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J Deshusses

Publications and source records attributed to J Deshusses.

At least 37 records · Page 2Linked to original sources
PubMed1986

Transport of gamma-butyrobetaine in an Agrobacterium species isolated from soil.

An Agrobacterium sp. isolated from soil by selective growth on gamma-butyrobetaine (gamma-trimethylaminobutyrate) as the sole source of both carbon and nitrogen has been shown to possess an inducible transport system for this growth substrate. This transport system has a Kt of 0.5 microM and a maximal velocity of 3.8 nmol/min per mg (dry weight). The influx of gamma-butyrobetaine is optimal at pH 8.5 and operates against a concentration gradient. The transport system shows a high specificity for trimethylamine carboxylic acid molecules of defined chain length. gamma-Butyrobetaine uptake was significantly reduced in osmotically shocked cells and a gamma-butyrobetaine binding activity was detected in the crude shock fluid. This suggests a transport mechanism involving a periplasmic gamma-butyrobetaine binding protein.

Bacterial Proteins↗
PubMed1985

Cloning of genes involved in myo-inositol transport in a Pseudomonas sp.

A soil isolate of a Pseudomonas sp. can utilize myo-inositol (MI) as the sole carbon source. In this strain, MI is transported through the membrane by a high-affinity transport system in which a periplasmic binding protein is involved. Mutants impaired in the transport system were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently identified by their slow growth rate at low MI concentrations. Strains with a low linear initial rate of MI uptake were analyzed. Using a broad-host-range cosmid cloning system, we have constructed a gene bank of the wild-type Pseudomonas sp. in an Escherichia coli recA-host. A rapid mating technique enabled us to screen the gene library for clones which are able to restore the active transport of MI in the mutant. An 11.5-kilobase segment containing genes involved in the MI transport has been isolated, and its restriction enzyme cleavage map has been determined.

Biological Transport↗
PubMed1984

Purification and properties of the myo-inositol-binding protein from a Pseudomonas sp.

A myo-inositol-binding protein was isolated from a Pseudomonas sp. soil isolate and was purified to homogeneity. Its molecular weight is 30,000, and it has a single binding site. The amino acid analysis showed that the protein contains three tryptophan residues and no cysteine. Tryptophan residues seem to be involved in the binding of the ligand, as shown by the modification of the fluorescence spectra and by the fact that oxidation of tryptophan residues with N-bromosuccinimide abolished the binding of myo-inositol. Sequence analysis of the N-terminal segment of 37 amino acids showed that 13 are conserved when compared with the galactose-binding protein of Escherichia coli.

Amino Acid Sequence↗
PubMed1983

Occurrence and role of phosphoenolpyruvate carboxykinase in procyclic Trypanosoma brucei brucei glycosomes.

Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) was detected in a particulate fraction of Trypanosoma brucei brucei procyclic culture form. It requires ADP rather than GDP for activity in the direction of carboxylation and is located in the glycosomes. Since phosphoenolpyruvate can serve to furnish ATP for glycolysis and can promote 3-phosphoglycerate or 1,3-bisphosphoglycerate formation without simultaneous alpha-glycerophosphate production, we suggest that the glycosomal phosphoenolpyruvate carboxykinase-malate dehydrogenase tandem contributes to ATP regeneration and NADH re-oxidation in the glycosome, and regulates alpha-glycerophosphate production.

Adenosine Diphosphate↗
PubMed1983

Stable cosmid vectors that enable the introduction of cloned fragments into a wide range of gram-negative bacteria.

A cosmid cloning system has been developed which is useful for the construction of genomic libraries and the introduction of clones into a broad range of bacterial species. The cosmids pMMB33 and pMMB34 allow selective cloning into their unique BamHI site of 36-kb DNA fragments generated by BamHI, Sau3A and MboI partial digestion. This selective cloning is achieved by a strategy that avoids formation of polycosmids without a dephosphorylation step. It uses two unique recognition sites within the vectors for endoncleases that generate blunt-ended DNA fragments for the preparation of left and right cosmid "arms". An alternative method that uses the unique EcoRI and SstI sites and dephosphorylation of the cosmid arms prior to BamHI digestion is also outlined and discussed. The DNA is first cloned with either vector into a rec- E. coli strain, where clones can be maintained stably, and can then be introduced by mobilization into a wide range of Gram-negative species to permit the study of gene expression and complementation. Because mobilization is much more efficient than transformation, the vector has the advantage that it can be transferred between bacterial species that specify different restriction systems, where transformation appears to be inefficient. The vectors have been used to generate gene libraries from the chromosomal DNA of several Pseudomonas and a Thiobacillus species. The genes specifying myo-inositol transport from Pseudomonas strain JD34 have been cloned with this system.

