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1.
The binding of [3H]AMPA (Dl--amino-3-hydroxy-5-methylisoxazole-4-propionic acid), a ligand for the putative quisqualate excitatory amino acid receptor subtype, was evaluated using centrifugation and filtration receptor binding techniques in rat brain crude synaptosomal membrane preparations. Maximal specific binding of [3H]AMPA occurred in Triton X-100 treated membranes in the presence of the chaotropic agent potassium thiocyanate (KSCN). The effects of KSCN on binding were reversible and optimal at 100 mM. Supernatant obtained from detergent-treated membranes inhibited specific [3H]AMPA and [3H]kainic acid binding, suggesting the presence of an inhibitory agent which was tentatively identified as glutamate. Using centrifugation, saturation analysis revealed two distinct binding sites in both the absence and presence of KSCN. The chaotrope was most effective in increasing binding at the low affinity binding site, enhancing the affinity (K
d) without a concommitant change in the total number of binding sites. Using filtration, a single binding site was detected in Triton-treated membranes. Like the data obtained by centrifugation, KSCN enhanced the affinity of the receptor (K
d value=10 nM) without altering the number of binding sites (B
max=1.2 pmol/mg protein). The rank order of potency of various glutamate analogs in the [3H]AMPA binding assay was quisqualate > AMPA >
l-glutamate > kainate >
d-glutamate, consistent with the labeling of a quisqualate-type excitatory amino acid receptor subtype.l-glutamic acid diethylester, and 2-amino-7-phosphonoheptanoic acid (AP7) were inactive. The present technique provides a rapid, reliable assay for the evaluation of quisqualate-type excitatory amino acid agonists and/or antagonists that may be used to discover more potent and selective agents. 相似文献
2.
Manfred Braun Jong Min Kim Rolf D. Schmid 《Applied microbiology and biotechnology》1992,37(5):594-598
Summary The soil isolate Cellulomonas cellulans AM8 produces an extracellular l-amino acid oxidase (L-AAO) with broad substrate specificity. The strain produced up to 0.35 unit (U)/ml of the extracellular L-AAO in a simple medium containing glycerol and yeast extract. The enzyme was easily purified up to 30 U/mg protein using Phenyl-Sepharose fast flow. The purified enzyme migrated as single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of 55 kDa. On native PAGE the molecular mass was approx. 300 000 kDa, which may be due to aggregation. With the exception of glycine, proline, and threonine, all the amino acids normally constituting proteins were oxidized. The V
max values from 0.7 to 35.2 U/mg for aspartic acid and lysine, respectively, and the K
m values from 0.007 to 7.1 mm for cysteine and valine, respectively, were obtained at 25° C and pH 7.0 in oxygen-saturated solutions. The L-AAO had a pH optimum of 6.5–7.5. It was stable for several months at — 30° C and for some days at 35° C. Ferricyanide served as an electron acceptor with a V
max of 50 U/mg and K
m for 0.3 mm with phenylalanine as the substrate.
Correspondence to: R. D. Schmid 相似文献
3.
Characteristics of amino acid uptake in barley 总被引:2,自引:0,他引:2
Plants have the ability to take up organic nitrogen (N) but this has not been thoroughly studied in agricultural plants. A
critical question is whether agricultural plants can acquire amino acids in a soil ecosystem. The aim of this study was to
characterize amino acid uptake capacity in barley (Hordeum vulgare L.) from a mixture of amino acids at concentrations relevant to field conditions. Amino acids in soil solution under barley
were collected in microlysimeters. The recorded amino acid composition, 0–8.2 μM of l-Serine, l-Glutamic acid, Glycine, l-Arginine and l-Alanine, was then used as a template for uptake studies in hydroponically grown barley plants. Amino acid uptake during 2 h
was studied at initial concentrations of 2–25 μM amino acids and recorded as amino acid disappearance from the incubation
solution, analysed with HPLC. The uptake was verified in control experiments using several other techniques. Uptake of all
five amino acids occurred at 2 μM and below. The concentration dependency of the uptake rate could be described by Michaelis–Menten
kinetics. The affinity constant (K
m) was in the range 19.6–33.2 μM. These K
m values are comparable to reported values for soil micro-organisms. 相似文献
4.
