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1.
Shveta Chaudhary J. David Van Horn 《Mutation Research - Genetic Toxicology and Environmental Mutagenesis》2006,610(1-2):56
Kinetics for the breakdown of the trinuclear chromium acetate cluster with a series of monoprotic and diprotic amino acid ligands and with glutathione in aqueous media have been investigated spectrophotometrically at pH 3.5–5.5 and in a temperature range of 45–60 °C. Under pseudo-first-order conditions, reactions with these ligands exhibited biphasic kinetic behavior that can be accounted for by a consecutive two-step reaction, A → B → C, where A is assumed to be a forced ion pair, B an intermediate and C is the product; experimental data fit to a biexponential equation for the transformation. Rates for kshort, klong, and kobs were determined by manual extrapolation of absorbance data or curve-fitting routines; associated activation parameters for each step of the reaction were calculated using the Eyring equation. Rates for the first and second steps of the reaction are on the order of 10−4 and 10−5 s−1, respectively. The large negative values of ΔS‡ and smaller ΔH‡ in the first step indicate an associative step, while high positive values of ΔS‡ in the second step indicate dissociation. To account for the results mechanistically, the results are interpreted to be a first step of ligand exchange with a pseudo-axial aqua ligand, followed by a dissociative step involving acetate or oxo ligand displacement. The dissociative step is the rate determining step, with kobs ≈ klong.The results demonstrate reaction pathways that are available to the Cr(III) metal centers that may be physiologically relevant in the ligand-rich environment of biological systems. Under general conditions Cr(III) clusters may be expected to be broken down, unless some unique biological environment stabilizes the cluster. The present study has application to the processes related to Cr(III) transport and excretion, to potential mechanisms of Cr(III) action in a biological setting, and to the pharmacokinetics of Cr(III) supplements for animal and human consumption. 相似文献
2.
In this work, we derive an analytical expression for the relaxation time τ as a function of temperature T for myoglobin protein (Mb, PDB:1MBN) in the high temperature limit (T > Tg = 200 K). The method is based on a modified version of the Adam–Gibbs theory (AG theory) for the glass transition in supercooled liquids and an implementation of differential geometry techniques. This modified version of the AG theory takes into account that the entropic component in protein's denaturation has two major sources: a configurational contribution ΔSc due to the unfolding of the highly ordered native state N and a hydration contribution ΔShyd arising from the exposure of non-polar residues to direct contact with solvent polar molecules. Our results show that the configurational contribution ΔSc is temperature-independent and one order of magnitude smaller than its hydration counterpart ΔShyd in the temperature range considered. The profile obtained for log τ(T) from T = 200 K to T = 300 K exhibits a non-Arrhenius behavior characteristic of α relaxation mechanisms in hydrated proteins and glassy systems. This result is in agreement with recent dielectric spectroscopy data obtained for hydrated myoglobin, where at least two fast relaxation processes in the high temperature limit have been observed. The connection between the relaxation process calculated here and the experimental results is outlined. 相似文献
3.
John W. Huffman John Liddle Shu Yu Mie Mie Aung Mary E. Abood Jenny L. Wiley Billy R. Martin 《Bioorganic & medicinal chemistry》1999,7(12):964
The synthesis and pharmacology of 15 1-deoxy-Δ8-THC analogues, several of which have high affinity for the CB2 receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ8-THC (5), 1-deoxy-Δ8-THC (6), 1-deoxy-3-butyl-Δ8-THC (7), 1-deoxy-3-hexyl-Δ8-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ8-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB1 and CB2 receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ8-THC analogues (2, n=1–5) have high affinity (Ki=<20 nM) for the CB2 receptor. Four of them (2, n=1–4) also have little affinity for the CB1 receptor (Ki=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ8-THC (2, n=2) has very high affinity for the CB2 receptor (Ki=3.4±1.0 nM) and little affinity for the CB1 receptor (Ki=677±132 nM).
