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1.
Astrocytes have been proposed to regulate the extracellular space in the brain, even if rather little is known about their specific functions. One possibility for obtaining more knowledge on the functions of astroglial cells is to examine how they respond on exposure to pharmacological agents. Na+-valproate is an anticonvulsive drug which is used in the treatment of several types of epilepsy. The mechanisms of action of the drug are not fully understood, but the GABA-ergic system, both in neurons and astrocytes, has been shown to be affected. In the present study, the effects of valproate were investigated on astroglial cells in primary cultures from newborn rat cerebral cortex. The transport of the drug itself and its effects on the transport of the amino acid transmitters glutamate, aspartate and -aminobutyric acid (GABA) into astrocytes were examined. The [3H]valproate transport into the astrocytes was increased after exposure tol-glutamate but notl-aspartate. On the other hand, after acute exposure for the drug, the transport of [3H]l-glutamate and [3H]l-aspartate decreased, as also did the affinity but not the transport capacity for the [3H]GABA uptake. However, after 5 days chronic valproate exposure, no effects could be seen on the uptake kinetics ofl-glutamate orl-aspartate. For GABA, the affinity decreased, while the transport capacity remained unchanged compared with controls. The results showed that valproate, glutamate, aspartate and GABA were capable of interacting significantly with each others transport into the astrocytes. 相似文献
2.
Hypotaurine uptake was compared to taurine and GABA uptakes in brain slices under identical experimental conditions. The slices effectively concentrated both hypotaurine and GABA from the medium, whereas taurine was taken up more slowly. The uptakes of these three structurally related amino acids were all saturable, consisting of one low-and one high-affinity transport component. The kinetic parameters of hypotaurine uptake were of the same order of magnitude as those of GABA uptake. All uptake systems were sensitive to temperature, metabolic poisons, and sodium omission. Hypotaurine uptake was inhibited by GABA,l-2,4-diaminobutyric acid (l-DABA), cysteic acid, and -alanine, but not by taurine. Taurine uptake was strongly reduced by hypotaurine, -alanine, andl-DABA, as well as by GABA, whereas GABA uptake was affected only by cystamine,l-DABA, and nipecotic acid.The uptake processes of hypotaurine, taurine, and GABA were thus fairly similar and showed properties characteristic for neurotransmitter uptake. Hypotaurine uptake resembled more GABA than taurine uptake. The present inhibition studies suggest that there may exist only one common two-component transport system for these three amino acids. 相似文献
3.
Summary Rapid unidirectional transport (15 sec) ofl-serine and 2-methylaminoisobutyric acid (MeAIB) was studied in the isolated perfused rat pancreas using a dual-tracer dilution technique. Time-course experiments in the presence of normal cation gradients revealed a time-dependent transstimulation ofl-serine influx and transinhibition of MeAIB influx. Transport of the model nonmetabolized System A analog MeAIB was Na+ dependent and significantly inhibited during perfusion with 1mm ouabain. Although transport ofl-serine was largely Na+ independent, ouabain caused a time-dependent inhibition of transport. Influx of both amino acids appeared to be inhibited by the ionophore monensin but unaffected by a lowered extracellular potassium concentration. Removal of extracellular calcium had no effect on influx of the natural substratel-serine, whereas stimulation of transport by exogenous insulin (100 U/ml) was entirely dependent upon extracellular calcium and unaffected by ouabain. Paradoxically, exogenous insulin had no effect on the time-course of MeAIB influx. The characteristics ofl-serine influx described in earlier studies together with our present findings suggest that insulin may modulate the activity of System asc in the exocrine pancreatic epithelium by a calcium-dependent mechanism. 相似文献
4.
