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1.
Lars-Åke Fransson Birgitta Havsmark Katsukiyo Sakurai Sakaru Suzuki 《Glycoconjugate journal》1992,9(1):45-55
To generate xyloside-primed dermatan sulfate suitable for sequence analysis, skin fibroblasts were incubated withp-hydroxyphenyl--d-xylopyranoside and [3H]galactose, and free [3H]glycosaminoglycan chains were isolated from the culture medium by ion exchange and gel chromatography. After125I labelling of their reducing-terminal hydroxyphenyl groups, chains were subjected to various chemical and enzymatic degradations, both partial and complete, followed by gradient polyacrylamide gel electrophoresis and autoradiographic identification of fragments extending from the labelled reducing-end to the point of cleavage. Results of periodate oxidation-alkaline scission indicated that the xylose moiety remained unsubstituted at C-2/C-3; exhaustive treatment with chondroitin AC-I lyase afforded the fragment HexA-Gal-Gal-Xyl-R (R = radio-iodinated hydroxyphenyl group), and complete degradations with chondroitin ABC lyase as well as testicular hyaluronidase yielded the fragments HexA/HexA-GalNAc-GlcA-Gal-Gal-Xyl-R with or without sulfate on theN-acetylgalactosamine. Partial digestions with testicular hyaluronidase or chondroitin B lyase indicated that glucuronic acid was common in the first three repeats after the linkage region and that iduronic acid could occupy any position thereafter. Hence, there were no indications of a repeated, periodic appearance of the clustered GlcA-GalNAc repeats which was previously observed in proteoglycan derived dermatan sulfate [Fransson L-Å, Havsmark B, Silverberg I (1990)Biochem J
269:381–8], suggesting a role for the protein part in controlling the formation of particular copolymeric features during glycosaminoglycan assembly.Abbreviations GAG
glycosaminoglycans
- CS
chondroitin sulfate
- DS
dermatan sulfate
- Ser
serine
- Xyl
d-xylose
- Gal
d-galactose
- GlcA
d-glucuronic acid
- IdoA
l-iduronic acid
- GalNAc
N-acetyl-d-galactosamine
- GlcNAc
N-acetyl-d-glucosamine
- HexA
4-deoxy-l-threo-hex-4-enopyranosyluronic acid
- HO-Phe
p-hydroxyphenyl group
- HO-Phe-Xyl
p-hydroxyphenyl-O--d-xylopyranoside
- O2N-Phe-Xyl
p-nitrophenyl--d-xylopyranoside
- OSO3
ester sulfate
- PAGE
polyacrylamide gel electrophoresis
- HPLC
high performance liquid chromatography
- FPLC
fast performance liquid chromatography
- LC
standard liquid chromatography 相似文献
2.
D. A. Theocharis N. Papageorgacopoulou D. H. Vynios S. Th. Anagnostides C. P. Tsiganos 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,754(2):297-309
Chondroitin sulfate and dermatan sulfate are galactosaminoglycans that have similar size and charge density thus making difficult their separation and accurate determination from tissue preparations. A procedure was developed, which was based on the specific action of chondroitinase B, that allowed the determination of dermatan sulfate content in a mixture of chondroitin sulfate/dermatan sulfate, its molecular mass (Mr), and iduronic acid content and distribution throughout the chain. According to this procedure, the galactosaminoglycan sample was treated with chondroitinase B and its profile, upon gel chromatography on Sepharose CL-6B, was compared to that of the initial sample. The differences in uronic acid content of the fractions of the gel chromatographies were plotted and a secondary profile was constructed, which corresponded to the elution profile of intact dermatan sulfate in the sample. From this profile, the size distribution of dermatan sulfate was obtained and its Mr was calculated. In addition, the accurate content of dermatan sulfate in the sample was determined. The digest contained oligosaccharides of variable size that were separated on BioGel P-10. From the separated oligosaccharides the distribution of iduronic acid throughout the dermatan sulfate chains was determined. The procedure was applied to the determination and partial characterisation of dermatan sulfate from sheep nasal cartilage, in which it is reported for the first time that it contains a significant proportion of dermatan sulfate chains of low iduronic acid content. 相似文献
3.
