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1.
Solute mobilities in cuticular membranes of six species (Hedera helix, Malus domestica, Populus alba, Pyrus communis, Stephanotis floribunda, Strophantus gratus) were measured using plant hormones, growth regulators and other organic model compounds varying in molar volumes from 99
to 349 mL · mol−1 The dependence of mobilities (k*) on molar volume (V
x
) was exponential and could be described with equations of the type log
k*=log
k*0−′
V
x
. The y-intercepts (log
k*0) represent mobilities of a hypothetical solute of zero molar volume. The parameter β′ is a measure of size selectivity of
cuticular membranes and no differences among the six species were observed. At 25 °C the average β′ was 0.0095 mol · mL−1. Solute mobility decreased by about a factor of 8.9 when molar volume increased by 100 mL · mol−1 and the mobility of a compound with V
x
= 100 mL · mol−1 was about 700-fold higher than the mobility of a compound with V
x
= 400 mL · mol−1. Size selectivity decreased with increasing temperatures and for Strophantusβ′-values of 1.6 × 10−2 to 8.0 × 10-4 mol · mL−1 were obtained for 10 and 30 °C, respectively. The-intercepts (log k*0) differed among plant species by 3 orders of magnitude and since size selectivity was the same for all species, solute mobilities
for solutes having zero molar volumes were the sole cause for differences among species in solute mobilities and permeabilities.
We argue that these differences in k*0 are related to tortuosity of the diffusion path. These results were used to derive an equation which predicts rates of cuticular
penetration on the basis of k*0, the average size selectivity of 9.5 × 10−3 mol · mL−1 and the driving forces of penetration.
Received: 25 November 1997 / Accepted: 9 March 1998 相似文献
2.
The binding and unbinding constants describing interaction of ω-CTx-GVIA with N-type Ca2+ channels were calculated based on the time course of the blocking action of the toxin. The experiments were carried out on
pyramidal neurons freshly dissociated from theCA3 region of the rat hippocampus using a “concentration-clamp” technique and a patch-clamp technique in the whole-cell configuration.
The bindingk
1 and unbindingk
−1 constants were evaluated as 0.32 (μM·sec)−1 and 0.004 sec−1, respectively. The dissociation constantK
D kinetically derived from the ratiok
−1/k
1 was 0.012 μM. These values allow us to interpret the apparent “irreversibility” of the toxin action. 相似文献
3.
Hiroshi Takashima Ayako Araki Keiko Takemoto Naokazu Yoshikawa Keiichi Tsukahara 《Journal of biological inorganic chemistry》2006,11(3):316-324
In order to understand the detailed mechanism of the stereoselective photoinduced electron-transfer (ET) reactions of zinc-substituted
myoglobin (ZnMb) with optically active molecules by flash photolysis, we designed and prepared new optically active agents,
such as N,N′-dimethylcinchoninium diiodide ([MCN]I2) and N,N′-dimethylcinchonidinium diiodide ([MCD]I2). The photoexcited triplet state of ZnMb, 3(ZnMb)*, was successfully quenched by [MCN]2+ and [MCD]2+ ions to form the radical pair of ZnMb cation (ZnMb·+) and reduced [MCN]·+ and [MCD]·+, followed by a thermal back ET reaction to the ground state. The rate constants (k
q) for the ET quenching at 25 °C were obtained as k
q(MCN)=(1.9±0.1)×106 M−1 s−1 and k
q(MCD)=(3.0±0.2)×106 M−1 s−1, respectively. The ratio of k
q(MCD)/k
q(MCN)=1.6 indicates that the [MCD]2+ preferentially quenches 3(ZnMb)*. The second-order rate constants (k
b) for the thermal back ET reaction from [MCN]·+ and [MCD]·+ to ZnMb·+ at 25 °C were k
b(MCN)=(0.79±0.04)×108 M−1 s−1 and k
b(MCD)=(1.0±0.1)×108 M−1 s−1, respectively, and the selectivity was k
q(MCD)/k
q(MCN)=1.3. Both quenching and thermal back ET reactions are controlled by the ET step. In the quenching reaction, the energy
differences of ΔΔH
≠(MCD–MCN) and ΔΔS
≠(MCD–MCN) at 25 °C were obtained as −1.1 and 0 kJ mol−1, respectively. On the other hand, ΔΔH
≠(MCD–MCN)=11±2 kJ mol−1 and TΔΔS
≠(MCD–MCN)=−10±2 kJ mol−1 were given in the thermal back ET reaction. The highest stereoselectivity of 1.7 for [MCD]·+ found at low temperature (10 °C) was due to the ΔΔS
≠ value obtained in the thermal back ET reaction.
Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users. 相似文献
4.
This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast
fractions of wheat-seedling roots. Lectin (40 μg mL−1) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most
stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase.
Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained
by change in the antigenic properties of the mutant lectin. 相似文献
5.
R. Toriano P. Ford V. Rivarola B.K. Tamarappoo A.S. Verkman M. Parisi 《The Journal of membrane biology》1998,161(2):141-149
Transepithelial water permeability was measured in LLC-PK1 cells stably transfected with aquaporins (AQPs): AQP1, AQP2, and
a chimera of AQP1 and AQP2 containing 41 amino acids of the C-terminus of AQP2. Transepithelial water fluxes (Jw) were not
previously reported in cells transfected with aquaporins. Jw were now recorded each minute using a specially developed experimental
device. A significant increase in Posm after forskolin (FK) plus vasopressin (VP) was found in AQP2 transfected cells (39.9
± 8.2 vs. 12.5 ± 3.3 cm · sec−1· 10−3), but not in cells transfected with AQP1 (15.3 ± 3.6 vs. 13.4 ± 3.6 cm · sec−1· 10−3). In the case of the AQP1/2 cells (chimera) the FK plus VP induced Posm was smaller than in AQP2 cells but significantly
higher than in mock cells at rest (18.1 ± 4.8 vs. 6.7 ± 1.0 cm · sec−1· 10−3). The increases in Posm values were not paralleled by increases in 14C-Mannitol permeability. HgCl2 inhibited the hydrosmotic response to FK plus VP in AQP2 transfected epithelia. Results were comparable to those observed,
in parallel experiments, in a native ADH-sensitive water channel containing epithelial barrier (the toad urinary bladder).
Electron microscopy showed confluent LLC-PK1 cells with microvilli at the mucosal border. The presence of spherical or elongated
intracellular vacuoles was observed in AQP2 transfected cells, specially after FK plus VP stimulus and under an osmotic gradient.
These results demonstrate regulated transepithelial water permeability in epithelial cells transfected with AQP2.
Received: 24 June 1997/Revised: 16 September 1997 相似文献
6.
Theodoros Goulas Athanasios Goulas George Tzortzis Glenn R. Gibson 《Applied microbiology and biotechnology》2009,82(6):1079-1088
Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming
a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351
and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas
the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na+ and/or K+ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg++ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence
or absence of cations, with Mg++, Mn++ and Ca++ ions exerting the most positive effect. Determination of the specificity constants (k
cat/K
m) clearly indicated that BbgI (6.11 × 104 s−1 M−1), BbgIII (2.36 × 104 s−1 M−1) and especially BbgIV (4.01 × 105 s−1 M−1) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 104 s−1 M−1) than lactose (1.48 × 103 s−1 M−1). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different
way to the bacterial physiology. 相似文献
7.
The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography.
It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k
cat of 1.65 min−1 and a K
m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol−1). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C30 seems to be essential for enzyme activity. 相似文献
8.
The effects of temperature (20, 24 and 28 °C) and irradiance (15 and 40 μmol photon m−2 s−1) on the nitrate and ammonium uptake rates of the subtropical red alga, Laurencia brongniartii, were investigated to prepare for tank cultivation. Nitrate uptake followed saturation kinetics and was faster at higher
irradiances and temperatures. In contrast, ammonium uptake was linear over the experimental range and was not affected by
an increase in temperature. A parameter, β, was calculated to compare substrate uptake rates of nitrate along the linear portion
of the uptake curve with that of ammonium. For nitrate, β was lower at low irradiance and higher at high irradiance (β = 0.007
± 0.003 and 0.030 ± 0.002 [μmol N L−1 (μmol N gww−1 d−)−1], respectively). However, β was 0.023 ± 0.002 and 0.034 ± 0.002 [μmol N L−1 (μmol N gww−1 d−1)−1] for ammonium, suggesting a preference for ammonium over nitrate. 相似文献
9.