We determined the extent of Na+-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na+-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na+-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, -alanine, -aminoisobutyric acid (AIB), -methylaminoisobutyric acid (MeAIB), -amino-n-butyric acid and l-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both d-serine and d-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and d-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2,7-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (I
sc) in voltage-clamped Caco-2 cell monolayers in Na+-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, d-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pHi and inward I
sc. In conclusion, Caco-2 cells express a Na+-independent, H+-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.This study was supported by a Wellcome Trust Fellowship (to DTT). Charlotte Ward, Maureen Sinclair and Ken Elliott provided excellent technical assistance. 相似文献
5.
An inherited amino acid transport deficiency results in low concentrations of glutathione (GSH) in the erythrocytes of certain sheep. Earlier studies based on phenotyping according to GSH concentrations indicated that the gene Tr
H, which controls normal levels of GSH, behaves as if dominant or incompletely dominant to the allele Tr
h, which controls the GSH deficiency. The present paper shows that when sheep are classified according to amino acid transport activity, the Tr
H gene behaves as if codominant to Tr
h. Erythrocytes from sheep homozygous for the Tr
H gene exhibit rapid saturable l-alanine influx (apparent K
m ,21.6mm; V
max, 22.4 mmol/liter cells/hr). Cells from sheep homozygous for the Tr
h gene exhibit slow nonsaturable l-alanine uptake (0.55 mmol/liter cells/hr at 50mm extracellular l-alanine). Cells from heterozygous sheep show saturable l-alanine uptake with a diminished V
max (apparent K
m, 19.1mm; V
max, 12.7 mmol/liter cells/hr). These erythrocytes have a significantly lower GSH concentration than cells from Tr
H, TrH sheep but similar intracellular levels of dibasic amino acids.The authors are grateful to the M.R.C. for a Project Grant. 相似文献
6.
[3H]Glutamate uptake into astrocytes in primary culture was potently inhibited by the aspartate analoguesl- andd-aspartic acid,Dl-threo--hydroxy-aspartic acid,l-aspartic acid--hydroxymate (IC50's: 136, 259, 168, and 560 M, respectively) and by -Dl-methylene-aspartate, a suicide inhibitor of asparate aminotransferase (IC50: 524 M), and by the endogenous sulphur-containing amino acidl-cysteinesulfinic acid (IC50: 114 M). [3H]Glutamate uptake was not significantly affected by either N-methyl-d-aspartate orDl-homocysteine thiolactone. These results demonstrate that other excitatory amino acids including aspartate andl-cysteinesulfinic acid (but excludingl-homocysteic acid) interact with the glutamate transport system of astrocytes. Inhibition of glutamate uptake may significantly increase the level of neuronal excitability. 相似文献
7.
Summary Amino acid and carbohydrate transport in normal and malignant transformed hamster cells was studied after binding of the protein Concanavalin A (Con. A) to the surface membrane. Experimental conditions were used so that a similar number of Con. A molecules were bound to both types of cells. The transport of amino acids was inhibited after Con. A binding in the transformed cells but not in normal cells. This was found with the metabolizable amino acidsl-leucine,l-arginine,l-glutamic acid, andl-glutamine, and with the non-metabolizable amino acids cycloleucine and -aminoisobutyric acid. Transport ofd-glucose andd-galactose was more inhibited by Con. A in transformed than in normal cells, and in both types of cellsd-glucose was inhibited more thand-galactose. The inhibition by Con. A on transport was specific, since there was no effect on the transport ofl-fucose in either normal or transformed cells. Con. A also did not effect the entry of 3-0-methyl-d-glucose.These observations can be used to locate amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A. The results indicate that Con. A sites are associated in normal cells with transport sites ford-glucose and to a lesser extentd-galactose, and in transformed cells with transport sites for amino acids and to a greater extent than in normal cells withd-glucose andd-galactose. Malignant transformation of normal cells therefore results in a change in the location of amino acid and carbohydrate transport sites in the surface membrane in relation to the binding sites for Con. A. 相似文献
8.