Synthesis of glycosaminoglycans by human embryonic lung fibroblasts. Different distribution of heparan sulphate, chondroitin sulphate and dermatan sulphate in various fractions of the cell culture
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Foetal human lung fibroblasts, grown in monolayer, were allowed to incorporate 35SO42− for various periods of time. 35S-labelled macromolecular anionic products were isolated from the medium, a trypsin digest of the cells in monolayer and the cell residue. The various radioactive polysaccharides were identified as heparan sulphate and a galactosaminoglycan population (chondroitin sulphate and dermatan sulphate) by ion-exchange chromatography and by differential degradations with HNO2 and chondroitinase ABC. Most of the heparan sulphate was found in the trypsin digest, whereas the galactosaminoglycan components were largely confined to the medium. Electrophoretic studies on the various 35S-labelled galactosaminoglycans suggested the presence of a separate chondroitin sulphate component (i.e. a glucuronic acid-rich galactosaminoglycan). The 35S-labelled galactosaminoglycans were subjected to periodate oxidation of l-iduronic acid residues followed by scission in alkali. A periodate-resistant polymer fraction was obtained, which could be degraded to disaccharides by chondroitinase AC. However, most of the 35S-labelled galactosaminoglycans were extensively degraded by periodate oxidation–alkaline elimination. The oligosaccharides obtained were essentially resistant to chondroitinase AC, indicating that the iduronic acid-rich galactosaminoglycans (i.e. dermatan sulphate) were composed largely of repeating units containing sulphated or non-sulphated l-iduronic acid residues. The l-iduronic acid residues present in dermatan sulphate derived from the medium and the trypsin digest contained twice as much ester sulphate as did material associated with the cells. The content of d-glucuronic acid was low and similar in all three fractions. The relative distribution of glycosaminoglycans among the various fractions obtained from cultured lung fibroblasts was distinctly different from that of skin fibroblasts [Malmström, Carlstedt, Åberg & Fransson (1975) Biochem. J. 151, 477–489]. Moreover, subtle differences in co-polymeric structure of dermatan sulphate isolated from the two cell types could be detected. 相似文献
4.
From cultures of human umbilical vein endothelial cells incubated with3H-glucosamine or35S-sulphate, we have purified three heparan sulphate proteoglycans: 1) a low density (1.31 g/ml) proteoglycan from the cell extract, 2) a low density proteoglycan from the medium, and 3) a high density (>1.4 g/ml) proteoglycan from the medium. The disaccharide composition of heparan sulphate chains from the low density proteoglycan of the medium was examined, using specific chemical and enzymic degradations followed by gel chromatography and strong anion exchange HPLC. Chains released from each of the different proteoglycan populations were then compared by gel chromatography and gradient polyacrylamide gel electrophoresis before and after various specific degradations. The results indicate that heparan sulphate from human endothelial cells are large polymers (MW>50,000) of low overall sulphation (32–35%N-sulphated glucosamine and an N/O-linked sulphate ratio of 2.0) with rare and solitary heparin-like disaccharides. Heparan sulphate from the different proteoglycan populations appeared to have similar structure except that chains from the high density fraction were larger polymers.Abbreviations HSPG
heparan sulphate proteoglycan
- DSPG
dermatan sulphate proteoglycan
- GlcNAc(6S)
N-acetylglucosamine 6-sulphate
- GlcNAc6R
glucosamine with either-OH or-OSO3 at C-6
- GlcNR
glucosamine with either-SO3 or-COCH3 as N-substituent
- GlcNSO3
N-sulphated glucosamine
- GlcNSO3(3S)
N-sulphated glucosamine 3-sulphate
- GlcA
d-glucuronic acid
- IdoA
l-iduronic acid
- IdoA(2S)
iduronic acid 2-sulphate
- HexA
hexuronic acid
- DHexA
hexuronic acid with a 4,5-double bond
- Xyl
xylose
- SAX
strong anion exchange
- d.p.
degree of polymerization (a disaccharide has d.p.=1 etc)
- AUFS
absorbance units full scale
The codes used for proteoglycans denote in turn: C 2, low-density (1.35–1.28 g/ml) HSPG from the cell extract; M 1a, high density (>1.4 g/ml) HSPG fraction from the spent medium; M 2a, low-density (1.31 g/ml) HSPG from the spent medium [6]. 相似文献
5.