The goal of this research was to measure in vitro the inhibitory constants of the antioxidants ascorbic and uric acid in urine, with lucigenin enhanced chemiluminescence (CL)
in Fenton’s system. Maximum CL emission is registered in urine containing H2O2 (5·10−4 M), Fe2+ (5·10−5 M), EDTA (5·10−5 M), and chemical enhancer lucigenin (10−4 M) at pH 5.5 and 36°C. Ascorbic acid exhibits up to 4-fold stronger antioxidant effect than uric acid. The constants of antioxidant
inhibition in urine were measured at concentrations 10−3 and 10−4 M: for ascorbic acid, 5.92 ± 0.04 and 24.05 ± 1.82 μmol·sec−1; for uric acid, 1.60 ± 0.02 and 21.45 ± 0.97 μmol·sec−1, respectively. Three phases of CL kinetics of urine are well observed: spontaneous CL (0–10 sec), fast flash of CL (10–50
sec), and latent period (50–300 sec). The antioxidant efficiency of ascorbic and uric acids in the final stage of catabolic
processes in the body is discussed.
Published in Russian in Biokhimiya, 2006, Vol. 71, No. 8, pp. 1062–1065. 相似文献
10.
Beal AM 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》2000,170(8):589-599
Intracarotid infusions of noradrenaline (0.15 nmol · kg−1 · min−1) either alone or accompanied by phentolamine (1.5 nmol · kg−1 · min−1) caused similar-sized increases in salivary protein, magnesium and bicarbonate, and decreases in osmolality, sodium, potassium
and chloride whereas intravenous noradrenaline stimulated much smaller responses. Concurrent infusions of the β1-antagonist, CGP20712A, blocked these noradrenaline-induced changes in salivary composition more effectively than equimolar
infusions of the β2-antagonist, ICI118551, thereby confirming the presence of β1-adrenoceptors. Intracarotid infusion of salbutamol at 0.15, 0.3 and 1.5 nmol · kg−1 · min−1 caused increasing but qualitatively similar changes in salivary composition, sodium excepted, to intracarotid noradrenaline
with 0.3 nmol being most similar quantitatively. Intravenous infusion of salbutamol caused larger changes in salivary composition
than equimolar intravenous noradrenaline thereby indicating that the response to salbutamol may, in part, be mediated by reflex
increases in general sympathetic tone triggered by lowered blood pressure. Eliminating this hypotensive effect by concurrent
intravenous and intracarotid infusions of β1-(CGP or atenolol) and β2-(ICI118551) antagonists with intracarotid salbutamol showed that ICI118551 was more potent than the β1-antagonists thereby demonstrating the presence of β2-receptors. It was concluded that the kangaroo mandibular has functional β1- and β2-adrenoceptor subtypes in both endpieces and excurrent ducts and that the duct system has two populations of cells, each expressing
one receptor subtype.
Accepted: 26 July 2000 相似文献
11.
