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D L Oxender

Publications and source records attributed to D L Oxender.

At least 73 records · Page 4Linked to original sources
PubMed1980

Regulation of high-affinity leucine transport in Escherichia coli.

Leucine is transported into E coli by two osmotic shock-sensitive, high-affinity systems (LIV-I and leucine-specific systems) and one membrane bound, low-affinity system (LIV-II). Expression of the high-affinity transport systems is altered by mutations in livR and 1stR, genes for negatively acting regulatory elements, and by mutations in rho, the gene for transcription termination. All four genes for high-affinity leucine transport (livJ, livK, livH, and livG) are closely linked and have been cloned on a plasmid vector, pOX1. Several subcloned fragments of this plasmid have been prepared and used in complementation and regulation studies. The results of these studies suggest that livJ and livK are separated by approximately one kilobase and give a gene order of livJ-livK-livH. livJ and livK appear to be regulated in an interdependent fashion; livK is expressed maximally when the livJ gene is activated by mutation or deletion. The results support the existence of separate promotors for the livJ and livK genes. The effects of mutations in the rho and livR genes are additive on one another and therefore appear to be involved in independent regulatory mechanisms. Mutations in the rho gene affect both the LIV-I and leucine-specific transport systems by increasing the expression of livJ and livK, genes for the LIV-specific and leucine-specific binding proteins, respectively.

Biological Transport↗
PubMed1980

Structural and functional analysis of cloned DNA containing genes responsible for branched-chain amino acid transport in Escherichia coli.

The four genes encoding the components of the high-affinity branched-chain amino acid transport systems in Escherichia coli (livH, livG, livJ, and livK) have been cloned into lambda phage and subsequently into the plasmid vector pACYC184. The presence of the four structural genes and their accompanying regulatory regions on the resultant plasmid, pOXI, was confirmed by genetic complementation and analysis and by transport studies carried out on the appropriate transformed mutant strains. When pOX1 DNA was used to direct an in vitro transcription/translation system, four major polypeptide products were produced. Immunoprecipitation with antibody directed against the LIV-binding protein identified the two leucine-binding proteins as products of in vitro synthesis. The binding proteins were produced in precursor forms and had molecular weights approximately 2500 higher than the processed, mature forms. A minicell-producing strain transformed with plasmid pOX1 produced the binding proteins in the processed form.

Amino Acids, Branched-Chain↗
PubMed1980

Amino-terminal sequence and processing of the precursor of the leucine-specific binding protein, and evidence for conformational differences between the precursor and the mature form.

A 2.1-kilobase Bgl II DNA fragment from Escherichia coli containing livK, the gene coding for the leucine-specific binding protein, has been cloned into the BamHI site of the plasmid vector pBR322. The DNA sequence of segments of the resulting plasmid, pOX7, established the location of the livK gene and the direction of its transcription. In vitro protein synthesis directed by pOX7DNA yielded the Mr 42,000 precursor of the leucine-specific binding protein and a small amount of the Mr 39,000 mature protein. Continued incubation of the in vitro reaction mixture after DNase and RNase treatment resulted in additional processing. The DNA sequence of the beginning of livK suggested that 23 additional amino acid residues are present as an extension of the NH2 terminus of the mature protein. Amino acid sequence analysis established that the precursor has the predicted 23-residue extension. Proteolytic digestion studies with the precursor and mature forms of the leucine-specific binding protein indicate that there are conformational differences between the two. This suggests a possible role for the signal sequence in determining the conformation of the binding protein precursor that is recognized by the membrane.

Amino Acid Sequence↗
PubMed1980

Complement-mediated cytotoxic effects on pancreatic islets with sera from diabetic patients.

Sera from 11/30 patients with juvenile-onset-type diabetes (JOD) had cytotoxic effects on isolated pancreatic islets from hamsters when guinea pig complement was present. The cytotoxic activity could be readily eliminated by heating the guinea pig complement at 56 degrees C for 30 min, thus demonstrating a complement-dependent process. Complement-dependent cytotoxicity toward pancreatic islets was not observed with sera from 11 patients with maturity-onset-type diabetes, 4 nondiabetic hospital patient controls, and 28 healthy adult subjects. The presence of circulating complement-mediated cytotoxic factor(s) toward isolated islets indicates the potential importance of autoimmune pathogenic damage to pancreatic islets in some patients with diabetes.

