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1.
Int J Pharm ; 231(1): 1-9, 2002 Jan 01.
Article in English | MEDLINE | ID: mdl-11719008

ABSTRACT

The ex vivo permeation of an acylated model dipeptide, Myristoyl-Tryptophan-Leucine (Myr-Trp-Leu) was studied using pig buccal mucosa. Myr-Trp-Leu, being lipophilic, did not readily penetrate across the membrane. Rather, it accumulated in the epithelial and connective tissue of the mucosal barrier. The topological distribution of Myr-Trp-Leu across the mucosa, following its application in ethanol/phosphate buffer (30/70 pH 7.4), was determinated by thin-sectioning of the tissue, extraction of the peptide, and high performance thin layer chromatography (HPTLC). The concentration profile depended, of course, on the duration of the experiment and appeared to be dependent upon the presence of sufficient ethanol in order that the peptide could be solubilized. This important role for ethanol then raised the question of the solvent's effect on tissue integrity. Light microscopic examination of the mucosa was, therefore, undertaken, under identical conditions to those used in the permeation experiments, to evaluate any perturbation induced by the ethanolic vehicle. No obvious effects were observed.


Subject(s)
Dipeptides/pharmacokinetics , Mouth Mucosa/metabolism , Myristic Acid/pharmacokinetics , Absorption , Administration, Topical , Animals , Central Nervous System Depressants/pharmacokinetics , Dipeptides/chemical synthesis , Ethanol/pharmacokinetics , Leucine/pharmacokinetics , Swine , Tissue Distribution , Tryptophan/pharmacokinetics
2.
Eur J Pharm Biopharm ; 51(2): 93-109, 2001 Mar.
Article in English | MEDLINE | ID: mdl-11226816

ABSTRACT

Peptides and polypeptides have important pharmacological properties but only a limited number (e.g. insulin, oxytocin, vasopressin) have been exploited as therapeutics because of problems related to their delivery. The buccal mucosa offers an alternative route to conventional, parenteral administration. Peptides are generally not well absorbed through mucosae because of their molecular size, hydrophilicity and the low permeability of the membrane. Peptide transport across buccal mucosa occurs via passive diffusion and is often accompanied by varying degrees of metabolism. This review describes various approaches to improve the buccal absorption of peptides including the use of penetration enhancers to increase membrane permeability and/or the addition of enzyme inhibitors to increase their stability. Other strategies including molecular modification with bioreversible chemical groups or specific formulations such as bioadhesive delivery systems are also discussed.


Subject(s)
Administration, Buccal , Mouth Mucosa/metabolism , Peptides/administration & dosage , Animals , Cell Membrane Permeability , Humans , Mouth Mucosa/chemistry , Mouth Mucosa/cytology , Peptides/chemistry , Peptides/metabolism , Peptides/pharmacokinetics
3.
Eur J Pharm Biopharm ; 48(1): 21-6, 1999 Jul.
Article in English | MEDLINE | ID: mdl-10477324

ABSTRACT

In order to improve the buccal absorption of a dipeptide model compound, Tryptophan-Leucine (Trp-Leu), we have synthesised a lipophilic derivative by myristoylation of the N- terminal amino group of Trp-Leu. The acylated peptide (Myr-Trp-Leu) was characterized by HPTLC, purified and isolated by chromatography on a silica gel column. Its structure was confirmed by (13)C and (1)H NMR and mass spectroscopy. The increased lipophilicity of the Myr-Trp-Leu was compared to that of the native peptide by chromatography and by its partition coefficient between n-octanol and saline phosphate buffer. In addition, the sensitivity towards hydrolytic enzymes was studied. The interaction of Trp-Leu with liposomes as model membranes was also studied. The phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) was lowered in the presence of Myr-Trp-Leu, while it was increased in the presence of native parent peptide. Permeation experiments performed in vitro with pig buccal mucosa showed that the Myr-Trp-Leu accumulated in the tissue at the various concentrations tested. In contrast, the native peptide was able to pass through the membrane at all concentrations used. Lipophilic modification of the peptide by acylation drastically changes its behaviour towards tissue systems.