Tatsuo Omata Xanja Andriesse Atsushi Hirano 《Molecular & general genetics : MGG》1993,236(2-3):193-202
Summary The nrtA gene, which has been proposed to be involved in nitrate transport of Synechococcus sp. PCC7942 (Anacystis nidulans R2), was mapped at 3.9 kb upstream of the nitrate reductase gene, narB. Three closely linked genes (designated nrtB, nrtC, and nrtD), which encode proteins of 279, 659, and 274 amino acids, respectively, were found between the nrtA and narB genes. NrtB is a hydrophobic protein having structural similarity to the integral membrane components of bacterial transport systems that are dependent on periplasmic substrate-binding proteins. The N-terminal portion of NrtC (amino acid residues 1–254) and NrtD are 58% identical to each other in their amino acid sequences, and resemble the ATP-binding components of binding protein-dependent transport systems. The C-terminal portion of NrtC is 30% identical to NrtA. Mutants constructed by interrupting each of nrtB and nrtC were unable to grow on nitrate, and the nrtD mutant required high concentration of nitrate for growth. The rate of nitrate-dependent O2 evolution (photosynthetic O2 evolution coupled to nitrate reduction) in wild-type cells measured in the presence of l-methionine d,l-sulfoximine and glycolaldehyde showed a dual-phase relationship with nitrate concentration. It followed saturation kinetics up to 10 mM nitrate (the concentration required for half-saturation = 1 M), and the reaction rate then increased above the saturation level of the first phase as the nitrate concentration increased. The high-affinity phase of nitrate-dependent O2 evolution was absent in the nrtD mutant. The results suggest that there are two independent mechanisms of nitrate uptake and that the nrtB-nrtC-nrtD cluster encodes a high-affinity nitrate transport system. 相似文献
5.
Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative
was replaced with amino acid amide (viz l-Leu–NH2) and amino acid (l-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and
were separated on a reversed-phase C18 column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method
was validated for limit of detection, linearity, accuracy, precision, and recovery. 相似文献
6.
The reaction catalyzed by chorismate mutase (EC 5.4.99.5) is a crucial step for biosynthesis of two aromatic amino acids as well as for the synthesis of phenylpropanoid compounds. The regulatory properties of two chorismate-mutase isoenzymes expressed in Nicotiana silvestris Speg. et Comes are consistent with their differential roles in pathway flow routes ending with l-phenylalanine and l-tyrosine on one hand (isoenzyme CM-1), and ending with secondary metabolites on the other hand (isoenzyme CM-2). Isoenzyme CM-1 was very sensitive to allosteric control by all three aromatic amino acids. At pH 6.1, l-tryptophan was a potent allosteric activator (K
a
=1.5 M), while feedback inhibition was effected by l-tyrosine (K
i
=15 M) or by l-phenylalanine (Ki=15 M). At pH 6.1, all three effectors acted competitively, influencing the apparent K
m
for chorismate. All three allosteric effectors protected isoenzyme CM-1 at pH 6.1 from thermal inactivation at 52° C. l-Tryptophan abolished the weak positive cooperativity of substrate binding found with isoenzyme CM-1 only at low pH. At pH 7.2, the allosteric effects of l-tyrosine and l-tryptophan were only modestly different, in striking contrast to results obtained with l-phenylalanine. At pH 7.2 (i) the K
i
for l-phenylalanine was elevated over 30-fold to 500 M, (ii) the kinetics of inhibition became non-competitive, and (iii) l-phenylalanine now failed to protect isoenzyme CM-1 against thermal inactivation. l-Phenylalanine may act at different binding sites depending upon the intracellular pH milieu. In-vitro data indicated that the relative ability of allosteric activation to dominate over allosteric inhibition increases markedly with both pH and temperature. The second isoenzyme, CM-2, was inhibited competitively by caffeic acid (K
i
=0.2 mM). Aromatic amino acids failed to affect CM-2 activity over a broad range of pH and temperature. Inhibition curves obtained in the presence of caffeic acid were sigmoid, yielding an interaction coefficient (from Hill plots) of n=1.8.Abbreviation DAHP synthase
3-deoxy-d-arabino-heptulosonate 7-phosphate synthase 相似文献
7.
