共查询到20条相似文献,搜索用时 31 毫秒
1.
The effect of aging and CDP-choline treatment (20 mg kg−1 body weight i.p. for 28 days) on the maximal rates (Vmax) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months. 相似文献
2.
RafaŁ Kamiński Ewa Kamler Jacek Wolnicki Justyna Sikorska Jakub WaŁowski 《Journal of thermal biology》2010
Populations of a rheophilic cyprinid Barbus barbus have declined in last decades, which created a need of conservation aquaculture. Production of stocking material in controlled conditions calls for optimization of the two major factors, temperature and diet. Condition, growth and food conversion ratio in fish fed a formulated diet Aller Futura were compared with those on natural food—frozen Chironomidae larvae at 17, 21 and 25 °C. Groups of 60 early juveniles (0.6–3.7 g) were reared in each of 18 aquaria in which six experimental groups were run in triplicate. Daily food ratios were adjusted according to fish biomass, differences in hydration between the two diets and rearing temperature. No mortality occurred during the experiment. Condition coefficient K was significantly higher in fish fed Aller Futura compared to those fed Chironomidae irrespective of temperature tested; body deformities were not recorded. Relative growth rate at the same temperature was always higher in fish on the formulated diet than in those fed Chironomidae, and food conversion ratio was always suppressed, both suggesting an efficient utilization of Aller Futura for growth in B. barbus early juveniles. On both diets the coefficient K was depressed at 21 °C. Relative growth rate (RGR) was accelerated with temperature according the Krogh’s “normal curve” within the range 21–25 °C, while at lower temperatures (17–21 °C) the observed values of temperature coefficient Q10 were much higher than the theoretical Q10 values based on Krogh’s “normal curve”. Food conversion ratios (FCR) were reduced on both diets at 21 and 25 °C. Theoretical optimum temperatures for food conversion were 22.0 and 23.6 °C. Summing up, responses of three independent indices: condition, growth and food utilization locate the optimum temperature for B. barbus between 21 and 25 °C. No evidence was found that the effect of temperature on these indices was substantially modified by the diet. 相似文献
3.
An activation gating switch in Kv1.2 is localized to a threonine residue in the S2-S3 linker
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The activation properties of Kv1.2 channels are highly variable, with reported half-activation (V1/2) values ranging from ∼−40 mV to ∼+30 mV. Here we show that this arises because Kv1.2 channels occupy two distinct gating modes (“fast” and “slow”). “Slow” gating (τact = 90 ± 6 ms at +35 mV) was associated with a V1/2 of activation of +16.6 ± 1.1 mV, whereas “fast” gating (τact = 4.5 ± 1.7 ms at +35 mV) was associated with a V1/2 of activation of −18.8 ± 2.3 mV. It was possible to switch between gating modes by applying a prepulse, which suggested that channels activate to a single open state along separate “fast” and “slow” activation pathways. Using chimeras and point mutants between Kv1.2 and Kv1.5 channels, we determined that introduction of a positive charge at or around threonine 252 in the S2-S3 linker of Kv1.2 abolished “slow” activation gating. Furthermore, dialysis of the cytoplasm or excision of cell-attached patches from cells expressing Kv1.2 channels switched gating from “slow” to “fast”, suggesting involvement of cytoplasmic regulators. Collectively, these results demonstrate two modes of activation gating in Kv1.2 and specific residues in the S2-S3 linker that act as a switch between these modes. 相似文献
4.
Lee Tin Sin Soo-Tueen Bee Tiam-Ting Tee A.A.H. Kadhum Chi Ma A.R. Rahmat P. Veerasamy 《Carbohydrate polymers》2013
In this study, the interactions of α-tocopherol (α-TOH) in PVOH–starch blends were investigated. α-TOH is an interacting agent possesses a unique molecule of polar chroman “head” and non-polar phytyl “tail” which can improve surface interaction of PVOH and starch. It showed favorable results when blending PVOH–starch with α-TOH, where the highest tensile strengths were achieved at 60 wt.% PVOH–starch blend for 1 phr α-TOH and 50 wt.% for 3 phr α-TOH, respectively. This due to the formation of miscible PVOH–starch as resulted by the compatibilizing effect of α-TOH. Moreover, the enthalpy of melting (ΔHm) of 60 wt.% PVOH–starch and 50 wt.% PVOH–starch added with 1 and 3 phr α-TOH respectively were higher than ΔHm of the neat PVOH–starch blends. The thermogravimetry analysis also showed that α-TOH can be used as thermal stabilizer to reduce weight losses at elevated temperature. The surface morphologies of the compatible blends formed large portion of continuous phase where the starch granules interacted well with α-TOH by acting as compatilizer to reduce surface energy of starch for embedment into PVOH matrix. 相似文献
5.