Subject(s)
Dipeptides/chemical synthesis , Dipeptides/pharmacokinetics , Chemical Phenomena , Chemistry, Physical , Chromatography, High Pressure Liquid , Chromatography, Thin Layer , Dipeptides/chemistry , Liposomes , Magnetic Resonance Spectroscopy , Mucous Membrane/metabolism , Myristic Acid/chemistry , Oxidation-Reduction , Permeability , Solubility
4.
Biol Chem ; 378(9): 1039-46, 1997 Sep.
Article in English | MEDLINE | ID: mdl-9348114

ABSTRACT

The mechanisms regulating the intracellular pH (pHi) in both forms of Trypanosoma brucei brucei (cultured cells) were investigated using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). The pHi values measured were 7.22+/-0.03 in the procyclics and 7.40+/-0.05 in the bloodstream form. In the presence of 24mM HCO3-, pHi values were slightly higher in both forms of trypanosomes suggesting a bicarbonate-linked pH regulation. pHi was more stable in procyclics (between 7.15 and 7.30 in the external pH range 6.4-7.6) than in the bloodstream forms. The amiloride analogue tested decreased pHi, suggesting Na+-driven Na+/H+ antiporters. H+-ATPases also seem to be involved in pHi regulation since the inhibitors N-ethylmaleimide (1 mM) and N,N'-dicyclohexylcarbodiimide (80 microM) induced a rapid acidification in both forms of trypanosomes. Addition of pyruvate caused a cytosol acidification in the bloodstream form only confirming the existence of a diffusion-facilitated carrier for pyruvate, with the cotransport of H+. Our results show that, although similar pH regulation mechanisms seem to exist in both forms of trypanosomes, the procyclics can regulate efficiently their pHi and consequently their plasma membrane potential whereas the bloodstream forms cannot always maintain their pHi and are easily depolarized following a small acid load.


Subject(s)
Trypanosoma brucei brucei/chemistry , Trypanosoma brucei brucei/metabolism , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/analogs & derivatives , 4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid/pharmacology , Amiloride/pharmacology , Animals , Bicarbonates/metabolism , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cytosol/metabolism , Diuretics/pharmacology , Enzyme Inhibitors/pharmacology , Ethylmaleimide/pharmacology , Fluoresceins , Fluorescent Dyes , Hydrogen-Ion Concentration , Nigericin/pharmacology , Proton-Translocating ATPases/antagonists & inhibitors , Pyruvic Acid/pharmacology , Sodium-Hydrogen Exchangers/metabolism
6.
Electrophoresis ; 18(1): 150-5, 1997 Jan.
Article in English | MEDLINE | ID: mdl-9059837

ABSTRACT

The translationally controlled tumor protein (TCTP) is a growth-related protein which is regulated at the translational level. It is present in mammals, higher plants and Saccharomyces cerevisiae. This study was undertaken to localize and further characterize the TCTP in human cell lysates using two-dimensional gel electrophoresis, monoclonal antibodies, and 45Ca-gel overlay. TCTP was found in several healthy and tumoral cells including erythrocytes, hepatocytes, macrophages, platelets, keratinocytes, erythroleukemia cells, gliomas, melanomas, hepatoblastomas, and lymphomas. It could not be detected in kidney and renal cell carcinoma (RCC). A monoclonal antibody raised against TCTP detected three isoforms likely due to post-translational modifications. A calcium binding property was found as well as heat stability and cytoplasmic localization. The high degree of homology from plants to man and its expression in many tissues suggests that TCTP most likely has a cell housekeeping function.


Subject(s)
Biomarkers, Tumor , Calcium-Binding Proteins/analysis , Erythrocytes/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Blood Platelets/chemistry , Calcium/metabolism , Calcium-Binding Proteins/chemistry , Chickens , Conserved Sequence , Electrophoresis, Gel, Two-Dimensional , Humans , Keratinocytes/chemistry , Liver/chemistry , Macrophages/chemistry , Mice , Molecular Sequence Data , Neoplasms/chemistry , Plants/chemistry , Rabbits , Saccharomyces cerevisiae/chemistry , Sequence Alignment , Tumor Protein, Translationally-Controlled 1
7.
Biochem J ; 314 ( Pt 2): 595-601, 1996 Mar 01.
Article in English | MEDLINE | ID: mdl-8670075