The purpose of this study was to identify the seleno-l-methionine (l-SeMet) α,γ-elimination enzyme that catalyzes l-SeMet to generate methylselenol (CH3SeH), a notable intermediate for the metabolism of selenium compounds, in mammalian tissues. The enzyme purified from ICR
mouse liver was separated by one-dimensional gel electrophoresis, and the specific band was subjected to in-gel trypsin digestion
followed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometric analysis. In the peptide mass fingerprinting
search, the mass numbers of 14 peptides produced by tryptic digestion of the enzyme were consistent with the theoretical mass
numbers calculated from the amino acid sequence of murine cystathionine γ-lyase (E.C. 4.4.1.1). The peptide sequence tags
search was also performed to obtain the amino acid sequence data of five tryptic peptides. These peptides were significantly
identical to the partial amino acid sequences of cystathionine γ-lyase. This enzyme was clearly shown to catalyze the α, γ-elimination
reaction of l-cystathionine by the enzymological research. The K
m
value for the catalysis of l-cystathionine was 0.81 mM and V
max was. 0.0013 unit/mg protein. These results suggested that cystathionine γ-lyase catalyzes l-SeMet to generate CH3SeH by its α,γ-elimination reaction. 相似文献
8.
Phenylalanine hydroxylase converts phenylalanine to tyrosine utilizing molecular oxygen and tetrahydropterin as a cofactor, and belongs to the aromatic amino acid hydroxylases family. The catalytic domains of these enzymes are structurally similar. According to recent crystallographic studies, residue Tyr179 in Chromobacterium violaceum phenylalanine hydroxylase is located in the active site and its hydroxyl oxygen is 5.1 Å from the iron, where it has been suggested to play a role in positioning the pterin cofactor. To determine the catalytic role of this residue, the point mutants Y179F and Y179A of phenylalanine hydroxylase were prepared and characterized. Both mutants displayed comparable stability and metal binding to the native enzyme, as determined by their melting temperatures in the presence and absence of iron. The catalytic activity (kcat) of the Y179F and Y179A proteins was lower than wild-type phenylalanine hydroxylase by an order of magnitude, suggesting that the hydroxyl group of Tyr179 plays a role in the rate-determining step in catalysis. The KM values for different tetrahydropterin cofactors and phenylalanine were decreased by a factor of 3–4 in the Y179F mutant. However, the KM values for different pterin cofactors were slightly higher in the Y179A mutant than those measured for the wild-type enzyme, and, more significantly, the KM value for phenylalanine was increased by 10-fold in the Y179A mutant. By the criterion of kcat/KPhe, the Y179F and Y179A mutants display 10% and 1%, respectively, of the activity of wild-type phenylalanine hydroxylase. These results are consistent with Tyr179 having a pronounced role in binding phenylalanine but a secondary effect in the formation of the hydroxylating species. In conjunction with recent crystallographic analyses of a ternary complex of phenylalanine hydroxylase, the reported findings establish that Tyr179 is essential in maintaining the catalytic integrity and phenylalanine binding of the enzyme via indirect interactions with the substrate, phenylalanine. A model that accounts for the role of Tyr179 in binding phenylalanine is proposed.Electronic Supplementary Material Supplementary material is available in the online version of this article at Abbreviations AAAHs aromatic amino acid hydroxylases - BH2 7,8-dihydro-l-biopterin - BH4 (6R)-5,6,7,8-tetrahydro-l-biopterin - CD circular dichroism - cPAH Chromobacterium violaceum phenylalanine hydroxylase - DMPH4 6,7-dimethyl-5,6,7,8-tetrahydropterin - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - ES-MS electrospray ionization mass spectrometry - hPAH human phenylalanine hydroxylase - ICP-AE inductively coupled plasma atomic emission - 6-MPH4 6-methyl-5,6,7,8-tetrahydropterin - PAH phenylalanine hydroxylase - PH4 tetrahydropterin - PKU phenylketonuria - RDS rate-determining step - TH tyrosine hydroxylase - THA 3-(2-thienyl)-l-alanine - TPH tryptophan hydroxylase - wt wild-type 相似文献
9.