A UDP-glucose: anthocyanin 3′,5′-O-glucosyltransferase (UA3′5′GT) (EC 2.4.1.-) was purified from the petals of Clitoria ternatea L. (Phaseoleae), which accumulate polyacylated anthocyanins named ternatins. In the biosynthesis of ternatins, delphinidin
3-O-(6″-O-malonyl)-β-glucoside (1) is first converted to delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′-O-β-glucoside (2). Then 2 is converted to ternatin C5 (3), which is delphinidin 3-O-(6″-O-malonyl)-β-glucoside-3′,5′-di-O-β-glucoside. UA3′5′GT is responsible for these two steps by transferring two glucosyl groups in a stepwise manner. Its substrate
specificity revealed the regioselectivity to the anthocyanin′s 3′- or 5′-OH groups. Its kinetic properties showed comparable
k
cat values for 1 and 2, suggesting the subequality of these anthocyanins as substrates. However, the apparent K
m value for 1 (3.89 × 10−5 M), which is lower than that for 2 (1.38 × 10−4 M), renders the k
cat/K
m value for 1 smaller, making 1 catalytically more efficient than 2. Although the apparent K
m value for UDP-glucose (6.18 × 10−3 M) with saturated 2 is larger than that for UDP-glucose (1.49 × 10−3 M) with saturated 1, the k
cat values are almost the same, suggesting the UDP-glucose binding inhibition by 2 as a product. UA3′5′GT turns the product 2 into a substrate possibly by reversing the B-ring of 2 along the C2-C1′ single bond axis so that the 5′-OH group of 2 can point toward the catalytic center.
K. Kogawa, N. Kato, K. Kazuma, and N. Noda contributed equally to this work. 相似文献
12.
Chaoning Liang Yanfen Xue Marco Fioroni Francisco Rodríguez-Ropero Cheng Zhou Ulrich Schwaneberg Yanhe Ma 《Applied microbiology and biotechnology》2011,89(2):315-326
A β-1,4-endoglucanase (Cel5A) was cloned from the genomic DNA of saccharolytic thermophilic eubacterium Thermoanaerobacter tengcongensis MB4 and functionally expressed in Escherichia coli. Substrate specificity analysis revealed that Cel5A cleaves specifically the β-1,4-glycosidic linkage in cellulose with high
activity (294 U mg−1; carboxymethyl cellulose sodium (CMC)). On CMC, kinetics of Cel5A was determined (K
m 1.39 ± 0.12 g l−1; k
cat/K
m 1.41 ± 0.13 g−1 s−1). Cel5A displays an activity optimum between 75 and 80 °C. Residues Glu187 and Glu289 were identified as key catalytic amino
acids by sequence alignment. Interestingly, derived from a non-halophilic bacterium, Cel5A exhibits high residual activities
in molar concentration of NaCl (3 M, 49.3%) and KCl (4 M, 48.6%). In 1 M NaCl, 82% of Cel5A activity is retained after 24
h incubation. Molecular Dynamics studies performed at 0 and 3 M NaCl, correlate the Cel5A stability to the formation of R-COO−···Na+ ···−OOC-R salt bridges within the Cel5A tertiary structure, while activity possibly relates to the number of Na+ ions trapped into the negatively charged active site, involving a competition mechanism between substrate and Na+. Additionally, Cel5A is remarkably resistant in ionic liquids 1-butyl-3-methyllimidazolium chloride (1 M, 54.4%) and 1-allyl-3-methylimidazolium
chloride (1 M, 65.1%) which are promising solvents for cellulose degradation and making Cel5A an attractive candidate for
industrial applications. 相似文献
13.
In search of means to reduce the by-catch of juvenile flatfish in the shrimp fishery, vibrations and changes in current velocity
caused by shrimp trawls were investigated in the field and in the laboratory. Buried as well as emerged shrimps (Crangon crangon) exhibit tailflips 5–10 cm before being touched by the rollers of a shrimp gear approaching them at a speed of 0.5 m·sec−1, as was revealed by slow motion video recordings in aquaria under artificial light. Hence, the signal effective in triggering
escape must be attenuated strongly with increasing distance. Sediment vibration, commonly assumed to be an important signal
in triggering escape of shrimps, was found to decrease by a factor 100·m−1. Signals from the rollers of a commercial shrimp gear in operation (towing speed 1 m·sec−1) were directly recorded with an accelerometer. Their frequency ranged from 50 to 500 Hz and reached an acceleration of 40
m·sec−2 on soft bottom or up to 100 m·sec−2 on hard substrate. Accelerometers, which had been buried right at the surface of a tidal sand flat during low tide, produced
only one sharp signal of 100 Hz with an acceleration of 24 m·sec−2, when a shrimp gear swept them on the submerged tidal flats. However, in aquaria short sinusoidal signals (<5 m·sec−2; 20 to 300 Hz) made buried shrimps and flatfish (Pleuronectes platessa, Solea solea, Microstomus kitt) hide rather than flee. The vibrations recorded directly at the rollers and the underlying jolting movements of the rollers
induce corresponding pulses in the water surrounding the rollers in a layer of approximately 10–15 cm. Similar water displacement
of high acceleration was experimentally produced by a spring loaded transparent lucite piston (7 cm in diameter) fitted to
an accelerometer. Accelerating this piston (12–116 m·sec−2, 50–200 Hz range) from 5 cm above towards the shrimp produced escape responses in up to 94% of the tests. Arthropods are
known to perceive medium displacement rather than pressure. Hence, strong and rapidly rising water currents caused by the
rollers rather than sediment vibration are assumed to mainly trigger the escape reaction, which makesCrangon accessible to the gear. 相似文献
14.