ABSTRACT

The characteristics of the plasma-membrane potential of procyclic and bloodstream forms of Trypanosoma brucei brucei (cultured cells) were investigated using the fluorescent anionic probe bisoxonol. Observation of a stable and representative plasma-membrane potential in the resting state required careful washing, centrifugation and maintenance of the cells at room temperature before measurement. Bloodstream forms were more prone to depolarization during washing at 4 degrees C than procyclic cells. The higher fluorescence observed in the presence of long slender cells than in the presence of procyclic cells shows that the plasma-membrane potential is more negative in the insect form. Healthy dilute cells can sustain their plasma-membrane potential for hours in the presence of external glucose. The presence of a high K+ concentration in the medium did not promote by itself the depolarization of either type of cell. Study of bisoxonol fluorescence as a function of time allowed us to follow the kinetics of the action of metabolic inhibitors in the presence of various ions. o-Vanadate (1 mM) was found to depolarize bloodstream-form cells rapidly but only in a phosphate-free NaCl buffer. Omeprazole and strophanthidin also specifically depolarized bloodstream-form trypanosomes. However, NN'-dicyclohexylcarbodi-imide depolarized both types of cell, but more rapidly for bloodstream-form cells. Bloodstream-form trypanosomes appear to use mainly a vanadate-sensitive Na+ pump to maintain their Na+-diffusion gradient. However, most of the ATPase inhibitors tested had little or no effect on the plasma-membrane potential of procyclics suggesting that this form of trypanosome may rely on several regulation mechanisms.


Subject(s)
Barbiturates/chemistry , Fluorescent Dyes/chemistry , Isoxazoles/chemistry , Membrane Potentials , Trypanosoma brucei brucei/physiology , Animals , Buffers , Cell Membrane/drug effects , Cell Membrane/physiology , Ethylmaleimide/pharmacology , Kinetics , Membrane Potentials/drug effects , Vanadates/pharmacology
8.
Eur Arch Otorhinolaryngol ; 252(8): 465-8, 1995.
Article in English | MEDLINE | ID: mdl-8719587

ABSTRACT

Human nasal mucosa biopsy samples were studied by biochemical and histological methods to determine whether the concentration of calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) as a marker of sensory nerves was dependent on the activity of neutral endopeptidase-like enzyme (NEP-LE). Mucosal samples from the middle turbinate were obtained from 32 patients undergoing functional endoscopic nasal surgery for non-allergic chronic rhinosinusitis. The degree of symptoms related to nasal obstruction, rhinorrhea and headaches was recorded. The number of inflammatory cells in each biopsy sample was evaluated by conventional histopathological examination. CGRP-LI was measured by radioimmunoassay. The activity of NEP-LE was evaluated in vitro using [3H] Leu5-enkephalin as substrate. A good correlation was observed between increased concentrations of CGRP, abundant inflammatory cells and the intensity of symptoms (R2 = 0.80). A low activity of NEP-LE was associated with a high concentration of both inflammatory cells and CGRP, suggesting that NEP-LE activity was reduced during inflammation. These observations further support the hypothesis that reduced degradation of sensory neuropeptides could be involved in the pathophysiological mechanisms of non-specific chronic rhinosinusitis.


Subject(s)
Calcitonin Gene-Related Peptide/analysis , Endopeptidases/metabolism , Nasal Mucosa/chemistry , Nasal Mucosa/enzymology , Culture Techniques , Humans , Nasal Mucosa/ultrastructure , Turbinates/enzymology , Turbinates/surgery
9.
FEBS Lett ; 320(3): 256-60, 1993 Apr 12.
Article in English | MEDLINE | ID: mdl-8385029

ABSTRACT

The nature of reactions catalysed by yeast phosphatidylinositol synthase expressed in E. coli has been investigated. The single enzyme is shown to carry both CDP-diacylglycerol-dependent incorporation of inositol into phosphatidylinositol (Km for inositol of 0.090 mM) and a CDP-diacylglycerol-independent exchange reaction between phosphatidylinositol and inositol (Km for inositol of 0.066 mM). The exchange reaction and reversal of phosphatidylinositol synthase were both stimulated by CMP, but had different optimum pH and requirements for substrates. These results suggest that CMP-stimulated exchange and CMP-dependent reverse reactions are distinct processes catalysed by the same enzyme, phosphatidylinositol synthase.