Cloning, Molecular↗
PubMed1981

Sugar Uptake in Lily Pollen : A PROTON SYMPORT.

The data presented here are consistent with a proton-sugar co-transport in germinated pollen of Lilium longiflorum Thunb. Optimal uptake occurs at pH 5.0. A K(m) of 1.7 to 1.8 millimolar is obtained from the initial rate of pH change induced by sucrose uptake as well as from uptake of [U-(14)C]-sucrose. The energy of activation is - 11 kilocalories mole(-1). The effect of several inhibitors and sugar competitors on [U-(14)C]sucrose and d-[U-(14)C] glucose uptake is given. The possibility of hydrolysis of sucrose prior to its transport into the pollen tube has been considered and reasons for choosing a sucrose-type uptake are presented. The possible in vivo significance of this co-transport process during pollen germination is discussed. Germinated pollen has features to recommend it as an experimental system of choice for studies of sugar uptake.

Journal Article↗
PubMed1981

Phosphate ion transport in rabbit brain synaptosomes.

Synaptosomes (vesicles of nerve endings) isolated from rabbit brain were studied as a model system for the uptake of inorganic phosphate. The phosphate uptake showed a sodium-dependent, saturable component with a Kt of 0.29 mM. The sodium-dependent component was larger at pH 6 than at pH 7.4 or 8. Application of potassium salts, ouabain, monensin, nigericin or FCCP decreased the uptake. The results indicate that the sodium-sensitive phosphate influx is dependent on the Na+ gradient and on the membrane potential, which might act, preferentially, on the transport of the monovalent phosphate ion.

Animals↗
PubMed1980

Labelling of the cyclitol permease in Klebsiella aerogenes.

The protein component(s) of the cyclitol-transport system in Klebsiella aerogenes has been labelled by using three different procedures. One method is based on differential alkylation with N-ethylmaleimide, and another on incorporation of amino acids during the induction process. A protein of mol.wt. 34 000 was labelled by both procedures; by alkylation with N-ethylmaleimide two other proteins of mol.wts. 55 000 and 67 000 were also labelled. The third uses diazotized [35S]sulphanilic acid after protection by substrate and the comparison of labelling of induced cells with non-induced cells; the label was also concentrated in a mol.wt.-33 000 peak. The labelled protein is, from the evidence, the cyclitol carrier.

Amino Acids↗
PubMed1980

Role of glycerol permeation in the bloodstream form of Trypanosoma brucei.

Under aerobic conditions, we have determined glycerol uptake in the long slender (LS) bloodstream form of Trypanosoma (Trypanozoon) brucei brucei by studying glycerophosphate accumulation in the parasites. The coupled enzyme theory applies to the permeation-phosphorylation sequence. Glycerol passage through the plasma membrane is asymmetric, the efflux process being favored over the influx process. No free diffusion of glycerol can be detected even under conditions under which free glycerol accumulates within the cells; most probably, glycerol permeation is mediated by a specific transport system. In the absence of respiratory activities, glycerol is known to be an end-product of T. brucei glycolysis; its production from glycerophosphate should allow ATP synthesis. The observed efflux of free glycerol following intracellular accumulation of glycerophosphate confirms the hypothesis that glycerol production occurs through reversal of glycerol kinase activity. We conclude that in vivo the role of the carrier-mediated asymmetric permeation process is to prevent inhibition of the reversal of the glycerol kinase-mediated reaction by removing free glycerol.

Anaerobiosis↗
PubMed1978

D-Glucose transport in Trypanosoma brucei. D-Glucose transport is the rate-limiting step of its metabolism.

D-Glucose and 2-deoxyglucose enter the 'long-slender' bloodstream form of Trypanosoma brucei only by means of a carrier-mediated process; no free diffusion can be observed. Permeation is not energy-dependent. The uptake is driven by the downhill concentration gradient of free substrate. The latter is maintained by the continual removal of sugar, due to the extremely high activities of the glycolytic enzymes. The permeation process is the rate-limiting step of glucose consumption, because permeation proceeds at a rate slower than metabolism. The inhibition of sugar uptake by glycerol was tested. Interactions at the carrier site can be ruled out since glucose and its 2-deoxy analog exhibit different inhibition kinetics.

Animals↗
PubMed1978

Purification and characterization of a long chain, fatty-acid-binding protein supplying the mitochondrial beta-oxidative system in the heart.

A fatty-acid-binding protein with a molecular weight of approximately 12 000 was purified from rat heart and the binding investigated by electron spin resonance. The stearic acid bound to the protein was found to be transferred to the mitochondrial beta-oxidative system, suggesting a role as transcytoplasmic fatty acid carrier for this protein. For the first time a physiological cytoplasmic protein was used as a carrier supplying the mitochondrial beta-oxidative system. A new mechanism of action is proposed to explain the control exerted by this type of protein in some membrane-linked enzymatic processes.