15.
One-hundred and five fungal strains, belonging to 46 different species, were screened for exopolysaccharide production. Phytopathogenicity
and, in particular, inability to produce conidia, were physiological characteristics positively associated and correlated
with the fungal ability to produce polysaccharides. Among the 29 positive strains, Botryosphaeria rhodina DABAC-P82 was the most interesting reaching, when grown on optimal nitrogen source and concentration (NaNO3 and 2.0 g l−1, respectively) and culture medium pH (3.7), 17.7 g l−1 of exopolysaccharide production after only 24 h of fermentation; yield and productivity were 0.69 g g−1 and 0.73 g l−1 h−1, respectively. The purified polysaccharide was characterised as a homopolysaccharide of glucose with a molecular weight of
4.875·106 Da. Studies of structural analysis indicated the presence of β-1,3 and β-1,6 linkages; the EPS structure was very similar
to that of scleroglucan.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
16.
Biomass, carbonate standing stock and production of the mediterranean coralCladocora caespitosa (L.) 总被引:1,自引:0,他引:1
Summary The Mediterranean coralCladocora caespitosa often occurs in large beds, i.e. populations of hemispherical clonies with stock densities varying between 1.9 and 4 coloneis
·m−2. Laboratory measurements of volume, skeleton weight, surface and number of corallites per colony, coupled with mean annual
growth rates evaluated through sclerochronology, allowed for the estimation of biomass, skeleton bulk density, calcimass (carbonate
standing stock) and secondary production (both organic and inorganic) of twoC. caespitosa beds at 4 and 9 m depth. The mean colony biomass varied between 0.73 and 0.99 kg dw ·m−2, corresponding to a calcimass between 2 and 5 kg CaCO3·m−2. Organic secondary production was 215.5–305.4 g dw of polyps ·m−2·y−1, while the potential (mineral) production was 1.1–1.7 kg CaCO3·m−2·y−1, for the year 1996–1997.
These values show thatC. caespitosa is one of the major carbonate producers within the Mediterranean and one of the major epibenthic species originating stable
carbonate frameworks both in recent and past times. 相似文献
17.
Kiyoshi Ohkawa Takashi Hatano Naoko Takizawa Kazue Shinmoto Kyosuke Yamada Makoto Matsuda Koji Takada Yutaka Tsukada 《In vitro cellular & developmental biology. Animal》1992,28(6):449-454
Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously
in a chemically defined medium supplemented with Na2SeO3 (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic
antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal
growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity
of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based
on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum
(FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h−1 · mg−1 protein) than cultured with FBS-containing media (18.2±1.2 pmol · h−1 · mg−1 protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media. 相似文献
18.
An extracellular β-agarase (AgaA34) was purified from a newly isolated marine bacterium, Agarivorans albus YKW-34 from the gut of a turban shell. AgaA34 was purified to homogeneity by ion exchange and gel filtration chromatographies
with a recovery of 30% and a fold of ten. AgaA34 was composed of a single polypeptide chain with the molecular mass of 50 kDa.