Subject(s)
Phosphatidylinositols/biosynthesis , Phosphotransferases/metabolism , Saccharomyces cerevisiae/enzymology , Transferases (Other Substituted Phosphate Groups) , CDP-Diacylglycerol-Inositol 3-Phosphatidyltransferase , Cytidine Diphosphate/metabolism , Diglycerides/metabolism , Genes, Fungal , Hydrogen-Ion Concentration , Inositol/metabolism , Recombinant Proteins/metabolism
10.
Biochem J ; 291 ( Pt 1): 297-301, 1993 Apr 01.
Article in English | MEDLINE | ID: mdl-8471048

ABSTRACT

The characteristics of L-proline transport in the procyclic form of Trypanosoma brucei were studied by using L-[14C]proline and a quick separation technique by centrifugation through an oil mixture. L-Proline uptake displayed typical Michaelis-Menten kinetics, with a Km of 19 microM and a maximum transport velocity of 17 nmol/min per 10(8) cells at 27 degrees C. The maximum concentration gradient factor obtained after 1 min of incubation was 270-fold in 0.02 mM proline. Cells permeabilized with 80 microM digitonin were still able to accumulate 14C label, but to a lower extent. The temperature-dependence of proline uptake gave an apparent activation energy of 74.9 kJ.mol-1. In competition studies with a 10-fold excess of structural analogues, L-alanine, L-cysteine and L-azetidine-2-carboxylate were found to inhibit L-proline uptake. Variation of pH or addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone ('CCCP') did not affect proline transport, showing that it is not driven by a protonmotive force. The absence of Na+, with or without monensin, did not affect proline transport. The absence of K+ and the addition of the Na+,K(+)-ATPase inhibitor ouabain had no significant effect on proline uptake activity. The thiol-modifying reagent iodoacetate (10 mM) decreased proline uptake by half. KCN (1 mM) inhibited proline uptake to a lesser extent, and the degree of inhibition was proportional to the intracellular ATP concentration. Preliminary experiments on proline transport in plasma-membrane vesicles of the cells, using a filtration technique, showed an uptake of proline (0.67 nmol/mg of protein) by the vesicles, but only in the presence of intravesicular ATP. The results thus obtained suggest that the proline carrier system in T. brucei is ATP-driven and independent of Na+, K+ or H+ co-transport.


Subject(s)
Proline/metabolism , Trypanosoma brucei brucei/metabolism , Adenosine Triphosphate/metabolism , Alanine/pharmacology , Animals , Azetidinecarboxylic Acid/pharmacology , Binding, Competitive , Biological Transport, Active/drug effects , Cell Membrane Permeability/drug effects , Cysteine/pharmacology , Digitonin/pharmacology , Hydrogen-Ion Concentration , Hydroxyproline/pharmacology , Iodoacetates/pharmacology , Iodoacetic Acid , Kinetics , Potassium Cyanide/pharmacology , Temperature , Thermodynamics
11.
Antimicrob Agents Chemother ; 36(11): 2506-11, 1992 Nov.
Article in English | MEDLINE | ID: mdl-1489194

ABSTRACT

Binding to phospholipids, uptake by simple diffusion, and an energy-dependent, carrier-mediated efflux are thought to characterize interactions between fluoroquinolones and bacterial cytoplasmic membranes. Here, we have found that an endogenous active efflux is unlikely in quinolone-susceptible Staphylococcus aureus. The protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), increased pefloxacin uptake in different membrane systems under conditions which excluded carrier-mediated transport, i.e., in bacterial cells at 4 degrees C and in protein-free phosphatidylglycerol liposomes. When plotted as a function of outer pH, the CCCP effect, both in S. aureus cells and in phosphatidylglycerol liposomes, correlated with pefloxacin labeling of everted S. aureus membrane vesicles, with all three profiles showing maximal effect at an acidic pH. So the CCCP effect may result not from inhibition of the proton motive force, as previously thought, but rather from acidification of the intramembrane space by the protonophore, leading to enhanced binding of the positive pefloxacin species to the inner leaflet of the bilayer. Moreover, antistaphylococcal potency and uptake profiles of pefloxacin in S. aureus and phosphatidylglycerol liposomes, assayed as a function of outer pH, peaked at a neutral pH. These observations suggest that zwitterionic and positive quinolone species are responsible for diffusion through and binding to the cytoplasmic membrane, respectively.


