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D L Oxender

Publications and source records attributed to D L Oxender.

At least 55 records · Page 3Linked to original sources
PubMed1985

Regulation of amino acid transport system L by amino acid availability in CHO-K1 cells. A special role for leucine.

Starvation of CHO-K1 cells for leucine leads to a 3-4-fold increase in transport system L activity, without modification of transport through systems A and ASC. The concentration of leucine must be below 10 microM before the enhancement of transport can be clearly seen. To achieve low concentrations of leucine such as 10 microM, extensive dialysis of fetal calf serum was required. The enhancement of transport was completed after 12-24 h of starvation and was fully reversed within 1 h of re-feeding with leucine. Starvation for isoleucine, valine or phenylalanine also produced an increase in system L transport activity, but the effect was only one half of that seen following leucine starvation.

Amino Acids↗
PubMed1985

The complete nucleotide sequences of the Escherichia coli LIV-BP and LS-BP genes. Implications for the mechanism of high-affinity branched-chain amino acid transport.

The Escherichia coli LIV-I and LS amino acid transport systems are high-affinity, periplasmic, binding protein-dependent systems that utilize the leucine-, isoleucine-, valine-binding protein (LIV-BP) and leucine-specific binding protein (LS-BP), respectively. These two binding proteins (BPs) interact with a common set of membrane proteins to transport branched-chain amino acids into the cytoplasm. The two BP genes are encoded in a regulon at minute 76 of the E. coli chromosome that also contains the genes for the common membrane protein components. We report here the nucleotide and deduced amino acid sequences for the LIV-BP and LS-BP genes and their protein products. Both BPs are encoded with a 23-amino acid signal sequence that is removed when the BPs are secreted into the periplasm. We have examined the pattern of amino acid sequence conservation between the two BP molecules and, by comparison of the predicted secondary structures to the 2.0-A crystal structure of the LIV-BP, have found regions of the BPs that may be involved in membrane protein interaction. We also analyzed the translational efficiency of the two BP mRNAs as determined by their nucleotide sequences.

Amino Acid Sequence↗
PubMed1985

The leucine binding proteins of Escherichia coli as models for studying the relationships between protein structure and function.

The genes encoding the leucine binding proteins in E coli have been cloned and their DNA sequences have been determined. One of the binding proteins (LIV-BP) binds leucine, isoleucine, valine, threonine, and alanine, whereas the other (LS-BP) binds only the D- and L-isomers of leucine. These proteins bind their solutes as they enter the periplasm, then interact with three membrane components, livH, livG, and livM, to achieve the translocation of the solute across the bacterial cell membrane. Another feature of the binding proteins is that they must be secreted into the periplasmic space where they carry out their function. The amino acid sequence of the two binding proteins is 80% homologous, indicating that they are the products of an ancestral gene duplication. Because of these characteristics of the leucine binding proteins, we are using them as models for studying the relationships between protein structure and function.

Amino Acid Sequence↗
PubMed1985

Identification of livG, a membrane-associated component of the branched-chain amino acid transport in Escherichia coli.

Branched-chain amino acids are transported into Escherichia coli by two osmotic shock-sensitive systems (leucine-isoleucine-valine and leucine-specific transport systems). These high-affinity systems consist of separate periplasmic binding protein components and at least three common membrane-bound components. In this study, one of the membrane-bound components, livG, was identified. A toxic analog of leucine, azaleucine, was used to isolate a large number of azaleucine-resistant mutants which were defective in branched-chain amino acid transport. Genetic complementation studies established that two classes of transport mutants with similar phenotypes, livH and livG, were obtained which were defective in one of the membrane-associated transport components. Since the previously cloned plasmid, pOX1, genetically complemented both livH and livG mutants, we were able to verify the physical location of the livG gene on this plasmid. Recombinant plasmids which carried different portions of the pOX1 plasmid were constructed and subjected to complementation analysis. These results established that livG was located downstream from livH with about 1 kilobase of DNA in between. The expression of these plasmids was studied in minicells; these studies indicate that livG appears to be membrane bound and to have a molecular weight of 22,000. These results establish that livG is a membrane-associated component of the branched-chain amino acid transport system in E. coli.

Amino Acids↗
PubMed1984

Genetic studies of leucine transport in mammalian cells.

We have taken two approaches to the study of the genetics of leucine transport in mammalian cells. First, from a mutant Chinese hamster ovary cell line that has a temperature-sensitive leucyl-tRNA synthetase, we isolated temperature-resistant revertants with increased leucine transport activity. This transport elevation is reflected by increased Vmax values of leucine uptake and unchanged Km values of uptake. The temperature resistance in each revertant appears to result from the increased transport and not from any change in the leucyl-tRNA synthetase. We conclude that in each revertant there is a stable derepression of amino acid transport system L. In a second approach, we started with a Chinese hamster-human hybrid strain formed by the fusion of a temperature-sensitive leucyl-tRNA synthetase mutant hamster cell line and normal human leukocytes. From this temperature-sensitive hybrid strain we selected temperature-resistant hybrids, one class of which we found to have greatly elevated leucine transport activity. We have allowed human chromosomes to segregate from these high-transport hybrids, promoted by the presence of low concentrations of colcemid. The loss of the high-transport phenotype coincides with the loss of a single small human chromosome, which we are attempting to identify by using G-11 and G-banding staining techniques.