Scheme 3. (a) (C6H5)3PCH3+ Br−, n-BuLi/THF, 65°C; (b) LiAlH4/THF, 25°C; (c) KBH(sec-Bu)3/THF, −78 to 25°C then H2O2/NaOH. 相似文献
4.
Madoka Takahashi Wakako Handa Hiromi Umeta Saki Ishikawa Kouwa Yamashita Mitsuteru Numazawa 《The Journal of steroid biochemistry and molecular biology》2009,116(3-5):191-199
To gain the structure–activity relationship of Δ1-androstenediones (Δ1-ADs) as mechanism-based inactivator of aromatase, series of 2-alkyl- and 2-alkoxy-substitiuted Δ1-ADs (6 and 9) as well as 2-bromo-Δ1-AD (14) were synthesized and tested. All of the inhibitors examined blocked aromatase in human placental microsomes in a competitive manner. In a series of 2-alkyl-Δ1-ADs (6), n-hexyl compound 6f was the most powerful inhibitor with an apparent Ki value of 31 nM. The inhibitory activities of 2-alkoxy steroids 9 decreased in relation to length of the alkyl chain up to n-hexyloxy group (Ki: 95 nM for methoxy 9a). All of the alkyl steroids 6 along with the alkoxy steroid 9, except for the ethyl and n-propyl compounds 6b and 6c, caused a time-dependent inactivation of aromatase. The inactivation rates (kinact: 0.020–0.084 min−1) were comparable to that of the parent compound Δ1-AD. The inactivation was prevented by the substrate AD, and no significant effect of l-cysteine on the inactivation was observed in each case. The results indicate that the 2-hexyl compound 6f act as the most powerful mechanism-based inactivator of aromatase among Δ1-AD analogs and may be submitted to the preclinical study in estrogen-dependent breast cancer. 相似文献
5.
Yoshiyuki Tanaka Toshihiro Miyajima Katsumasa Yamada Masakazu Hori Natsuki Hasegawa Yu Umezawa Isao Koike 《Aquatic Botany》2008,89(3):331-336
The seasonal variability of specific growth rate and the carbon stable isotope ratio (δ13C) of leaf blades (δ13Cleaf) of a temperate seagrass, Zostera marina (within 10 days old) were measured simultaneously, together with the δ13C of dissolved inorganic carbon (δ13CDIC) at three sites in the semi-closed Akkeshi estuary system, northeastern Japan, in June, September, and November 2004. The δ13Cleaf ranged from −16.2 to −6.3‰ and decreased from summer to winter. The simultaneous measurement of the δ13Cleaf, growth rate, and morphological parameters (mean leaf length and width, mean number of leaves per shoot, and sheath length) of the seagrass and δ13CDIC in the surrounding water allowed us to compare directly the δ13Cleaf and specific growth rate of seagrass. The difference in the δ13C of seagrass leaves relative to the source DIC (Δδ13Cleaf − DIC) was the least negative (−11 to −7‰) in June at all three sites and became more negative (−17 to −8‰) as the specific growth rate decreased. This positive correlation between Δδ13Cleaf − DIC and specific growth rate can be used to diagnose the growth of seagrasses. Δδ13Cleaf − DIC changed by −1.7 ± 0.2‰ when the leaf specific growth rate decreased by 1% d−1. 相似文献
6.
