. The glycine, d-serine and d-alanine transport system is defective in a d-serine-resistant mutant, strain EM1302. The mutation, dagA, is recessive in dagA/dagA(+) merodiploids and is 7 to 12% linked by phage P1 transduction to the pyrB locus of E. coli. E. coli with the dagA mutation are unable to utilize d-alanine as a carbon source, providing an additional basis for selecting such mutants. The remaining l-alanine uptake in dagA mutants is subject to inhibition by l-serine, l-threonine, and l-leucine. It is also sensitive to osmotic shock treatment and repressed by growth of the cells on l-leucine. It appears from a comparison of the properties of the second l-alanine system with those of the leucine, isoleucine, and valine system (LIV system) that the LIV system also serves for the transport of l-alanine and l-threonine and perhaps l-serine.

Alanine↗
PubMed1973

Multiplicity of leucine transport systems in Escherichia coli K-12.

The major component of leucine uptake in Escherichia coli K-12 is a common system for l-leucine, l-isoleucine, and l-valine (LIV-I) with a Michaelis constant (K(m)) value of 0.2 muM (LIV-I system). The LIV-binding protein appears to be associated with this system. It now appears that the LIV-I transport system and LIV-binding protein also serve for the entry of l-alanine, l-threonine, and possibly l-serine. A minor component of l-leucine entry occurs by a leucine-specific system (L-system) for which a specific leucine-binding protein has been isolated. A mutant has been obtained that shows increased levels of the LIV-I transport activity and increased levels of both of the binding proteins. Another mutant has been isolated that shows only a major increase in the levels of the leucine-specific transport system and the leucine-specific binding protein. A third binding protein that binds all three branched-chain amino acids but binds isoleucine preferentially has been identified. The relationship of the binding proteins to each other and to transport activity is discussed. A second general transport system (LIV-II system) with a K(m) value of 2 muM and a relatively low V(max) can be observed in E. coli. The LIV-II system is not sensitive to osmotic shock treatment nor to growth of cells in the presence of leucine. This high K(m) system, which is specific for the branched-chain amino acids, can be observed in membrane vesicle preparations.

Alanine↗