Amino Acyl-tRNA Synthetases↗
PubMed1984

Secretion and degradation of mutant leucine-specific binding protein molecules containing C-terminal deletions.

The leucine-specific binding protein (LS-BP), a periplasmic component of the Escherichia coli high-affinity leucine transport system, is initially synthesized in a precursor form with a 23 amino acid N-terminal leader sequence that is removed during secretion of the protein into the periplasm. Using in vitro mutagenesis, deletion mutants of the LS-BP gene have been constructed with altered or missing amino acid sequences in the C-terminal portion of the protein. These altered binding proteins exhibited normal processing and secretion but were rapidly degraded in the periplasmic space. In the presence of an uncoupler of the transmembrane potential (CCCP) the precursor forms accumulated in the membrane and were protected from degradation. The altered binding proteins also were secreted by spheroplasts of E coli, after which they were easily detected.

Bacterial Proteins↗
PubMed1984

Role of membrane potential in protein folding and domain formation during secretion in Escherichia coli.

The synthesis and processing of the periplasmic components of the leucine transport system of E coli have been studied to determine the role played by transmembrane potential in protein secretion. Both the leucine-isoleucine-valine binding protein and the leucine-specific binding protein are synthesized as precursors with 23 amino acid N-terminal leader sequences. The processing of these precursors is sensitive to the transmembrane potential. Since the amino acid sequence and the crystal structure have been determined for the leucine-isoleucine-valine binding protein, it and the closely related leucine-specific binding protein represent convenient models in which to examine the mechanism of protein secretion in E coli. A model for secretion has been proposed, suggesting a role for transmembrane potential. In this model, the N-terminal amino acid sequence of the precursor is assumed to form a hairpin of two helices. The membrane potential may orient this structure to make it accessible to processing. In addition, the model suggests that a negatively charged, folded domain of the secretory protein may electrophorese toward the trans-positive side of the membrane, thus providing an additional role for the transmembrane potential.

Bacterial Proteins↗
PubMed1984

A Chinese hamster ovary leucyl-tRNA synthetase mutant with a uniquely altered high molecular weight leucyl-tRNA synthetase complex.

The Chinese hamster ovary (CHO) cell culture temperature-sensitive mutant ts025Cl with a defect in leucyl-tRNA synthetase ( LeuRS ) does not have an inherently more thermolabile LeuRS , but instead the mutation causes the complete loss of the LeuRS high molecular weight complexes which are present in normal wild-type cells. The mutant cell LeuRS has a single 8 S enzyme form which corresponds hydrodynamically to the 8 S free form of wild-type enzyme. Both 8 S forms have the same thermostability and the same Km for leucine, indicating that there is no inherent defect in the catalytic activity of the enzyme. The temperature-sensitive phenotype can be explained by the lack of thermostable high molecular weight forms of LeuRS .

Amino Acyl-tRNA Synthetases↗
PubMed1984

Characterization of a Chinese hamster-human hybrid cell line with increased system L amino acid transport activity.

We have studied leucine transport in several Chinese hamster-human hybrid cell lines obtained by fusion of a temperature-sensitive line of Chinese hamster ovary cells, ts025C1, and normal human leukocytes. A hybrid cell line exhibiting a twofold increase in L-leucine uptake over that in the parental cell line was found. This hybrid cell line, 158CnpT-1, was temperature resistant, whereas the parental Chinese hamster ovary mutant, ts025C1, contained a temperature-sensitive leucyl-tRNA synthetase mutation. An examination of the different amino acid transport systems in this hybrid cell line revealed a specific increase of system L activity with no significant changes in systems A and ASC. The Vmax for L-leucine uptake exhibited by the hybrid 158CnpT-1 was twice that in the CHO parental mutant, ts025C1. Cytogenetic analysis showed that the hybrid 158CnpT-1 contains four complete human chromosomes (numbers 4, 5, 10, and 21) and three interspecific chromosomal translocations in a total complement of 34 chromosomes. Biochemical and cytogenetic analysis of segregant clones obtained from hybrid 158CnpT-1 showed that the primary temperature resistance and high system L transport phenotypes can be segregated from this hybrid independently. The loss of the primary temperature resistance was associated with the loss of the human chromosome 5, as previously reported by other laboratories, whereas the loss of the high leucine transport phenotype, which is associated with a lesser degree of temperature resistance, was correlated with the loss of human chromosome 20.

Amino Acids↗
PubMed1983

Isolation of Chinese hamster ovary cell mutants defective in the regulation of leucine transport.