Purification and properties of thermostable N-acylamino acid racemase from Amycolatopsis sp. TS-1-60
Thermostable N-acylamino acid recemase from Amycolatopsis sp. TS-1-60, a rare actinomycete strain selected for its ability to grow on agar plates incubated at 40° C, was purified to homogeneity and characterized. The relative molecular mass (M
r) of the native enzyme and the subunit was estimated to be 300 000 and 40 000 on gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis respectively. The isoelectric point (pI) of the enzyme was 4.2. The optimum temperature and pH were 50° C and 7.5 respectively. The enzyme was stable at 55° C for 30 min. The enzyme catalyzed the racemization of optically active N-acylamino acids such as N-acetyl-l-or d-methionine, N-acetyl-l-valine, N-acetyl-l-tyrosine and N-chloroacetyl-l-valine. In addition, the enzyme also catalyzed the recemization of the dipeptide l-alanyl-l-methionine. By contrast, the optically active amino acids, N-alkyl-amino acids and methyl and athyl ester derivatives of N-acetyl-d- and l-methionine were not racemized. The apparent K
m values for N-acetyl-l-methionine and N-acetyl-d-methionine were calculated to be 18.5 mM and 11.3 mM respectively. The enzyme activity was markedly enhanced by the addition of divalent metal ions such as Co2+, Mn2+ and Fe2+ and was inhibited by addition of EDTA and P-chloromercuribenzoic acid. The similarity between the NH2-terminal amino acid sequence of the enzyme and that of Streptomyces atratus Y-53 [Tokuyama et al. (1994) Appl Microbiol Biotechnol 40:835–840] was above 80%. 相似文献
6.
Gregory S. Harper Tina Rozaklis Julie Bielicki John J. Hopwood 《Glycoconjugate journal》1993,10(5):407-415
Studies using lysosomal membrane vesicles have suggested that efflux of the sulfate that results from lysosomal glycosaminoglycan degradation is carrier-mediated. In this study, glycosaminoglycan degradation and sulfate efflux were examined using cultured skin fibroblasts and lysosomes deficient in the lysosomal enzymeN-acetylgalactosamine-4-sulfatase. Such fibroblasts store dermatan sulfate lysosomally, which could be labelled biosynthetically with Na
2
35
SO4. The addition of recombinantN-acetylgalactosamine-4-sulfatase to the media of35S labelled fibroblasts degraded up to 82% of the stored dermatan [35S] sulfate over a subsequent 96 h chase and released inorganic [35S] sulfate into the medium. In the presence of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS), sulfate was reused to a minor extent in newly synthesized proteoglycan. Isolated granules from recombinant enzyme supplemented fibroblasts degraded stored dermatan [35S]sulfate to sulfate which was rapidly released into the medium at a rate that was reduced by the extra-lysosomal presence of the lysosomal sulfate transport inhibitors SITS, Na2SO4 and Na2MoO4. SITS also inhibited dermatan sulfate turnover, although it had no effect on the action of purified recombinant enzymein vitro. These data imply that sulfate clearance occurred concomitantly with dermatan sulfate turnover in the lysosome even at high substrate loading, and that lysosome-derived sulfate, while available, is reutilized minimally in synthetic pathways.Abbreviations SITS
4-acetamido-4-isothiocyanatostilbene-2,-2-disulfonic acid
- GAG
glycosaminoglycan
- 4S
N-acetylgalactosamine-4-sulfatase
- r4S
recombinant humanN-acetylgalactosamine-4-sulfatase
- PBS
phosphate buffered saline
- BME
basal modified Eagle's medium
- FBS
fetal bovine serum
- GalNAc4S-GlcA-GalitolNAc4S
-(N-acetyl-d-galactosamine-4-sulfate)-(1–4)--d-glucuronic acid)-(1–3)-N-acetyl-d-[1-3H]galactosaminitol-4-sulfate
- DS
dermatan sulfate
- MPS
mucopolysaccharidosis 相似文献
7.
The relative proportion of L-iduronic acid (IdoA) and D-glucuronic acid (GlcA) is of great importance for the structure–function relationship of chondroitin sulfate (CS)/dermatan
sulfate (DS). However, determination of the isotypes of uronic acid residues in CS/DS is still a challenge, due to the instability
of free uronic acid released by chemical degradation and its conversion to unsaturated uronic acid by digestion with bacterial
eliminase. 1H-Nuclear magnetic resonance (NMR) spectroscopy is a promising tool with which to address this issue, but the traditional
method based on the assignment of the ring proton signals of IdoA and GlcA residues still has drawbacks such as the serious
overlap of signals in the 1H-NMR spectrum of CS/DS polysaccharides. We found that the proton signals of the N-acetyl group of N-acetyl-D-galactosamines in CS and DS could be clearly distinguished and accurately integrated in the one-dimensional (1D) 1H-NMR spectrum. Based on this finding, here we report a novel, sensitive, and nondestructive 1D 1H-NMR-based method to determine the proportion of IdoA and GlcA residues in CS/DS hybrid chains.
The contributions of Fuchuan Li and Shuhei Yamada should be considered equal. 相似文献
8.
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
9.