Sujeet S. Bhoite Yilin Han Brandon T. Ruotolo Matthew R. Chapman 《The Journal of biological chemistry》2022,298(7)
The gut microbiome has been shown to have key implications in the pathogenesis of Parkinson’s disease (PD). The Escherichia coli functional amyloid CsgA is known to accelerate α-synuclein aggregation in vitro and induce PD symptoms in mice. However, the mechanism governing CsgA-mediated acceleration of α-synuclein aggregation is unclear. Here, we show that CsgA can form stable homodimeric species that correlate with faster α-synuclein amyloid aggregation. Furthermore, we identify and characterize new CsgA homologs encoded by bacteria present in the human microbiome. These CsgA homologs display diverse aggregation kinetics, and they differ in their ability to modulate α-synuclein aggregation. Remarkably, we demonstrate that slowing down CsgA aggregation leads to an increased acceleration of α-synuclein aggregation, suggesting that the intrinsic amyloidogenicity of gut bacterial CsgA homologs affects their ability to accelerate α-synuclein aggregation. Finally, we identify a complex between CsgA and α-synuclein that functions as a platform to accelerate α-synuclein aggregation. Taken together, our work reveals complex interplay between bacterial amyloids and α-synuclein that better informs our understanding of PD causation. 相似文献
7.
Manoel Cardoso de Barros Roberto do Nascimento Silva Marcelo Henrique Soller Ramada Alexsandro Sobreira Galdino Lídia Maria Pepe de Moraes Fernando Araripe Gonalves Torres Cirano Jos Ulhoa 《Carbohydrate research》2009,344(13):1682-1686
The yeast Cryptococcus flavus secretes a glycosylated α-amylase (Amy1) when grown in a starch-containing medium. The effects of N-glycosylation on secretion, enzyme activity, and stability of this glycoprotein were studied. Addition of tunicamycin (TM) to the medium at a concentration higher than 0.5 μg mL−1 affected C. flavus growth. Amy1 activity increased by 55% in the intracellular fraction after C. flavus growth in the presence of 0.5 μg mL−1 TM. SDS–PAGE and gel activity detection showed that native enzyme and deglycosylated enzyme had apparent molecular mass of 68 and 64.5 kDa, respectively. The N-glycosylation process did not affect either optimum pH or optimum temperature. The KM values of native and non-glycosylated α-amylases were 0.052 and 0.098 mg mL−1, and Vmax values were 0.038 and 0.047 mg min−1, respectively. However, the non-glycosylated form was more sensitive to inactivation by both the proteolytic enzyme trypsin and high temperature. Furthermore, the activity of the non-glycosylated enzyme was affected by Hg2+ and Cu2+ suggesting that N-glycosylation is involved in the folding of Amy1. 相似文献
8.
Wu X Oppermann M Berndt KD Bergman T Jörnvall H Knapp S Oppermann U 《Biochemical and biophysical research communications》2008,373(4):482-487
The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5–7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 °C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of ΔHcal is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (ΔG (T)) and results in a maximal ΔG of only 9.5 kcal/mol at 320 K and a ΔG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus. 相似文献
9.
Decarboxylation kinetics for the carbonatotris(pyridine)cobalt(III) ion in perchloric acid solutions
The kinetics of the decomposition reactions of the CO(py)3(CO3)(H2O)+ ion have been investigated in aqueous perchloric acid solutions over a range of hydrogen ion concentrations (0.10 to 5.0 M) and at two ionic strengths (I = 1.0 and 5.0 M). At the lower ionic strength, plots of ln (At–A∞ versus time show a nonlinearity that is consistent with that expected for consecutive first-order reactions. The rates of the faster reaction are similar to those reported for the spontaneous reduction of aquopyridine-cobalt(III) cations. At the higher ionic strength, the above noted curvature is not apparent and the decarboxylation kinetics of the title complex may be described by a pseudo-first-order rate constant: kobs = k[H3O+]. At 20°C, k = (1.75−+0.09) s−1 M−1 with activation parameters ofΔH‡ = (97 −+ 4) kJ mol−1 and ΔS‡ = −(54 −+ 32) J deg−1 mol−1. These kinetic parameters are compared with those previously reported for the similar complexes, Co(py)4CO3+ and Co(py)2(CO3)(H2O)2+. 相似文献
10.