Addison Disease↗
PubMed1979

Attenuation in the Escherichia coli tryptophan operon: role of RNA secondary structure involving the tryptophan codon region.

The secondary structure of the terminated trp leader transcript from Escherichia coli was analyzed by RNase T1 partial digestion. Base-paired regions were recovered by nondenaturing gel electrophoresis and identified by denaturing gel electrophoresis and fingerprinting. The tandem tryptophan codons in the leader peptide coding region were found to be base paired with a more distal region of the transcript. This and other secondary structures that the trp leader RNA can form help explain the physiological response of the operon as well as the behavior of regulatory mutants.

Codon↗
PubMed1979

The relA locus specifies a positive effector in branched-chain amino acid transport regulation.

The regulation of branched-chain amino acid transport and periplasmic binding proteins was studied in Escherichia coli strains which were isogenic except for the relA locus, the gene for the "stringent factor," which is responsible for guanosine tetraphosphate synthesis. The strain containing the relA mutation could not be derepressed for the synthesis of leucine transport or binding proteins when shifted from a medium containing all 20 amino acids in excess to one in which leucine was limiting. The relA+ strain showed normal derepression under these conditions.

Amino Acids, Branched-Chain↗
PubMed1979

Defective transport and other phenotypes of a periplasmic "leaky" mutant of Escherichia coli K-12.

A mutant of Escherichia coli K-12 deficient in high-affinity leucine transport and related binding proteins was obtained by selecting for azaleucine resistance after bacteriophage Mu mutagenesis. We determined that the cause was a generalized loss of periplasmic binding proteins and a sharp decrease in the activity of transport systems requiring them. Other transport systems resistant to osmotic shock and present in membrane vesicles, were affected to a lesser degree or not at all. The mutation, designated lky::Mucts, was shown to be a pleiotropic envelope mutation, rendering the mutant sensitive to ionic and nonionic detergents, antibiotics, and ethylenediaminetetraacetic acid: the strain had also acquired tolerance to colicins E1, E2, and E3, while remaining normally sensitive to a variety of bacteriophages. An analysis of the lipopolysaccharide of parent and mutant strains revealed a twofold reduction in the neutral sugar content of the core oligosaccharide of the lky strain, but no change in sensitivities to phages which utilize lipopolysaccharide or outer membrane proteins for absorption. The lky::Mucts locus was mapped by transduction and found to be located near, or in, the tolPAB gene cluster linked to gal. Secondary mutations suppressing the detergent sensitivity of lky arose at a frequency of 10(-7), yielding a variety of new phenotypes. The lky::Mucts mutation did not give rise to obvious alterations in the gross morphology of the cell or in cell division.

Bacterial Proteins↗
PubMed1978

Branched-chain amino acid transport regulation in mutants blocked in tRNA maturation and transcriptional termination.

The regulation of branched-chain amino acid transport and binding protein biosynthesis was studied in Escherichia coli strains containing hisT (the structural gene for pseudouridine synthetase) and rho (the structural gene for the mRNA transcriptional termination factor rho) mutations. The results indicate that the hisT strain cannot be fully derepressed for transport and that the hisT rho double mutant is partially derepressed under excess leucine conditions, but cannot be further derepressed by leucine deprivation. These data are consistent with a model in which fully mature tRNALeu is required for derepression and in which rho interacts with tRNALeu in regulating transport by terminating transcription, especially in excess-leucine growth conditions.

Amino Acids↗
PubMed1978

Genetic separation of high- and low-affinity transport systems for branched-chain amino acids in Escherichia coli K-12.

The Escherichia coli K-12 mutant strain AE4107 (livH::Mu) is defective in the high-affinity binding protein-mediated uptake system for L-leucine, L-valine, and L-isoleucine (LIV-I). We have used this strain to produce mutations in the residual LIV-II membrane-bound branched-chain amino acid uptake system. Mutants selected for their inability to utilize exogenous L-leucine were found to be defective in the LIV-II system and fell into two classes. One class, represented by strain AE410709 (livP9), showed a complete loss of saturable uptake for L-leucine, L-valine, and L-isoleucine up to 50 muM, and a second class, represented by strain AE4017012 (liv-12), showed a residual component of saturable leucine uptake with increased Km. These mutations, livP9 and liv-12, were closely linked and mapped in the 74 to 78 min region of the E. coli genetic map. Strains constructed so that they lacked both LIV-I and LIV-II transport systems excreted leucine. Strains of the genotype livH+ livP were found to have normal high-affinity binding protein-mediated transport (LIV-I and leucine specific), whereas the low-affinity (LIV-II) transport was completely missing. We concluded from these studies that the high-affinity binding protein-mediated transport systems (LIV-I and leucine specific) can operate independently of the membrane-bound LIV-II system.