Carvalho de Souza A Ganchev DN Snel MM van der Eerden JP Vliegenthart JF Kamerling JP 《Glycoconjugate journal》2009,26(4):457-465
Cell aggregation in the marine sponge Microciona prolifera is mediated by a multimillion molecular-mass aggregation factor, termed MAF. Earlier investigations revealed that the cell
aggregation activity of MAF depends on two functional domains: (i) a Ca2+-independent cell-binding domain and (ii) a Ca2+-dependent proteoglycan self-interaction domain. Structural analysis of involved carbohydrate fragments of the proteoglycan
in the self-association established a sulfated disaccharide β-d-GlcpNAc3S-(1→3)-α-l-Fucp and a pyruvated trisaccharide β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp. Recent UV, SPR, and TEM studies, using BSA conjugates and gold nanoparticles of the synthetic sulfated disaccharide, clearly
demonstrated self-recognition on the disaccharide level in the presence of Ca2+-ions. To determine binding forces of the carbohydrate–carbohydrate interactions for both synthetic MAF oligosaccharides,
atomic force microscopy (AFM) studies were carried out. It turned out that, in the presence of Ca2+-ions, the force required to separate the tip and sample coated with a self-assembling monolayer of thiol-spacer-containing
β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH2)3S(CH2)6S- was found to be quantized in integer multiples of 30 ± 6 pN. No binding was observed between the two monolayers in the
absence of Ca2+-ions. Cd2+-ions could partially induce the self-interaction. In contrast, similar AFM experiments with thiol-spacer-containing β-d-Galp4,6(R)Pyr-(1→4)-β-d-GlcpNAc-(1→3)-α-l-Fucp-(1→O)(CH2)3S(CH2)6S- did not show a binding in the presence of Ca2+-ions. Also TEM experiments of gold nanoparticles coated with the pyruvated trisaccharide could not make visible aggregation
in the presence of Ca2+-ions. It is suggested that the self-interaction between the sulfated disaccharide fragments is stronger than that between
the pyruvated trisaccharide. 相似文献
10.
Whole-genome sequence analysis of Bacillus halodurans ATCC BAA-125 revealed an isomerase gene (rhaA) encoding an l-rhamnose isomerase (l-RhI). The identified l
-RhI gene was cloned from B. halodurans and over-expressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,257 bp capable of encoding a polypeptide of 418 amino acid residues
with a molecular mass of 48,178 Da. The molecular mass of the purified enzyme was estimated to be ∼48 kDa by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and 121 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer.
The enzyme had an optimal pH and temperature of 7 and 70°C, respectively, with a k
cat of 8,971 min−1 and a k
cat/K
m of 17 min−1 mM−1 for l-rhamnose. Although l-RhIs have been characterized from several other sources, B. halodurans
l-RhI is distinguished from other l-RhIs by its high temperature optimum (70°C) with high thermal stability of showing 100% activity for 10 h at 60°C. The half-life
of the enzyme was more than 900 min and ∼25 min at 60°C and 70°C, respectively, making B. halodurans
l-RhI a good choice for industrial applications. This work describes one of the most thermostable l-RhI characterized thus far. 相似文献
11.
K. Mochizuki 《Archives of microbiology》1999,172(3):182-185
An enzyme cleaving l-2-oxothiazolidine-4-carboxylic acid to l-cysteine was purified 75-fold with 8% recovery to near homogeneity from crude extracts of Paecilomyces varioti F-1, which had been isolated as a fungus able to assimilate l-2-oxothiazolidine-4-carboxylic acid. The molecular mass was estimated to be 260 kDa by gel filtration. The purified preparation
migrated as a single band of molecular mass 140 kDa upon SDS-PAGE. The maximum activity was observed at a range of pH 7.0–8.0
and at 50 °C. The enzyme activity was completely inhibited by SH-blocking reagents such as AgNO3, p-chloromercuribenzoic acid, N-ethylmaleimide, and N-bromosuccinimide. The enzyme required ATP, Mg2+, and KCl for the cleavage of l-2-oxothiazolidine-4-carboxylic acid. The enzyme also cleaved 5-oxo-l-proline to l-glutamic acid and is considered to be 5-oxo-l-prolinase.
Received: 23 March 1999 / Accepted: 22 June 1999 相似文献
12.
l-2-Amino-Δ2-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu2+ and Mn2+ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K
m (0.35 mM) and V
max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine. 相似文献
13.