The transport specificity of system y+L of human erythrocytes was investigated and the carrier was found to accept a wide range of amino acids as substrates. Relative rates of entry for various amino acids were estimated from their trans-effects on the unidirectional efflux of l-[14C]-lysine. Some neutral amino acids, l-lysine and l-glutamic acid induced marked trans-acceleration of labeled lysine efflux; saturating concentrations of external l-leucine and l-lysine increased the rate by 5.3±0.63 and 6.2±0.54, respectively. The rate of translocation of the carrier-substrate complex is less dependent on the structure of the amino acid than binding. Translocation is slower for the bulkier analogues (l-tryptophan, l-phenylalanine); smaller amino acids, although weakly bound, are rapidly transported (l-alanine, l-serine). Half-saturation constants (±sem) calculated from this effect (l-lysine, 10.32±0.49 m and l-leucine, 11.50±0.50 m) agreed with those previously measured in cis-inhibition experiments. The degree of trans-acceleration caused by neutral amino acids did not differ significantly in Na+, Li+ or K+ medium, whereas the affinity for neutral amino acids was dramatically decreased if Na+ or Li+ were replaced by K+. The observation that specificity is principally expressed in substrate binding indicates that the carrier reorientation step is largely independent of the forces of interaction between the carrier and the transport site.We wish to thank Dr C.A.R. Boyd for helpful discussions and Prof. H.N. Christensen for sharing with us very relevant bibliographic material. We are grateful to FONDECYT (1282/91) and DTI (B 2674) (Chile) for financial assistance. 相似文献
9.
The yeastRhodotorula glutinis was found to transport amino acids against a concentration gradient (100∶1 for 10−6
m l-lysine and 1500∶1 for 10−6
m α-aminoisobutyric acid). Anaerobically, the concentration gradients of free amino acids were occasionally higher than aerobically.
The influx is saturable with an apparentK
m of 1mm forl-lysine and 2mm for α-aminoisobutyric acid. The pH optimum for AIB uptake was 5.0, the apparent activation energy between 5° and 30° was
13,200 cal/mole. Competition of an asymmetric nature among various amino acids for uptake was observed. Intracellular amino
acids did not leave the cell under any conditions of incubation, short of breaking up the plasma membrane, but they showed
a powerful “trans” inhibitory effect on the uptake of amino acids. 相似文献
10.
Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to
nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were
under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently
employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10−4
m for glycine, 2.5×10−4
m forl-cysteine, 6×10−5 and 4×10−4
m forl-lysine, 3×10−5 and 6×10−4
m forl-methionine, 7–18×10−5 and 1.6×10−3
m forl-aspartic acid and 6×10−5 and 2×10−3
m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for
glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan
(and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine,
arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids
tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic
inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical
activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine. 相似文献
11.
Robert H. White 《Archives of microbiology》1985,142(1):1-5
In an attempt to establish the nature of the ammonium-assimilation products which mediate the inhibition by ammonium of nitrate uptake in cyanobacteria, the effect of different amino acids on nitrate utilization by intact Anacystis nidulans cells has been assayed. To exclude an indirect inhibition of nitrate uptake through the ammonium which the amino acids might release, the cells were pretreated with l-methionine-d,l-sulfoximine (MSX), a potent inactivator of glutamine synthetase. Under these conditions, several l-amino acids, but not the corresponding d-isomers, affected nitrate utilization to a variable extent, causing inhibitions ranging between 20 and 80% when added at 20 mM concentration.For most of the inhibitory amino acids, including l-isoleucine, l-leucine and l-valine, a correlation was found between their ability to act as amino group donors to -ketoglutarate, in reactions catalyzed by A. nidulans cell-free extracts, and their inhibitory effect on nitrate utilization. l-Glutamine, l-asparagine and glycine, being effective inhibitors of nitrate utilization, were poor substrates for the transaminating activity to -ketoglutarate, however. The possible role of the latter amino acids as mediators in the ammonium-promoted inhibition of nitrate uptake is discussed.Abbreviations MSX
l-methionine-d,l-sulfoximine
- MTA-5
mixed alkyltrimethylammonium bromide
- Mops
morpholinopropane sulfonic acid 相似文献
12.