Biological Transport, Active↗
PubMed1978

Reconstitution of neutral amino acid transport systems from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine were reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1mM dithiothreitol, and 0.5 mM EDTA a mixture that solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue-staining bands on sodim dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.

Alanine↗
PubMed1978

Reconstitution of neutral amino acid transport system from Ehrlich ascites tumor cells.

Amino acid transport systems for alanine and leucine have been reconstituted into artificial lipid vesicles. Purified plasma membrane vesicles from Ehrlich ascites cells were dissolved in 2% sodium cholate, 1 mM dithiothreitol, 0.5 mM EDTA, a mixture which solubilized approximately 50% of the membrane protein. This solubilized protein fraction was further purified by a combination of ammonium sulfate precipitations, gel filtration, and DEAE-cellulose chromatography. A fraction containing approximately 15 Coomassie blue staining bands on sodium dodecyl sulfate gels was obtained. This material was reconstituted into liposomes, and preliminary results demonstrated transport of alanine and leucine dependent on a sodium gradient. In addition, an electrogenic gradient mediated by valinomycin-induced potassium diffusion seemed to stimulate alanine uptake further.

Alanine↗
PubMed1977

A role for aminoacyl-tRNA synthetases in the regulation of amino acid transport in mammalian cell lines.

The regulation of neutral amino acid transport was examined utilizing two temperature-sensitive aminoacyl-tRNA synthetase mutants, by comparison of their transport activities at permissive and marginally permissive temperatures. When the temperature-sensitive leucyl-tRNA -synthetase mutant tsH1 was shifted from a normal growth temperature of 34 degrees to a marginally permissive temperature for growth of 38%, a significant enhancement in the initial rate of uptake of leucine and other L system amino acids was observed when compared with that of the parental cells (CHO-S). In contrast, the uptake of A system-specific amino acids (alanine, glycine, alpha-aminoisobutyric acid) showed no significant differences relative to the parent strain. In a similar manner, a temperature-sensitive asparaginyl-tRNA synthetase mutant RJK-4 exhibited a significant enhancement of the transport activity of A system amino acids relative to its parental strain RJK-0 when the growth temperature was shifted from 33 degrees to 39.5 degrees. Preliminary kinetic data suggest that the Vmax for transport is increased when the mutants are grown under conditions of amino acid limitation. These experiments suggest that the aminoacyl-tRNA synthetase or a related product may function in the regulation of neutral amino acid transport in Chinese hamster cells.

Amino Acids↗
PubMed1977

Neutral amino acid transport systems of tissue culture cells.

We have studied the interactions among a varied group of neutral amino acids for uptake into two different animal tissue culture cell lines. 3T3 mouse cells and Chinese hamster ovary cells were used in these studies. The results showed that the affinities of the amino acids clustered into two groups, one including amino acids such as glycine, alanine, and alpha-aminoisobutyric acid and the other group included amino acids such as leucine and phenylalanine. The specificities and properties of these systems are similar to those described earlier for the Ehrlich ascites cell, System A (alanine-preferring) and System L (leucine-preferring). System A is highly concentrative, sensitive to sodium ion, and serves only poorly for exchange while System L is not very concentrative or sensitive to alkali metal ion, although it serves very well for exchange with endogenous amino acids. The kinetic parameters for several amino acids are reported and other properties of the transport systems are discussed.

Alanine↗
PubMed1977

Regulation of amino acid transport activity and growth rate of animal cells in culture.