Animals↗
PubMed1977

Stereospecific ring opening of conduritol-B-epoxide by an active site asparatate residue of sucrase-isomaltase.

Conduritol-B-epoxide inactivates sucrase-isomaltase (sucrose alpha-glucohydrolase, EC 3.2.1.48-dextrin 6-alpha-glucohydrolase, EC 3.2.1.10) irreversibly with incorporation of 1 mol inhibitor/mol subunit, the affinity label being bound in both subunits to a beta-carboxyl group of an aspartic acid (Quaroni, A. and Semnza; G. (1976) J. Biol. Chem. 251, 3250-3253). Conduritol-B-epoxide is a racemic mixture of 1-L-1,2-anhydro-myo-inositol and 1-D-1,2-anhydro-myo-inositol, but only the latter one is the reactive component, since 1-L-1,2-anhydro-myo-inositol alone did not inactivate the enzyme. After inactivation by 1-D-1,2-anhydro-myo-inositol the label was released by hydroxylamine and identified as scyllo-inositol. One can decide now which C atom of the epoxide ring has been attacked by the enzyme's aspartate residue. This explains why only the D-enantiomer is the reactive species and provides further information about the role of the carboxylate residue during enzymic hydrolysis.

Animals↗
PubMed1977

Myo-Inositol transport in Klebsiella aerogenes. scyllo-Inositol, a non-metabolizable substrate for the study of the myo-inositol transport system.

1. Scyllo-Inositol is transported against a concentration gradient by Klebsiella aerogenes, but is not metabolized. 2. The apparent KT of scyllo-inositol is 0.05 X 10(-3) M while that of myo-inositol is 0.23 X 10(-3) M. The V values are respectively 32 and 12 nmol min(-1) mg cells(-1) (dry weight). 3. Both cyclitols bind to the same carrier, since typical competitive inhibition kinetics are observed. 4. Neither phosphoenolpyruvate phosphotransferase nor a periplasmic binding protein seems to be involved in the scyllo-inositol transport system. 5. The induction of the transport system is not repressed by malate, furmarate, citrate, succinate or pyruvate; however, acetate represses the induction. The activity of the transport system is increased when the growth medium is supplemented with adenosine 3':5'-monophosphate.

Biological Transport, Active↗
PubMed1977

Transport of cyclitols by a proton symport in Klebsiella aerogenes.

The respiration and the ATP content of Klebsiella aerogenes in the presence of various inhibitors were compared to the transport of scyllo-inositol. The ATPase was found to be inhibited by dicyclohexyl carbodiimide. The transport has been tested in anaerobiosis and aerobiosis. From the results obtained it is concluded that either ATP or respiration can sustain the transport activity in independent manner. 2. The energy derived from the respiratory chain reactions or the ATP hydrolysis results in electrogenic extrusion of protons. The electrochemical potential created drives the accumulation of scyllo-inositol, as shown by an increase of pH of the medium on addition of the substrate to cells in anaerobiosis. With non-induced cells no change in pH occurs, which demonstrates that proton flow is really linked to the transport. No H+/Na+ or K+ exchange is observed and the proton conductor carbonylcyanide m-chlorophenylhydrazone abolishes the pH shift caused by substrate addition. The stoichiometry of the symport H+/cyclitol is 1 and the half-maximum value of the pH variation as a function of the amount of scyllo-inositol added corresponds to a concentration of scyllo-inositol very close to the KT of influx.

Adenosine Triphosphatases↗
PubMed1977

Assymetry of the myo-inositol transport system in Klebsiella aerogenes. Energy is necessary to create the assymetry of the transport system.

1. In Klebsiella aerogenes the influx of myo-inositol proceeds during the steady state at a rate equal to that of efflux. 2. The kinetic parameters of influx during the steady state are similar to those observed at the initial time of uptake. 3. Efflux and influx processes share the same transport system. 4. The ability of other cyclitols to chase the accumulated scyllo-inositol is dependent on their affinity for the transport system. 5. A counter transport can be observed in preloaded cells only in poisoned cells and under anaerobiosis. 6. The efflux process is not temperature-dependent. 7. The KT of influx in poisoned cells is larger than that in normal cells. 8. Although no saturation kinetics of efflux could be observed, it can be inferred that the KT of efflux is at least 50-fold larger than the KT of influx; 9. The results suggest that in the transport of myo-inositol by K. aerogenes energy coupling enhances the affinity of the carrier for the substrate at the outer side of the cytolplasmic membrane by lowering the KT.

Aerobiosis↗