Lactate dehydrogenases which convert lactate to pyruvate are found in almost every organism and comprise a group of highly
divergent proteins in amino acid sequence, catalytic properties, and substrate specificity. While the l-lactate dehydrogenases are among the most studied enzymes, very little is known about the structure and function of d-lactate dehydrogenases (d-LDHs) which include two discrete classes of enzymes that are classified based on their ability to transfer electrons and/or
protons to NAD in NAD-dependent lactate dehydrogenases (nLDHs), and FAD in NAD-independent lactate dehydrogenases (iLDHs).
In this study, we used a combination of structural and phylogenomic approaches to reveal the likely evolutionary events in
the history of the recently described FAD binding oxidoreductase/transferase type 4 family that led to the evolution of d-iLDHs (commonly referred as DLD). Our phylogenetic reconstructions reveal that DLD genes from eukaryotes form a paraphyletic group with respect to d-2-hydroxyglutarate dehydrogenase (D2HGDH). All phylogenetic reconstructions recovered two divergent yeast DLD phylogroups. While the first group (DLD1) showed close phylogenetic relationships with the animal and plant DLDs, the second yeast group (DLD2) revealed strong phylogenetic and structural similarities to the plant and animal D2HGDH group. Our data strongly suggest that the functional assignment of the yeast DLD2 group should be carefully revisited. The present study demonstrates that structural phylogenomic approach can be used to
resolve important evolutionary events in functionally diverse superfamilies and to provide reliable functional predictions
to poorly characterized genes. 相似文献
10.
Two family GH10 xylanases with different thermostability, the Cex (optimum temperature 40°C) from Cellulomonas fimi and the XylA (optimum temperature 80°C) from Thermomonospora alba, were used to construct a chimeric xylanase by module shuffling for investigating the structural determinants responsible
for the difference. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase
Chain Reaction (PCR)s. The shuffled construct, designated as CXC-X4,5, was cloned and its nucleotide sequence was confirmed.
The chimera CXC-X4,5 showed activity against 4-O-methyl-d-glucurono-d-xylan–Remazol Brilliant Blue R (RBB-xylan) and over-expressed as His-tag fusion proteins. The homogeneous chimeric protein
CXC-X4,5 showed significantly improved thermal profiles (optimum temperature 65°C) compared to those of one of the parents,
Cex. This was apparently due to the influence of amino acids in the modules M4 and M5 inherited from thermophilic XylA. Measured
K
m and k
cat values for the substrate p-nitrophenyl-β-d-cellobioside (PNP-G2) were closer to those of the other parent, Cex; however the K
m and k
cat values for the substrate p-nitrophenyl-β-d-xylobioside (PNP-X2) were between two parental xylanases. The ability of the chimeric enzyme to produce reducing sugar from
xylan was enhanced in comparison with the parental enzymes. These results indicated that the amino acid residues in the modules
M4 and M5 of XylA play an important role in determining enzyme characteristics such as thermal stability, and xylanases with
improved properties can be prepared by manipulating this segment. 相似文献
11.
The TES ether of the C6-hydroxy derivative of naturally occurring epi-jasmonic acid (epi-JA) was designed as epimerization-free equivalent of epi-JA. The TES ether was synthesized from (1R,4S)-4-hydroxycyclopent-2-enyl acetate in 13 steps. The acid part of the ether was activated with ClCO2Bu
i
and subjected to condensation with l-amino acid at room temperature for 48 h. The TES group in the condensation product was removed in HCO2H (0°C, 30 min) and the resulting hydroxyl group was oxidized with Jones reagent (acetone, 0°C, 30 min) to furnish the amino
acid conjugate of epi-JA. The amino acids examined are l-isoleucine, l-leucine, l-alanine, l-valine, and d-allo-isoleucine, which afforded the conjugates in 48–68% yields with 89–96% diastereomeric purity over the trans isomers.
Similarly, the possible three stereoisomers of epi-JA were condensed with l-isoleucine successfully, producing the corresponding stereoisomers in good yields. 相似文献
12.