Katsumi Higashi Hiroshi Kamada Hiroshi Harada 《Plant Cell, Tissue and Organ Culture》1996,45(2):109-114
The addition of l-glutamine, -alanine or l-glutamic acid strongly stimulates somatic embryo formation in carrot, not only in the number of somatic embryos formed but also with respect to their development. The effects of the amino acids on somatic embryogenesis were stronger than that of ammonium ion. In particular, l-glutamine strongly stimulated the development of somatic embryos. To clarify the different effects of amino acids and ammonium ion, the activity of glutamine synthetase (GS; EC 6.3.1.2), a key enzyme involved in nitrogen assimilation, was measured. Its activity decreased during the later stages of embryo development.Abbreviations -Ala
-alanine
- Glu
l-glutamic acid
- Gln
l-glutamine
- 2,4-D
2, 4-dichlorophenoxyacetic acid
- -GHA
l-glutamic acid -monohydroxamate
- GS
glutamine synthetase
- MS medium
Murashige & Skoog (1962) medium
- MS-NH4 medium
MS medium without NH4NO3
- MS+NH4 medium
MS-NH4 medium with 10 mM NH4Cl
- MS+ala medium
MS-NH4 medium with 10 mM -alanine
- MS+GLU medium
MS-NH4 medium with 10 mM l-glutamic acid
- MS+GLN medium
MS-NH4 medium with 10 mM l-glutamine
- NIR
nitrite reductase
- NR
nitrate reductase 相似文献
13.
A chemically defined medium was developed for the biosynthesis of cephalosporin C by Paecilomyces persicinus Nicot strain P-10. Glucose served as the major carbon source and nitrogen was supplied by five amino acids, l-arginine, l-aspartic acid, l-glutamic acid, glycine and dl-methionine. Omission of any of the first four diminished or prevented production of cephalosporin C; omission of methionine did not. Methionine is not critical for the production of cephalosporin C in this defined medium. Production of the antibiotic was affected by the concentrations of inorganic salts employed. Biotin was required for growth and cephalosporin C synthesis. The addition of l-lysine precursors to the medium did not influence cephalosporin C levels and l-lysine itself inhibited antibiotic production. Known precursors of -lactam antibiotics as well as oleic acid did not affect biosynthesis of cephalosporin C. Chemical changes occurring in the defined medium revealed that glucose was efficiently utilized after 96 hours incubation whereas total soluble nitrogen levels increased following an initial sharp decrease. Mycelial weight and cephalosporin C production were both maximal after 96 hours incubation. Mycelial nitrogen was highest after 48 hours incubation whereas mycelial lipid levels were greatest after 72 hours. 相似文献
14.
Austin K. Mircheff Carel H. van Os Ernest M. Wright 《The Journal of membrane biology》1980,52(1):83-92
Summary Membrane vesicles obtained from the basal lateral membranes of the rat intestinal epithelium were used to study the pathways for neutral amino acid transport.In the absence of sodium there was a stereospecific uptake ofl-alanine which exhibited saturation kinetics (K
m 0.73mm andV
max 5.3 nmol/mg min at 22°C). The activation energy for this process was 8.1 kcal/mole between 5 and 25°C. Preloading the vesicles with alanine increased the unidirectional influx of alanine into the vesicle. Competition experiments indicated that the affinity of the sodium-independent transport system was glutamine > threonine > alanine > phenylalanine > valine > methionine > glycine > histidine > proline, N-MeAIB. These are the characteristics of the classical L transport system.External sodium increased the rate of the stereospecificl-alanine uptake. The Na-dependent flux had aK
m of 0.04mm and aV
max of 0.26 nmol/mg min at 22°, and an activation energy of 9.1 kcal/mole between 5 and 25°C. Competition experiments suggest the existence of three separate pathways for alanine transport in the presence of sodium. A major pathway is shared by all other amino acids tested (i.e., threonine, glutamine, methionine, phenylalanine, valine, proline and N-MeAIB). This resembles the classical A system. A second pathway is unavailable to either phenylalanine or N-MeAIB; this is reminiscent of the classical ASC system; and the third is a novel pathway which is shared by N-MeAIB but not phenylalanine.The sodium-independent and the sodium-dependent transport ofl-alanine was blocked by PCMBS and significantly inhibited by DTP and NEM. It is concluded that the sodium-independent system (the L-like system) accounts for the efflux of neutral amino acids from the epithelium to the blood during the absorption of amino acids from the gut, and that the sodium-dependent transport processes may play an important role in the supply of amino acids to the epithelium in the absence of amino acids from the gut lumen. 相似文献
15.