Hye Kyung Ju Su-Jeong Hwang Choon-Ju Jeon Gyun Min Lee Sung Kwan Yoon 《Journal of biotechnology》2009,143(2):145-150
To investigate the effect of hyperosmotic medium on production and aggregation of the variant of Angiopoietin-1 (Ang1), cartilage oligomeric matrix protein (COMP)–Ang1, in recombinant Chinese hamster ovary (CHO) cells, CHO cells were cultivated in shaking flasks. NaCl and/or sorbitol were used to raise medium osmolality in the range of 300–450 mOsm/kg. The specific productivity of COMP–Ang1, qCOMP–Ang1, increased as medium osmolality increased. At NaCl-450 mOsm/kg, the qCOMP–Ang1 was 7.7-fold higher than that at NaCl-300 mOsm/kg, while, at sorbitol-450 mOsm/kg, it was 2.9-fold higher than that at sorbitol-300 mOsm/kg. This can be attributed to the increased relative mRNA level of COMP–Ang1 at NaCl-450 mOsm/kg which was approximately 2.4-fold higher than that at sorbitol-450 mOsm/kg. Western blot analysis showed that COMP–Ang1 aggregates started to occur in the late-exponential phase of cell growth. When sorbitol was used to raise the medium osmolality, a severe aggregation of COMP–Ang1 was observed. On the other hand, when NaCl was used, the aggregation of COMP–Ang1 was drastically reduced at NaCl-400 mOsm/kg. At NaCl-450 mOsm/kg, the aggregation of COMP–Ang1 was hardly observed. This suggests that environmental conditions are critical for the aggregation of COMP–Ang1. Taken together, the use of NaCl-induced hyperosmotic medium to cell culture process turns out to be an efficient strategy for enhancing COMP–Ang1 production and reducing COMP–Ang1 aggregation. 相似文献
11.
Jian Hua Liang Jing Du Lian Deng Xu Tao Jiang Shuang Hao Jing Bi Bo Jiang 《Neurochemistry international》2009,55(8):741-746
It has been reported that catalpol, an iridoid glucoside, isolated from the root of Rehmannia glutinosa, protected cells from damage induced by a variety of toxic stimulus such as LPS, MPP+ and rotenone. Here, we further evaluated the effect of catalpol against Aβ1–42-induced apoptosis in primary cortical neuron cultures. In the present study, the primary cortical neuron culture treated with Aβ1–42 was severed as cell model of Alzheimer's disease (AD) in vitro. By exposure to Aβ1–42 (5 μM) for 72 h in cultures, neuronal apoptosis occurred characterized by enhancement of activities of caspases and reactive oxygen species (ROS) as well as Bax increase, loss of mitochondrial membrane potential and cytochrome c release. Pretreatment with catalpol (0.5 mM) for 30 min prior to Aβ1–42 treatment attenuated neuronal apoptosis not only by reversing intracellular ROS accumulation, Bax level, mitochondrial membrane potential and, cytochrome c release to some extent, but also through regulating the activity and cleavage of caspase-3 and caspase-9. Thus, catalpol protects primary cultured cortical neurons induced by Aβ1–42 through a mitochondrial-dependent caspase pathway. 相似文献
12.
Bruce C. Spalding Patricia Taber John G. Swift Paul Horowicz 《The Journal of membrane biology》1991,123(3):223-233
Summary Efflux of36Cl– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl– efflux. The pH-dependent Cl– efflux can be described by the relationu=1/(1+10n(pK
a
-pH)), whereu is the Cl– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK
a
is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK
a
at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK
a
suggests that the critical pH-sensitive moiety producing the pH-dependent Cl– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k
–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q10 of 2.5. The Cl– efflux inhibitor SCN– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl– efflux by DEPC. The findings that the apparent pK
a
is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl– efflux, and that this action is retarded by SCN– supports the notion that protonation of histidine groups associated with Cl– channels is the controlling reaction for the pH-dependent Cl– efflux. 相似文献
13.