The essential amino acid histidine performs critical roles in health and disease. These functions are generally attributed
to the amino acid itself, but could also be mediated by a positive effect on trace element bioavailability. Mechanistic information
regarding the absorption of histidine across the gastrointestinal tract is essential for understanding the interplay between
amino acid and mineral nutrients and the implications of these interactions for nutrition and toxicology. Using intestinal
brush-border membrane vesicles obtained from freshwater rainbow trout, absorption of histidine over the range 0.78–780 μm was found to be saturable, with a maximal transport rate (J
max) of 9.1 ± 0.8 nmol mg protein−1 min−1 and a K
m (histidine concentration required to reach 50% of this level) of 339 ± 68 μm. Histidine uptake was highly specific as 10-fold elevated levels of a variety of amino acids with putative shared transporters
failed to significantly inhibit uptake. Elevated levels of d-histidine, however, impaired uptake of the natural l-isomer. The presence of “luminal” copper (8.3 μm) significantly increased both the J
max and K
m of histidine transport. This suggests that chelated copper–histidine species cross the brush-border epithelium through transport
pathways distinct from those used by histidine alone. 相似文献
13.
Brevundimonas diminuta TPU 5720 produces an amidase acting l-stereoselectively on phenylalaninamide. The enzyme (LaaABd) was purified to electrophoretic homogeneity by ammonium sulfate fractionation and four steps of column chromatography. The
final preparation gave a single band on SDS-PAGE with a molecular weight of ≈53,000. The native molecular weight of the enzyme
was about 288,000 based on gel filtration chromatography, suggesting that the enzyme is active as a homohexamer. It had maximal
activity at 50°C and pH 7.5. LaaABd lost its activity almost completely on dialysis against potassium phosphate buffer (pH 7.0), and the amidase activity was
largely restored by the addition of Co2+ ions. The enzyme was, however, inactivated in the presence of ethylenediaminetetraacetic acid even in the presence of Co2+, suggesting that LaaABd is a Co2+-dependent enzyme. LaaABd had hydrolyzing activity toward a broad range of l-amino acid amides including l-phenylalaninamide, l-glutaminamide, l-leucinamide, l-methioninamide, l-argininamide, and l-2-aminobutyric acid amide. Using information on the N-terminal amino acid sequence of the enzyme, the gene encoding LaaABd was cloned from the chromosomal DNA of the strain and sequenced. Analysis of 4,446 bp of the cloned DNA revealed the presence
of seven open-reading frames (ORFs), one of which (laaA
Bd
) encodes the amidase. LaaABd is composed of 491 amino acid residues (calculated molecular weight 51,127), and the deduced amino acid sequence exhibits
significant similarity to that of ORFs encoding hypothetical cytosol aminopeptidases found in the genomes of Caulobacter crescentus, Bradyrhizobium japonicum, Rhodopseudomonas palustris, Mesorhizobium loti, and Agrobacterium tumefaciens, and leucine aminopeptidases, PepA, from Rickettsia prowazekii, Pseudomonas putida ATCC 12633, and Escherichia coli K-12. The laaA
Bd
gene modified in the nucleotide sequence upstream from its start codon was overexpressed in an E. coli transformant. The activity of the recombinant LaaABd in cell-free extracts of the E. coli transformant was 25.9 units mg−1 with l-phenylalaninamide as substrate, which was 50 times higher than that of B. diminuta TPU 5720. 相似文献
14.
We isolated RNAs by selection–amplification, selecting for affinity to Phe–Sepharose and elution with free l-phenylalanine. Constant sequences did not contain Phe condons or anticodons, to avoid any possible confounding influence
on initially randomized sequences. We examined the eight most frequent Phe-binding RNAs for inclusion of coding triplets.
Binding sites were defined by nucleotide conservation, protection, and interference data. Together these RNAs comprise 70%
of the 105 sequenced RNAs. The K
D for the strongest sites is ≈50 μM free amino acid, with strong stereoselectivity. One site strongly distinguishes free Phe from Trp and Tyr, a specificity
not observed previously. In these eight Phe-binding RNAs, Phe codons are not significantly associated with Phe binding sites.