N-terminal amino acid sequencing revealed a sequence of ASLVTSFEEA, which exhibited a high similarity (90%) with those of
agarases from glycoside hydrolase family 50. The pH and temperature optima of AgaA34 were pH 8.0 and 40°C, respectively. It
was stable over pH 6.0–11.0 and at temperature up to 50°C. Hydrolysis of agarose by AgaA34 produced neoagarobiose (75 mol%)
and neoagarotetraose (25 mol%), whose structures were identified by matrix-assisted laser desorption ionization time-of-flight
mass spectroscopy and 13C NMR. AgaA34 cleaved both neoagarohexaose and neoagarotetraose into neoagarobiose. The k
cat/K
m values for hydrolysis agarose and neoagarotetraose were 4.04 × 103 and 8.1 × 102 s−1 M−1, respectively. AgaA34 was resistant to denaturing reagents (sodium dodecyl sulfate and urea). Metal ions were not required
for its activity, while reducing reagents (β-Me and dithiothreitol, DTT) increased its activity by 30%. 相似文献
19.
The importance of various parameters such as sugarcane juice concentration, pH of the medium, and effects of different solid
supports for maximum secretion of pectin lyase from Penicillium citrinum MTCC 8897 has been studied. The enzyme was purified to homogeneity by Sephadex G-100 and DEAE-cellulose chromatography. The
molecular mass determined by SDS-PAGE was 31 kDa. The K
m and k
cat values were found to be 1 mg/ml and 76 sec−1, respectively. The optimum pH of the purified pectin lyase was 9.0, though it retains activity in the pH 9.0–12.0 range when
exposed for 24 h. The optimum temperature was 50°C, and the pectin lyase was found to be completely stable up to 40°C when
exposed for 1 h. The purified pectin lyase was found efficient in retting of Linum usitatissimum, Cannabis sativa, and Crotalaria juncea.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 985–992. 相似文献
20.
This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO2Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve
continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations
as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics.
When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A4 fromGriffonia simplicifolia is a simple bimolecular association with parametersk
+
= 9.4 × 104 M-1 s-1 andk
-1 = 5.3 s-1 at 23°C, but binding to the GSAI-B4 lectin is biphasic.
The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk
-1 = 0.24 s-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is 100 times smaller than for other mono-and disaccharides
investigated. The binding mechanism is simple and the derivatisation of Galβ1 → 3GalNAc does not affect the affinity to a considerable degree.
The binding preference of tetravalentsoybean agglutinin for MeαGalNAc over MeαGal by a factor of 25 is mainly of enthalpic origin with an additional 7 kJ mol-1; the NAc group causes perturbation of a tryptophanyl residue, evidenced by protein difference absorption spectrometry. In
the glycosides, a large aglycon likeβpNO2 Phe orβMeUmb hardly affects the affinity of SBA but a largeN-dansyl group increases the affinity by a factor 20 as compared to GalNAc. The 10-fold increase in carbohydrate-specificN-dansylgalactosamine fluorescence, together with a very favourable entropic contribution point at the presence of a hydrophobic
region in the vicinity of the carbohydrate-binding site. The dissociation-rate parameter of the MeUmbβGalNAc SBA complex is slower than for any reported monosaccharide-lectin complex: 0.4 s-1.
The divalent lectin fromErythrina cristagalli preferentially binds the Galβ1 → 4GlcNAc structure that occurs in manyN-glycoproteins. The combining site was mapped thermodynamically with carbohydrates ranging from mono-to pentasaccharides as
derived fromN-glycoproteins. Here, N-dansylgalactosamine was used as a fluorescent indicator ligand in substitution titrations. When Galβ1 → 4GlcNAc was linkedα1 → 2 orα1 → 6 to Man, the binding enthalpy and entropy remained practically constant. Application of stopped flow kinetics and pressure-jump
relaxation withN-dansylgalactosamine gave mono-exponential signal changes with a concentration dependence corresponding tok
+ = 4.8 x 104 M-1 s-1
k
- = 0.4 to 0.66 s-1 and a change in reaction volume of+7ml/mol. 相似文献