P. Campíns-Falc C. Molins-Legua A. Sevillano-Cabeza L. A. Tortajada Genaro 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2001,759(2):1162
A comparative study of different derivatization procedures has been performed in order to improve the stability of the reaction products o-phthalaldehyde–N-acetylcysteine (OPA–NAC) polyamines. Procedures such as solution derivatization, solution derivatization followed by retention on a packing support, derivatization on different packing supports and on-column derivatization, have been optimized and compared. The degradation rate constant (k) of the derivative was dependent on the procedure used and on the analyte. For the spermine (the most unstable isoindol tested) k was 8±2×10−2 min−1 in solution versus 7.7±1.1×10−4 min−1 on the (C18) solid support. The results obtained showed that forming the derivative on the packing support (C18) gave the best results following this procedure: conditioning the cartridges with borate buffer (1 ml, 0.5 M, pH 8), retention of the analyte, addition of 0.8 ml of OPA–NAC reagent, 0.2 ml borate buffer 0.8 M (pH 8) and elution of the isoindol with 3 ml of MeOH–borate buffer (9:1). The different derivatization procedures have been used to study the stability of the reaction products OPA–NAC polyamines formed in urine matrix using spermine as model compound. Similar results were obtained for standard solutions and urine samples. 相似文献
14.
Jeffrey A. Seminoff T. Todd Jones Tomoharu Eguchi Mervin Hastings David R. Jones 《Journal of experimental marine biology and ecology》2009,381(1):33-41
The trophic ecology of marine vertebrates has been increasingly studied via stable isotope analysis of body tissues. However, the theoretical basis for using stable isotopes to elucidate consumer–prey relationships remains poorly validated for most taxa despite numerous studies using this technique in natural systems. In this study, we measured stable carbon and stable nitrogen diet-tissue discrimination (Δdt) in whole blood, red blood cells, blood plasma solutes, and skin of leatherback sea turtles (Dermochelys coriacea; N = 7) maintained in captivity for up to 424 days and fed an isotopically consistent control diet with a mean C:N ratio of 2.94:1.00 and an energetic content of 20.16 ± 0.39 kJ g− 1 Dry Mass. We used a random-effect repeated measure model to evaluate isotopic consistency among tissue samples collected on days 276, 348, and 424. Both δ13C and δ15N remained consistent among sampling events in all tissues (all 95% posterior intervals for the slopes of a linear model included zero), indicating that all tissues had fully integrated diet-derived stable isotope compositions. Mean tissue-specific δ13C ranged from − 18.30 ± 0.16‰ (plasma solutes) to − 15.54 ± 0.14‰ (skin), whereas mean δ15N was from 10.06 ± 0.22‰ (whole blood) to 11.46 ± 0.10‰ (plasma solutes). The computed Δdt factors for carbon ranged from − 0.58‰ (plasma solutes) to + 2.25‰ (skin), whereas Δdt for nitrogen was from + 1.49 (red blood cells) to + 2.85 (plasma solutes). As the only discrimination factors available for leatherback turtles, our data will be useful for future interpretations of field-derived stable isotope data for this species. The inherent variability in Δdt values among individuals was low, which supports the value of these data for dietary reconstructions. However, it is important to note that tissue-specific discrimination factors for leatherbacks contrast with the widely accepted values for endothermic species (0–1‰ for C, 3–5‰ for N), and are also different from values established for hard-shelled turtles. This underscores the need for species- and tissue-specific discrimination factors before interpreting trophic studies of wild animals, including marine turtles. 相似文献
15.