Summary The sodium-dependentl-alanine transport across the plasma membrane of oocytes ofXenopus laevis was studied by means of [14C]-l-alanine,22Na+ and electrophysiological measurements. At fixed sodium concentrations, the dependence of alanine transport on alanine concentration follows Michaelis-Menten kinetics; at fixed alanine concentrations, the transport varies with sodium concentration with a Hill coefficient of 2. In the presence of sodium the uptake of alanine is accompanied by a depolarization of the membrane. Under voltage-clamp conditions this depolarization can be compensated by an inward-directed current. Assuming that this current is carried by sodium we arrive at a 21 stoichiometry for the sodium-alanine cotransport. The assumption was confirmed by direct measurements of both sodium and alanine fluxes at saturating concentrations of the two substrates, which also yielded a stoichiometry close to 21. The sodium-l-alanine cotransport is neither inhibited by furosemide (0.5 mmol/liter) nor by N-methyl amino isobutyric acid (5 mmol/liter). A 20-fold excess ofd-alanine overl-alanine caused about 60% inhibition. 相似文献
16.
Enchytraeus albidus is able to absorb dissolved14C-labeled neutral amino acids (glycine, L-alanine, L-valine,-aminoisobutyric acid) and an amino-acid mixture from ambient water across the body surface against considerable concentration gradients. Saturation kinetics and susceptibility of glycine uptake to competitive inhibition by alanine suggest mediated transport. Absorption of neutral amino acids is an active process. Exchange diffusion of preloaded-aminoisobutyric acid against external glycine or-aminoisobutyric acid could not be detected. Results on inhibition of glycine uptake by a variety of low-molecular-weight substances indicate that glycine absorption is highly specific for neutral amino acids and somewhat less for basic amino acids; it is unspecific for non--amino acids, acidic amino acids, carbohydrates, and organic acids. Rates of transintegumentary net influx of glycine are nearly identical to14C-glycine influx, suggesting that only small amounts of amino acids are released, as compared with the capacity for uptake. Thus,14C-amino-acid influx data are used for characterization of the uptake system. Glycine uptake is positively correlated to external salinity. In fresh water, absorption is nearly zero; between 10 and 20 S, uptake increases markedly reaching maximum values at 30 S; these remain almost constant at 40 S. Transport constants and maximum uptake rates increase with rising salinities. Since absorption of glycine and L-valine is susceptible to sodium depletion, similar mechanisms presumably underly salinity-dependent uptake of amino acids and sodium-dependent solute transport. Oxygen consumption is not significantly modified by different external salinities. Estimates of nutritional profit gained from absorption of amino acids vary between 4 and 15 % of metabolic rate for glycine absorption and between 10 and 39 % for uptake of an amino-acid mixture, according to external concentrations (10 and 50 µM) and salinities (20 and 30 S). 相似文献
17.