We have isolated mutants defective in the regulation of leucine transport by selecting temperature-resistant revertants from the CHO-tsH1 strain, a temperature-sensitive leucyl-tRNA synthetase mutant of the Chinese hamster ovary cell line. In each revertant, there is a stable 2- to 3-fold enhancement in the activity of transport System L, which serves for the uptake of branched-chain amino acids. This increased transport is reflected by an increase in the Vmax for System L transport without a significant change in the Km value and results in increased intracellular pools of leucine. The thermal stability of the leucyl-tRNA synthetase activity in each of these revertants is not changed significantly from that of the starting strain, CHO-tsH1. We conclude from these studies that the temperature resistance in the revertants arises from alterations in the regulation of transport System L, leading to constitutively high System L transport and increased intracellular pools of leucine, complementing the leucyl-tRNA synthetase defect.

Amino Acyl-tRNA Synthetases↗
PubMed1982

Regulation of amino acid transport system L in Chinese hamster ovary cells.

The regulation of the activity of transport System L for neutral amino acids has been investigated in Chinese hamster ovary cells. Incubation of the temperature-sensitive leucyl-tRNA synthetase mutant CHO-tsH1 at marginally permissive temperatures results in leucine-limited growth and increased transport of branched chain and aromatic amino acids. This temperature-dependent transport enhancement is restricted to transport System L and results in increased Vmax values of uptake of System L substrates with unchanged Km values of uptake. Several lines of evidence suggest that trans-stimulation by intracellular amino acids cannot account for the increased System L activity in CHO-tsH1. Other temperature-sensitive aminoacyl-tRNA synthetase mutants of the Chinese hamster ovary line show increased transport of the amino acid corresponding to the synthetase defect after incubation at elevated temperatures. Increased transport by System L occurs in CHO-S, the parental line of CHO-tsH1, after growth in limiting levels of leucine. The System L enhancement can be prevented by cycloheximide but not, at early times, by actinomycin D. We conclude that the activity of transport System L in Chinese hamster ovary cells is regulated by a mechanism which appears to act at the level of translation.

Amino Acids↗
PubMed1981

Neutral amino acid transport systems in Chinese hamster ovary cells.

The neutral amino acid transport systems A, ASC, and L have been characterized in the Chinese hamster ovary cell. System A, defined by its sodium ion dependency and inhibition by 2-methylaminoisobutyric acid, was found to be extremely sensitive to the pH of the external medium and to increase in response to starvation for amino acids. System ASC, identified by its sodium ion dependency and intolerance of N-methylation of substrates, was found to be relatively insensitive to external pH and nutrient limitation. System ASC in Chinese hamster ovary cells has been shown to be the major mode of entry of neutral amino acids. A much broader substrate specificity was observed for System ASC than has been reported for other mammalian cell types, with nearly every amino acid tested showing significant uptake by this system. In addition, the highest observed velocities of uptake were for System ASC. System L, defined by its sodium ion independency and inhibition by 2-aminobicyclo-[2,2,2]-heptane-2-carboxylic acid, was enhanced in activity by lowered pH. The starvation-induced enhancement in System A activity was prevented by the presence of a single substrate of this system, the nonmetabolizable analog 2-methylaminoisobutyric, or by the presence of the protein synthesis inhibitor cycloheximide.

Alanine↗
PubMed1981

Role for membrane potential in the secretion of protein into the periplasm of Escherichia coli.

The leucine-specific binding protein of Escherichia coli is a periplasmic protein that is synthesized as a precursor and subsequently is processed during its secretion into the periplasmic space. The processing of both the leucine-specific binding protein and a plasmid-coded beta-lactamase is inhibited by phenethyl alcohol and by the proton ionophore, carbonylcyanide m-chlorophenylhydrazone (CCCP). The levels of CCCP that inhibit processing also produce significant decreases in the membrane potential. Valinomycin, a potassium ionophore, also inhibits processing of the leucine-specific binding protein in spheroplasts. Processing can be restored in CCCP-treated cells and in valinomycin-treated spheroplasts by dilution of the treated cells in fresh medium. These results suggest a role for membrane potential in the secretion of periplasmic proteins. A model is presented which suggests that membrane potential plays a primary role in the proper orientation of the precursor signal sequence within the membrane, thus promoting processing and secretion.

Bacterial Proteins↗
PubMed1980

The in vitro synthesis and processing of the branched-chain amino acid binding proteins.

The synthesis of the leucine-specific and LIV-binding proteins was examined in vitro in a coupled transcription/translation system using the hybrid plasmids pOX7 and pOX13 as templates. Plasmid pOX7 contains the livK gene coding for the leucine-specific binding protein and pOX13 contains the livJ gene coding for the LIV-binding protein. Both binding proteins were synthesized in vitro as precursor forms with molecular weights approximately 2,500 greater than their respective mature forms. Conversion of the precursor forms to their mature forms occurred during post-translational incubation following synthesis in the presence of membrane. The precursor of the LIV-binding protein was processed more rapidly than the leucine-specific binding protein precursor. Processing activity could be removed from the in vitro synthesis system by centrifugation, suggesting that the processing activity was membrane associated. Restoration of post-translational processing activity was achieved by adding inside-out membrane vesicles to membrane-depleted reaction mixtures.

Amino Acids, Branched-Chain↗