Victoria Dorovska-Taran Diana Raykova Sava Tishkov Blagoy Blagoev 《Bioorganic chemistry》1980,9(4):482-490
The temperature dependence of the hydrolysis of p-nitrophenyl carboxylates with general formula H(CH2)nCOOC6H4NO2 catalyzed by alkaline mesentericopeptidase has been studied (n varying from 1 to 7, temperature range 2–30°C, pH 8.80, 5 vol% dimethylsulfoxide). The activation parameters of the deacylation step depend on the length of the hydrophobic side chain of the substrate molecule (
,
, and
decrease by 2.0 kcal/mol, 4.9 kcal/mol, and 10 eu, respectively, as the length of the acyl carbon chain increases from n = 1 to n = 4). The following criteria were applied to establish a chemical enthalpy-entropy compensation effect: (a) Exner's plot of log
vs
: (b) Petersen's plot of log, k/T vs 1/T; (c) Exner's statistical treatment in coordinates log k vs 1/T; (d) according to Krug et al. (ΔH≠ vs ΔGThm≠). By use of all the above-mentioned criteria the existence of a chemical enthalpy-entropy compensation effect was proved with an isokinetic temperature β of about 470°K, which is significantly higher than the average experimental temperature. 相似文献
16.
Padmarajaiah Nagaraja Anantharaman Shivakumar Ashwinee Kumar Shrestha 《Analytical biochemistry》2009,395(2):231-236
A sensitive, selective, and rapid enzymatic method is proposed for the quantification of hydrogen peroxide (H2O2) using 3-methyl-2-benzothiazolinonehydrazone hydrochloride (MBTH) and 10,11-dihydro-5H-benz(b,f)azepine (DBZ) as chromogenic cosubstrates catalyzed by horseradish peroxidase (HRP) enzyme. MBTH traps free radical released during oxidation of H2O2 by HRP and gets oxidized to electrophilic cation, which couples with DBZ to give an intense blue-colored product with maximum absorbance at 620 nm. The linear response for H2O2 is found between 5 × 10−6 and 45 × 10−6 mol L−1 at pH 4.0 and a temperature of 25 °C. Catalytic efficiency and catalytic power of the commercial peroxidase were found to be 0.415 × 106 M−1 min−1 and 9.81 × 10−4 min−1, respectively. The catalytic constant (kcat) and specificity constant (kcat/Km) at saturated concentration of the cosubstrates were 163.2 min−1 and 4.156 × 106 L mol−1 min−1, respectively. This method can be incorporated into biochemical analysis where H2O2 undergoes catalytic oxidation by oxidase. Its applicability in the biological samples was tested for glucose quantification in human serum. 相似文献
17.
Meng Wang Dingqin Li Xuepiao Sun Yun Judy Zhu Han Nong Jiaming Zhang 《Plant science》2009,177(6):716-723
Plant β-thioglucoside glucohydrolases (TGGs or myrosinases) are a young class of enzymes in the glycosyl hydrolase family 1 and have a narrow distribution. TGG genes have mainly been cloned from crucifers, while TGGs in other species have received little attention. The TGG gene CpTGG2 and its recombinant protein from papaya were characterized in this paper. This is the first plant TGG gene without unusual intron splicing borders, as present in all other available TGG genes. Phylogenetic analysis indicated that plant myrosinases are divided into two major lineages. CpTGG2 is located in the lineage constituted by AtTGG4–6 from Arabidopsis thaliana, while the rest of myrosinases (including MA, MB and MC subfamilies) are grouped into another lineage. RT-PCR analysis indicated that CpTGG2 was specifically expressed in the root. The recombinant CpTGG2 expressed in yeast had a subunit mass of 70 kDa, and had low basal TGG activity without addition of ascorbate. Low concentrations of ascorbate stimulated CpTGG2 activity, while high concentrations were inhibitory. CpTGG2 was active in broad pH and temperature ranges, similar to AtTGG4 and AtTGG5. The apparent Km and Vmax were 2.24 mM and 24.3 μmol min−1 mg−1 when sinigrin was the substrate. The calculated kcat/Km value was 1.3 × 104 S−1 M−1. Our results reshaped and expanded the myrosinase family structure and provided clues to the evolution of myrosinase genes. 相似文献
18.