Roger Griffiths Angus Grieve John Dunlop Inge Damgaard Hanne Fosmark Arne Schousboe 《Neurochemical research》1989,14(4):333-343
A detailed kinetic study of the inhibitory effects ofl- andd-enantiomers of cysteate, cysteine sulphinate, homocysteine sulphinate, homocysteate, and S-sulpho-cysteine on the neuronal, astroglial and synaptosomal high-affinity glutamate transport system was undertaken.d-[3H] Aspartate was used as the transport substrate. Kinetic characterisation of uptake in the absence of sulphur compounds confirmed the high-affinity nature of the transport systems, the Michaelis constant (K
m) ford-aspartate uptake being 6 M, 21 M and 84 M, respectively, in rat brain cortical synaptosomes and primary cultures of mouse cerebellar granule cells and cortical astrocytes. In those cases where significant effects could be demonstrated, the nature of the inhibition was competitive irrespective of the neuronal versus glial systems. The rank order of inhibition was essentially similar in synaptosomes, neurons and astrocytes. Potent inhibition (K
iK
m) of transport in each system was exhibited byl-cysteate, andl- andd-cysteine sulphinate whereas substantially weaker inhibitory effects (K
i>10–1000 times the appropriateK
m value) were exhibited by the remaining sulphur amino acids. In general, inhibition: (i) was markedly stereospecific in favor of thel-enantiomers (except for cysteine sulphinate) and (ii) was found to decrease with increasing chain length. Computer-assisted molecular modelling studies, in which volume contour maps of the sulphur compounds were superimposed on those ofd-aspartate andl-glutamate, demonstrated an order of inhibitory potency which was, qualitatively, in agreement with that obtained quantitatively by in vitro kinetic studies.Special issue dedicated to Dr. Elling Kvamme 相似文献
18.
The uptake of pipecolic acid by the mouse brain was compared to that of several amino acids and amines, following an injection of a double-labeled mixture into the carotid artery. In general, BUI (brain uptake index) values were lower in the mouse than those previously reported in the rat. The only exception was proline. Lysine, a precursor of pipecolic acid biosynthesis in brain, showed a higher BUI than pipecolic acid. The BUI ofD,l-[3H]pipecolic acid was found to be 3.39 (at 0.114 mM). This was saturable between a concentration of 0.114 and 3.44 mM. Kinetic analysis suggests the presence of two kinds of transport systems. Substances structurally related to pipecolic acid, such as nipecotic acid, isonipecotic acid,l-proline, and piperidine show a significant inhibitory effect. Among the amino acids tested, only GABA showed an inhibitory effect. Data are reported which, when considered with other findings (5), present evidence that pipecolic acid is (1) synthesized both in vitro and in vivo in the mouse brain, (2) actively transported in vivo into the brain, and (3) taken up in vitro by synaptosomal preparations. 相似文献
19.
l-Glutamic acid (l-Glu) and other excitatory amino acids and amino acid analogs enhanced [35S]thiocyanate (SCN–) uptake in isolated-resealed synaptic membrane vesicles. The SCN– uptake was used as a measure of membrane depolarization to evaluate the characteristics of functional excitatory amino acid receptors in the synaptic membranes.N-Methyl-d-aspartate (NMDA) andl-Glu produced additive effects on SCN– accumulation indicating the presence of distinctl-Glu and NMDA receptors. On the other hand, kainic acid (KA) andl-Glu shared either common receptor sites or ion channels. The effects of antagonists on NMDA,l-Glu, and KA stimulation of SCN– influx were consistent with previously reported electrophysiologic observations in intact neurons. 相似文献
20.
In Nocardia sp. 239 d-phenylalanine is converted into l-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent l-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on l- or d-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and l-phenylalanine. Highest rates of l-phenylalanine dehydrogenase production were observed with increasing ratios of l-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from l- and d-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42 000 and consisted of a single subunit; it showed activity with l-phenylalanine, phenylpyruvate, 4-hydroxyphenyl-pyruvate, indole-3-pyruvate and -ketoisocaproate, but not with imidazolepyruvate, d-phenylalanine and other l-amino acids tested. Maximum activities with phenylpyruvate (310 mol min-1 mg-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.Abbreviations RuMP
ribulose monophosphate
- HPS
hexulose-6-phosphate synthase
- HPT
hexulose-6-phosphate isomerase
- FPLC
fast protein liquid chromatography 相似文献