When Pseudomonas mendocina NK-01 was cultivated in a 200-L fermentor using glucose as carbon source, 0.316 g L−1 medium-chain-length polyhydroxyalkanoate (PHAMCL) and 0.57 g L−1 alginate oligosaccharides (AO) were obtained at the end of the process. GC/MS was used to characterize the PHAMCL, which was found to be a polymer mainly consisting of 3HO (3-hydroxyoctanoate) and 3HD (3-hydroxydecanoate). T
m and T
g values for the PHAMCL were 51.03°C and −41.21°C, respectively, by DSC. Its decomposition temperature was about 300°C. The elongation at break was
700% under 12 MPa stress. MS and GPC were also carried out to characterize the AO which had weight-average molecular weights
of 1,546 and 1,029 Da, respectively, for the two main components at the end of the fermentation process. MS analysis revealed
that the AO were consisted of β-d-mannuronic acid and/or α-l-guluronic acid, and the β-d-mannuronic acid and/or α-l-guluronic acid residues were partially acetylated at position C2 or C3. 相似文献
14.
During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l–1 to 0.6 g l–1 KH2PO4 reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l–1 to 71 g l–1 and from 1.36 g l–1 to 0.18 g l–1, respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l–1 KH2PO4 was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production. 相似文献
15.
Marta E. Farías Ana M. Strasser de Saad Aída A. Pesce de Ruiz Holgado Dr. Guillermo Oliver 《Current microbiology》1991,22(4):205-211
l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM
l-cysteine in 50 mM phosphate buffer. Mr=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (Mr=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent Km forl-serine was 65 mM. Fe++ was required for the enzymatic activity, and the apparent Km value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit. 相似文献
16.
J. Custot Jean-Luc Boucher Sandrine Vadon Catherine Guedes Sylvie Dijols Marcel Delaforge Daniel Mansuy 《Journal of biological inorganic chemistry》1996,1(1):73-82
The effects of various compounds bearing an N-OH group such as N-hydroxy-guanidines, amidoximes, and hydroxylamines, on bovine and rat liver arginases was studied. Some of these compounds
with an l-α-amino acid function at an appropriate distance from the N-OH group acted as strong competitive liver arginase inhibitors,
displaying Ki values between 4 and 150 μM. Two compounds, N
ε-hydroxy-l-lysine and N
ω-hydroxy-d,l-indospicine, which exhibited Ki values of 4 and 20 μM (at pH 7.4), were the most potent inhibitors of arginase described
to date. The distance between the α-amino acid and N-OH functions appeared to be crucial for potent inhibition of arginase,
as N
δ-hydroxy-l-ornithine, which has one -CH2 group less than N
ε-hydroxy-l-lysine, exhibited a 37-fold higher Ki value than N
ε-hydroxy-l-lysine. Based on these results, a model for the interaction of N
ω-hydroxyamino-l-α-amino acids with the arginase active site is proposed. This model involves the binding of the N-OH group of the inhibitors
to the arginase Mn(II) center and suggests that N
ε-hydroxy-l-lysine is a good transition state analog of arginase. 相似文献
17.
d-Gluconate dehydratase fromAchromobacter, grown ond-gluconate, was purified 100-fold by a procedure involving ammonium sulfate fractionation and preparative acrylamide gel electrophoresis. The purified enzyme appeared to be homogeneous by disc gel electrophoresis. It is an inducible enzyme with an optimal activity in the pH region 8.4–8.8, a Km value of 2.08 × 10–2 m ford-gluconate and a molecular weight of 270,000 ± 25,000. Only C5 and C6 aldonic acids possessing al-threo configuration at C2 and C3 are dehydrated. The dehydration products ofd-gluconate,d-xylonate,d-galactonate,d-fuconate andl-arabonate were identified as 2-keto-3-deoxy compounds by specific colour reactions and thin layer chromatography. Onemm Mg+ + is a powerful activator, 0.1 mm Mn+ + activates poorly and EDTA inhibits. Glutatione, dithiothreitol and mercaptoethanol had no effect, althoughp-chloromercuribenzoate (0.01 mm) decreased enzyme activity.We wish to thank Mr D. Dewettinck for skilful technical assistance. The senior author (J.D.L.) is indebted to the Fonds voor Kollektief en Fundamenteel Onderzoek (Belgium) for research and personnel grants. J.K.-M. is indebted to the Belgian government for a travel and study grant. 相似文献
18.
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
19.
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
20.
Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K
m
= 3 m and low capacity (Vmax) 0.4 mol l-Glu · Kg–1 dry cell mass · 10 min–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X
AG
–
family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K
m 39 m but high capacity (V
max) 1.8 mol l-Glu · Kg–1 dry cell mass · 10 min–1. 相似文献