Subject(s)
Anti-Infective Agents/pharmacology , Pefloxacin/pharmacokinetics , Staphylococcus aureus/metabolism , Anti-Infective Agents/pharmacokinetics , Binding Sites , Biological Transport , Carbon Radioisotopes , Carbonyl Cyanide m-Chlorophenyl Hydrazone/pharmacology , Cytoplasm/metabolism , Dinitrophenols/pharmacology , Hydrogen-Ion Concentration , Intracellular Membranes/metabolism , Kinetics , Liposomes/metabolism , Microbial Sensitivity Tests , Phospholipids/metabolism , Staphylococcus aureus/drug effects , Staphylococcus aureus/ultrastructure
13.
J Biol Chem ; 265(25): 15003-9, 1990 Sep 05.
Article in English | MEDLINE | ID: mdl-2203766

ABSTRACT

The hamster islet B cell line HIT retains the ability to secret insulin in response to glucose and several receptor agonists. We used HIT cells to study the initial signaling events in glucose or receptor agonist-stimulated insulin secretion. Glucose stimulated insulin release from HIT cells in a dose-dependent manner with a half-maximal effect seen already at 1 mM. Insulin release was also stimulated by carbachol in a glucose-dependent manner. Glucose depolarized the HIT cell membrane potential as assessed with the fluorescent probe bisoxonol and raised intracellular Ca2+ as revealed by fura-2 measurements. Using a Mn2+ fura-2 quenching technique, we could show that the rise in intracellular Ca2+ was due to Ca2+ influx following opening of voltage-gated Ca2+ channels. Glucose is thought to increase the diacylglycerol (DAG) content of insulin-secreting cells. However, although HIT cells respond to glucose in terms of insulin secretion, membrane depolarization, and Ca2+ rise, the hexose was unable to increase the proportion of protein kinase C activity associated with membranes. In contrast, the membrane-associated protein kinase C activity increased in HIT cells exposed to the two receptor agonists carbachol and bombesin. Bombesin was shown to generate DAG with the expected fatty acid composition of activators of phospholipase C. Glucose, in contrast, only caused minor increases in DAG containing myristic and palmitic acid without affecting total DAG mass. The failure to detect stimulation of protein kinase C by glucose could be due to both the limited amount and to the different fatty acid composition of the metabolically generated DAG. The latter was in part supported by experiments performed on protein kinase C partially purified from HIT cells. Indeed, 1,2-dipalmitoylglycerol, presumed to be the main DAG species generated by glucose, was only one-third as active as 1,2-dioleoylglycerol and 1-stearoyl-2-arachidonylglycerol in stimulating the isolated enzyme at physiological Ca2+ concentration. It is therefore unlikely that DAG and protein kinase C play a major role in glucose-stimulated insulin secretion.


Subject(s)
Calcium/metabolism , Carbachol/pharmacology , Diglycerides/metabolism , Glucose/pharmacology , Glycerides/metabolism , Insulin/metabolism , Islets of Langerhans/drug effects , Protein Kinase C/metabolism , Animals , Bombesin/pharmacology , Cell Line , Cricetinae , Cytosol/drug effects , Cytosol/metabolism , Fluorescent Dyes , Insulin Secretion , Islets of Langerhans/metabolism , Kinetics , Manganese/metabolism
14.
Mol Cell Biochem ; 97(2): 137-44, 1990 Sep 21.
Article in English | MEDLINE | ID: mdl-2280762

ABSTRACT

Cadmium speciation of the intestinal compartment of the earthworm species, Lumbricus terrestris, has been investigated using polyacrylamide gel electrophoresis under non-denaturing conditions. Worms exposed to Cd(NO3)2 supplemented soils have been studied and compared to control samples. Prior to electrophoresis, the worm intestines were removed and dissected. Proteins in the crude intestinal extracts were separated using polyacrylamide gel electrophoresis. The cadmium distribution in the proteins has also been described. In a second set of experiments, cadmium bound to proteins was first isotopically exchanged with labelled cadmium (109Cd) and then cadmium speciation was performed using gel electrophoresis. Autoradiography of this gel shows an intense band in the contaminated sample whereas this band was absent in the control sample. These results show that one type of major protein has a strong affinity for cadmium in the worm intestinal extract. This type of protein had a migration close of that of rabbit liver metallothionein used for comparison.


Subject(s)
Cadmium/metabolism , Intestinal Mucosa/metabolism , Metallothionein/metabolism , Animals , Autoradiography , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Oligochaeta , Rabbits
15.
J Bacteriol ; 170(11): 5236-40, 1988 Nov.
Article in English | MEDLINE | ID: mdl-3182728

ABSTRACT

A binding protein for gamma-butyrobetaine was purified from osmotic shock fluid of an Agrobacterium sp. It was a monomeric protein with an apparent molecular weight of 52,000 or 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The isoelectric point was 4.3, as determined by isoelectric focusing. Amino acid analysis of the protein showed that Asx and Glx were predominant components and that the protein contained no cysteine. The dissociation constant of this protein for gamma-butyrobetaine was found to be 0.7 microM by equilibrium dialysis. Attempts to sequence the amino-terminal end with the Edman method failed, suggesting that this region of the protein is blocked.