Balb/3T3 mouse cells grown in cell culture contain the A and L systems for neutral amino acid transport. L-Alanine was used as a test amino acid for the A system and L-leucine for the L system. Transport activities for both transport systems are altered as the growth conditions for the cells are altered. When cell growth was arrested by allowing cells to approach confluency, or by the removal of serum or by the addition of protein synthesis inhibitors, System A transport activity decreased and System L activity increased significantly. The content of the cellular amino acids increased 2- to 3-fold whenever cell growth was arrested by the conditions stated above. Increased cellular amino acids, therefore, are associated with decreased transport activity of System A and increased activity of System L. Altering cellular amino acids by loading or depleting the cells produced the predicted responses for the rapidly exchanging L-system but System A was less responsive. It is possible that regulatory mechanisms operate for the A system other than the direct action of alterations in cellular amino acid levels. The implications of the amino acid transport activity changes observed here on various studies on the role of transport in cell growth regulation and malignant transformation are discussed.

Alanine↗
PubMed1977

Reconstitution of neutral amino acid transport from partially purified membrane components from Ehrlich ascites tumor cells.

Solubilized protein fractions have been obtained from plasma membranes of Ehrlich ascites cells either by extraction with 0.5% Triton X-100 or by extraction with 2% cholate. Partial purification of the solubilized protein fraction has been obtained by utilizing a combination of ammonium sulfate precipitation and column chromatography. Leucine-binding activity has been detected in the Triton X-100 solubilized membrane fraction. The leucine-binding activity was measured by equilibrium dialysis and was saturable with high levels of leucine or phenylalanine and is not strongly effected by alanine. These properties are similar to those previously identified as System L. In addition, the cholate extracted protein fraction was partially purified and reconstituted into liposomes. Sodium dependent uptake of alanine and leucine could be demonstrated in the reconstituted vesicles. Concentrative uptake was dependent upon a sodium gradient. A membrane potential produced by valinomycin mediated potassium diffusion in the presence of sodium also stimulated amino acid transport in reconstituted liposomes.

Alanine↗
PubMed1977

Role of transport systems in amino acid metabolism: leucine toxicity and the branched-chain amino acid transport systems.

The livR locus, which leads to a trans-recessive derepression of branched-chain amino acid transport and periplasmic branched-chain amino acid-binding proteins, is responsible for greatly increased sensitivity toward growth inhibition by leucine, valine, and serine and, as shown previously, for increased sensitivity toward toxicity by branched-chain amino acid analogues, such as 4-azaleucine or 5',5',5'-trifluoroleucine. These phenotypes are similar to those of relA mutants; however, the livR mutants retain the stringent response of ribonucleic acid synthesis. However, an increase in the rate of transport or in the steady-state intracellular level of amino acids in the livR strain cannot completely account for this sensitivity. The ability of the LIV-I transport system to carry out exchange of pool amino acids for extracellular leucine is a major factor in leucine sensitivity. The previous finding that inhibition of threonine deaminase by leucine contributes to growth inhibition is confirmed by simulating the in vivo conditions using a toluene-treated cell preparation with added amino acids at levels corresponding to the internal pool. The relationship between transport systems and corresponding biosynthetic pathways is discussed and the general principle of a coordination in the regulation of transport and biosynthetic pathways is forwarded. The finding that the LIV-I transport system functions well for amino acid exchange in contrast to the LIV-II system provides another feature that distinguishes these systems in addition to previously described differences in regulation and energetics.

Biological Transport, Active↗
PubMed1977

Escherichia coli transport mutants lacking binding protein and other components of the branched-chain amino acid transport systems.

Further evidence on the role of binding proteins in branched-chain amino acid transport in Escherichia coli was obtained by selecting mutants with altered expression of the binding proteins. The mutator phage Mu was used to induce E. coli L-valine-resistant mutants defective in branched-chain amino acid transport. By making use of mild selective conditions and strain backgrounds with derepressed high-affinity, binding protein-mediated transport systems, we were able to isolate a new class of transport mutants defective in these systems. Mutant strains AE84084 (livK::Mucts) and AE840102 (livJ) were found to be defective in leucine-specific and LIV binding proteins, respectively, by transport assay, in vitro binding activity, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mutant strain AE4107 (livH::Mu), although lacking high-affinity, branched-chain amino acid transport, retained functional binding proteins and therefore evidently codes for an additional component of high-affinity transport. The livH, livJ, and livK mutations were mapped by transduction and shown to be closely linked to each other in the malT region (min 74) of the E. coli genetic map.

Amino Acids↗