Hepatitis B surface antibody (HBsAb) was immobilized to the surface of a gold electrode modified with cysteamine and colloidal gold as matrices to detect hepatitis B surface antigen (HBsAg). Differential pulse voltammetry (DPV) method was used for the investigation of the specific interaction between the immobilized HBsAb and HBsAg in solution, which was followed as a change of peak current in DPV with time. With the modified gold electrode, the differences in affinity of HBsAb with HBsAg at the temperatures of 37 and 40 °C were easily distinguished and the kinetic rate constants (kass and kdiss) and kinetic affinity constant K were determined from the curves of current versus time. In addition, the thermodynamic constants, ΔG, ΔH and ΔS, of the interaction at 37 °C were calculated, which were −56.65, −64.54 and −25.45 kJ mol−1, respectively. 相似文献
19.
The role of Try-81 in the reaction catalyzed by Saccharomyces cerevisiae sterol 24-C-methyltransferase (Erg6p) was investigated kinetically and for product differences against a panel of position-81 mutants in which Tyr was substituted with Trp, Phe, Ile, Leu, Val and Ala. The residue chosen for mutation is one that was reported previously to accept fecosterol and yield a set 24-ethyl (idene) sterol products typical of plants, showing the amino acid residue is located close to the transient C25 carbocation intermediate in the active site. One group of mutants (aromatic) tested with the natural substrate zymosterol accelerated the C-methylation reaction (kcat/Km) whereas the other group of mutants (aliphatics) decreased catalytic competence as the amino acid side chain was downsized. Mutating to aromatic and assaying with the substrate analog designed as a suicide substrate 26,27-dehydrozymosterol favored C26-monol formation, whereas mutating to the aliphatic of smaller size favored C26-diol formation (a measure of enzyme alkylation). In no case was zymosterol converted to an intermediate that formed a C25-diol. Thermodynamic analysis (determination of Ea, ΔG‡, ΔH‡ and TΔS‡) for the C-methylation reaction performed by these enzymes assayed with the substrate and its analog or zymosterol paired with the “charged’ high energy intermediate (HEI) analogs 24(R,S)25,epiminolanosterol and 25-azalanosterol or “neutral” membrane insert ergosterol showed that mutation to aromatics can reduce inhibitor potency (measured as Km/Ki), yet catalysis can improve in Trp81 by the introduction of a gain in free energy associated with stabilization of the transition state of a rate-controlling step directed toward turnover. Alternatively, mutation to the smaller aliphatic amino acid side chains led to a destabilization in the active site structure which was accompanied by increases in the partition ratios associated with abortive complex formation. The results are explained by consideration of the functional differences attributed to Tyr81 substitution to aromatics and aliphatics of different size involved with cation-π or hydrogen bonding interactions and in the activation barriers required of differing side chain conformations to orient the reactants in the direction of turnover versus enzyme inactivation. 相似文献
20.
Petros Katapodis Wim Nerinckx Marc Claeyssens Paul Christakopoulos 《Process Biochemistry》2006,41(12):2402-2409
An intracellular β-xylosidase from the thermophilic fungus Sporotricum thermophile strain ATCC 34628 was purified to homogeneity by Q-Sepharose and Mono-Q column chromatographies. The protein properties correspond to molecular mass and pI values of 45 kDa and 4.2, respectively. The enzyme is optimally active at pH 7.0 and 50 °C. The purified β-xylosidase is fully stable at pH 6.0–8.0 and temperatures up to 50 °C and retained over 58% of its activity after 1 h at 60 °C. The enzyme hydrolyzes β-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 6, releasing xylose from the non-reducing end, but is inactive against xylan substrates. The apparent Km and Vmax values from p-nitrophenyl β-d-xylopyranoside are 1.1 mM and 114 μmol p-nitrophenol min−1 mg−1, respectively. Alcohols inactivate the enzyme, ethanol at 10% (v/v) yields a 30% decrease of its activity. The enzyme is irreversibly inhibited by 2,3-epoxypropyl β-d-xylobioside while alkyl epoxides derived from d-xylose were not inhibitors of the enzyme. The enzyme catalyses the condensation reaction using high donor concentration, up to 60% (w/v) xylose. 相似文献