However, among 21 characterized RNAs binding Phe, Tyr, Arg, and Ile, containing 1342 total nucleotides, codons are 2.7-fold
more frequent within binding sites than in surrounding sequences in the same molecules. If triplets were not specifically related to binding sites, the probability of this distribution would be 4.8 × 10−11. Therefore, triplet concentration within amino acid binding sites taken together is highly likely. In binding sites for Arg,
Tyr, and Ile cognate codons are overrepresented. Thus Arg, Tyr, and Ile may be amino acids whose codons were assigned during
an era of direct RNA–amino acid affinity. In contrast, Phe codons arguably were assigned by another criterion, perhaps during
later code evolution. 相似文献
15.
l-DOPA is an amino acid derivative and most potent drug used against Parkinson’s disease, generally obtained from Mucuna pruriens seeds. In present communication, we have studied the in vitro production of l-DOPA from l-tyrosine by novel bacterium Bacillus sp. JPJ. This bacterium produced 99.4% of l-DOPA from l-tyrosine in buffer (pH 8) containing 1 mg ml−1 cell mass incubated at 40°C for 60 min. The combination of CuSO4 and l-ascorbic acid showed the inducing effect at concentrations of 0.06 and 0.04 mg ml−1, respectively. The activated charcoal 2 mg ml−1 was essential for maximum bioconversion of l-tyrosine to l-DOPA and the crude tyrosinase activity was 2.7 U mg−1 of tyrosinase. Kinetic studies showed significant values of Y
p/s (0.994), Q
s (0.500) and q
s (0.994) after optimization of the process. The production of l-DOPA was confirmed by analytical techniques such as HPTLC, HPLC and GC–MS. This is the first report on rapid and efficient
production of l-DOPA from l-tyrosine by bacterial source which is more effective than the plant, fungal and yeast systems. 相似文献
16.
The effect of l-arginine on transepithelial ion transport was examined in cultured M-1 mouse renal cortical collecting duct (CCD) cells using
continuous short circuit current (I
SC
) measurements in HCO3
−/CO2 buffered solution. Steady state I
SC
averaged 73.8 ± 3.2 μA/cm2 (n= 126) and was reduced by 94 ± 0.6% (n= 16) by the apical addition of 100 μm amiloride. This confirms that the predominant electrogenic ion transport in M-1 cells is Na+ absorption via the epithelial sodium channel (ENaC). Experiments using the cationic amino acid l-lysine (radiolabeled) as a stable arginine analogue show that the combined activity of an apical system y+ and a basal amino acid transport system y+L are responsible for most cationic amino acid transport across M-1 cells. Together they generate net absorptive cationic
amino acid flux. Application of l-arginine (10 mm) either apically or basolaterally induced a transient peak increase in I
SC
averaging 36.6 ± 5.4 μA/cm2 (n= 19) and 32.0 ± 7.2 μA/cm2 (n= 8), respectively. The response was preserved in the absence of bath Cl− (n= 4), but was abolished either in the absence of apical Na+ (n= 4) or by apical addition of 100 μm amiloride (n= 6). l-lysine, which cannot serve as a precursor of NO, caused a response similar to that of l-arginine (n= 4); neither L-NMMA (100 μm; n= 3) nor L-NAME (1 mm; n= 4) (both NO-synthase inhibitors) affected the I
SC
response to l-arginine. The effects of arginine or lysine were replicated by alkalinization that mimicked the transient alkalinization
of the bath solution upon addition of these amino acids. We conclude that in M-1 cells l-arginine stimulates Na+ absorption via a pH-dependent, but NO-independent mechanism. The observed net cationic amino acid absorption will counteract
passive cationic amino acid leak into the CCD in the presence of electrogenic Na+ transport, consistent with reports of stimulated expression of Na+ and cationic amino acid transporters by aldosterone.
Received: 11 September 2000/Revised: 6 December 2000 相似文献
17.
The polyamines stimulated tyrosine hydroxylase in whole homogenates of bovine caudate nuclei approximately 2 fold. TheV
max forl-tyrosine increased by 2.3 fold while theK
m
s forl-tyrosine and for the cofactor (DMPH4) were unchanged.l-Aromatic amino acid decarboxylase from whole rat brain homogenate was stimulated by about 40% in the presence of polyamines. These findings suggest that increased polyamine levels associated with increased cellular synthetic activity can modify the synthesis of neurotransmitters. 相似文献
18.