Subject(s)
Bacterial Proteins , Betaine/analogs & derivatives , Carnitine , Carrier Proteins/isolation & purification , Rhizobium/metabolism , Amino Acid Sequence , Amino Acids/analysis , Betaine/metabolism , Carrier Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Molecular Sequence Data , Molecular Weight , Peptide Fragments/analysis , Trypsin
16.
Biochimie ; 70(10): 1411-6, 1988 Oct.
Article in English | MEDLINE | ID: mdl-3148329

ABSTRACT

An Agrobacterium sp. isolated from soil is able to use gamma-butyrobetaine as its sole source of carbon and nitrogen. The involvement of thiol groups for active transport of gamma-butyrobetaine was investigated by use of the thiol alkylating reagent N-ethylmaleimide (NEM) and the dithiol specific reagent phenylarsine oxide (PAO). Both reagents strongly inhibited gamma-butyrobetaine uptake, but also induced the release of the accumulated substrate, suggesting that the transport system either contains a dithiol-dependent protein or that a small thiol-containing molecule is implicated in the uptake phenomenon.


Subject(s)
Betaine/analogs & derivatives , Carnitine , Rhizobium/metabolism , Sulfhydryl Reagents/pharmacology , Arsenicals/pharmacology , Betaine/metabolism , Biological Transport, Active/drug effects , Ethylmaleimide/pharmacology
18.
Mol Gen Genet ; 213(1): 36-41, 1988 Jul.
Article in English | MEDLINE | ID: mdl-2851708

ABSTRACT

The plasmid ColD-CA23, a high copy number plasmid of 5.12 kb, contains genes for colicin D (cda), for immunity colicin D (cdi), and for a lysis function (cdl). These genes are arranged on a contiguous 2.4 kb fragment in the following sequence: cda, cdi, cdl. They are transcribed in two operons, one transcribing cda and cdl from a SOS inducible promoter, the other transcribing cdi in the opposite direction. The expression of cda and cdl is modulated by a repressor, cdr, which is encoded on the same transcript as cda and cdl. In the absence of this repressor, transcription from the SOS inducible colicin D promoter is exceptionally strong and leads to protein contents up to 50% of total cellular proteins. This autoregulative repressor is a new finding in the control mechanisms of expression of colicins. We have also identified the gene product of cdl to be a 10,000 dalton protein.


Subject(s)
Colicins/genetics , Escherichia coli/genetics , Gene Expression Regulation , Genes, Bacterial , Plasmids , Repressor Proteins/metabolism , Transcription Factors/metabolism , Binding Sites , DNA Restriction Enzymes , DNA-Directed RNA Polymerases/metabolism , Genes , Operon , Protein Binding , Transcription, Genetic
19.
FEBS Lett ; 210(2): 137-41, 1987 Jan 05.
Article in English | MEDLINE | ID: mdl-3792559

ABSTRACT

The high efficiency of glycolysis in Trypanosoma brucei has been attributed to impermeability of the glycosomal membrane to most metabolites. However, the strong stimulation of the glycolytic rate by exogenous metabolites and coenzymes in intact glycosomes is only compatible with their accessibility to the internal space. The accessibility of glycosomal enzymes to protein labeling agents of varying charge and size has been investigated. The results show that the glycosomal membrane is permeable to small molecules of the size of metabolites, but impermeable to larger molecules.


Subject(s)
Enzymes/metabolism , Glycolysis , Intracellular Membranes/enzymology , Organoids/enzymology , Trypanosoma brucei brucei/enzymology , Animals , Enzymes/isolation & purification , Indicators and Reagents , Molecular Weight
20.
J Bacteriol ; 168(2): 780-4, 1986 Nov.
Article in English | MEDLINE | ID: mdl-3782024

ABSTRACT

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.


Subject(s)
Betaine/analogs & derivatives , Carnitine , Rhizobium/metabolism , Bacterial Proteins/metabolism , Betaine/metabolism , Biological Transport , Carrier Proteins/metabolism , Hydrogen-Ion Concentration , Osmotic Pressure , Soil Microbiology
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