Oenococcus oeni has numerous amino acid requirements for growth and dipeptides could be important for its nutrition. In this paper the individual or combined effect of dipeptides on growth of O. oeni X2L in synthetic media deficient in one or more amino acids with L-malic acid was investigated. Utilization of dipeptides, glucose, and L-malic acid was also analyzed. Dipeptides were constituted by at least one essential amino acid for growth. Dipeptides containing two essential amino acids, except leucine, had a more favorable effect than free amino acids on the growth rate. Gly-Gly was consumed to a greater extent than Leu-Leu and a rapid exodus of glycine to the extracellular medium accompanied it. The microorganism could use glycine in exchange for other essential amino acids outside the cell, favoring growth. In the presence of Leu-Leu, the increase in glucose consumption rate could be related to the additional energy required for dipeptide uptake. 相似文献
19.
The killer toxin produced by Pichia kluyveri 1002 kills yeast strains of the genera Candida, Saccharomyces and Torulopsis, including several S. cerevisiae killer strains.Binding of a lethal amount of the toxin to cells of S. cerevisiae SCF 1717 occurs rapidly after toxin addition. After treatment with the toxin for 10 min sensitive cells partially recovered when incubated under conditions that favor protein synthesis. Only after a lag time of 50–90 min sensitive cells changed physiologically. Killing of sensitive cells was characterized by leakage of potassium and adenosine 5-triphosphate, decrease of intracellular pH, and inhibition of the active uptake of amino acids. These effects coincided with cell shrinkage and varied with incubation conditions.Uptake of the amino acid leucine in sensitive cells involved two apparently distinct transport systems (Km1=0.04mm; Km2=0.46mm). The toxin showed different effects on these transport systems. 相似文献
20.
Summary The effects of several sulfamoyl benzoic acid derivatives on Na–K–Cl cotransport were investigated in winter flounder intestine. The relative efficacy (IC50 values) and order of potency of these derivatives were benzmetanide, 5×10–8
m> bumetanide 3×10–7
m>piretanide 3×10–6
m>furosemide 7×10–6
m> amino piretanide 1×10–5 3-amino-4-penoxy-5-sulfamoyl benzoic acid. Binding of [3H] bumetanide was studied in microsomal membranes from winter flounder intestine and compared to that in bovine kidney outer medulla. Binding was also studied in brush-border membranes from winter flounder intestine. The estimated values forK
d
and number of binding sites (n) were: bovine kidney,K
d
=1.6×10–7,n=10.5 pmol/mg protein; winter flounder intestine,K
d
1.2×10–7,n=7.3 pmol/mg protein, and brush-border membranes from winter flounder,K
d
=5.3×10–7,n=20.4 pmol/mg protein. The estimatedK
d
for bumetamide binding to winter flounder brush-border membranes derived from association and dissociation kinetics was 6.8×10–7
m. The similarity in magnitudes of IC50 andK
d
for bumetanide suggests that the brush-border cotransporter is ordinarily rate-limiting for transmural salt absorption and that bumetanide specifically binds to the cotransporter. Measurement of bumetanide binding at various concentrations of Na, K and Cl showed that optimal binding required all three ions to be present at about 5mm concentrations. Higher Na and K concentrations did not diminish binding but higher Cl concentrations (up to 100mm Cl) inhibited bumetanide binding by as much as 50%. Still higher Cl concentrations (500 and 900mm) did not further inhibit bumetanide binding. Scatchard analysis of bumetanide binding at 5 and 100mm Cl concentrations showed that bothK
d
andn were lower at the higher Cl concentration (5mm Cl:K
d
=5.29×10–7
m,n=20.4 pmol/mg protein; 100mm Cl:K
d
=2.3×10–7
m,n=8.8 pmol/mg protein). These data suggest two possibilities: that bumetanide and Cl binding are not mutually exclusive (in contrast to pure competitive inhibition) and that they each bind to separate sites or that two distinct bumetanide binding sites exist, only one of which exhibits Cl inhibition of binding. This inhibition would then be consistent with a competitive interaction with Cl. 相似文献