Alexandre A. Tonin Aleksandro S. Da Silva Jader B. Ruchel João F.P. Rezer Giovana Camillo Luciana Faccio Raqueli T. França Daniela B.R. Leal Marta M.M.F. Duarte Fernada F. Vogel Mario L. de la Rue Sonia T.A. Lopes 《Experimental parasitology》2013
An investigation of E-NTPDase and E-ADA activities in lymphocytes from rats experimentally infected with Toxoplasma gondii was carried out in this study. For this purpose, twenty four adult male Wistar rats were divided in two groups/four subgroups (A1 and A2; B1 and B2–6 animal/each group), with “A” as uninfected and “B” inoculated with T. gondii (RH strain). Sampling was performed on days 5 and 10 post-infection (p.i.), with evaluation of hemogram, immunoglobulins (IgM and IgG) and activity of E-NTPDase and E-ADA in lymphocytes. Enzymes essays showed ATP hydrolysis increased on days 5 (P < 0.05) and 10 (P < 0.01) p.i., as well as an increase of ADP hydrolysis on day 10 (P < 0.01) p.i. E-ADA activity on lymphocytes was also increased in both evaluated periods (P < 0.01). Based on E-NTPDase and E-ADA increased activities observed on lymphocytes, it is possible to suggest their involvement in an anti-inflammatory response, consisting of a modulatory response, preventing excessive tissue damage caused by the infection with Toxoplasma gondii. 相似文献
6.
Nay Chi Htun Koichi Miyaki Chenxi Zhao Masaaki Muramatsu Noriko Sato 《Biochemical and biophysical research communications》2014
Higher cognitive performance, maintenance of mental health and psychological well-being require adequate prefrontal cortex (PFC) function. “Inverted U-shaped” dopamine model indicates optimal PFC dopamine level is important to attain its function while high or low levels have adverse effects. Catechol-O-methyltransferase (COMT) and methylenetetrahydrofolate reductase (MTHFR) may be involved in this complex non-linear PFC dopamine regulation. We addressed whether genetic variation reflecting COMT and MTHFR activities can explain the inter-individual mental health differences in healthy Japanese men (n = 188). The mental health was measured by Mental Health Inventory (MHI)-5 score. The rs4633–rs4818–rs4680 haplotypes were used to represent the multilevel COMT activities, while for MTHFR, the functional single polymorphism, rs1801133 (C677T), was used. We examined the effectiveness of haplotype-based association analysis of COMT on mental health together with studying its interaction with MTHFR-C677T. As a result, the relation between activity-ranked COMT genotype and MHI-5 score showed a tendency to fit into an “inverted U-shaped” quadratic curve (P = 0.054). This curvilinear correlation was significant in the subjects with MTHFR-CC (P < 0.001), but not with MTHFR T-allele carriers (P = 0.793). Our pilot study implies a potential influence of COMT and MTHFR genotypic combination on normal variation of mental health. 相似文献
7.
Harald Bär Michael Schopferer Sarika Sharma Bernhard Hochstein Harald Herrmann 《Journal of molecular biology》2010,397(5):1188-2266
Inherited mutations in the gene coding for the intermediate filament protein desmin have been demonstrated to cause severe skeletal and cardiac myopathies. Unexpectedly, some of the mutated desmins, in particular those carrying single amino acid alterations in the non-α-helical carboxy-terminal domain (“tail”), have been demonstrated to form apparently normal filaments both in vitro and in transfected cells. Thus, it is not clear if filament properties are affected by these mutations at all. For this reason, we performed oscillatory shear experiments with six different desmin “tail” mutants in order to characterize the mesh size of filament networks and their strain stiffening properties. Moreover, we have carried out high-frequency oscillatory squeeze flow measurements to determine the bending stiffness of the respective filaments, characterized by the persistence length lp. Interestingly, mesh size was not altered for the mutant filament networks, except for the mutant DesR454W, which apparently did not form proper filament networks. Also, the values for bending stiffness were in the same range for both the “tail” mutants (lp = 1.0-2.0 μm) and the wild-type desmin (lp = 1.1 ± 0.5 μm). However, most investigated desmin mutants exhibited a distinct reduction in strain stiffening compared to wild-type desmin and promoted nonaffine network deformation. Therefore, we conclude that the mutated amino acids affect intrafilamentous architecture and colloidal interactions along the filament in such a way that the response to applied strain is significantly altered.In order to explore the importance of the “tail” domain as such for filament network properties, we employed a “tail”-truncated desmin. Under standard conditions, it formed extended regular filaments, but failed to generate strain stiffening. Hence, these data strongly indicate that the “tail” domain is responsible for attractive filament-filament interactions. Moreover, these types of interactions may also be relevant to the network properties of the desmin cytoskeleton in patient muscle. 相似文献
8.
Paola Manini Roberta Andreoli Stefano Sforza Chiara Dall’Asta Gianni Galaverna Antonio Mutti Wilfried M.A. Niessen 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2010,878(27):2616-2622
We present the application of a novel isotope dilution method, named Alternate Isotope-Coded Derivatization Assay (AIDA), to the quantitative analysis of hydrazone derivatives of malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) in exhaled breath condensate (EBC) samples using liquid chromatography–tandem mass spectrometry. AIDA is based on the alternate derivatization of the analyte(s) with reagents that are available in two pure isotopic forms, respectively “light” and “heavy”, by using light-derivatized standards for the quantification of the heavy-derivatized analytes, and vice versa. To this purpose, 2,4-dinitro-3,5,6-trideuterophenylhydrazine (d3-DNPH) has been synthesized and used as “heavy” reagent in combination with commercial “light” DNPH. Using the AIDA method, any unknown concentration of the analyte in the matrix can be calculated without the need of a calibration curve. An external calibration method has been also investigated for comparative purpose. The stability of DNPH and d3-DNPH derivatives was verified by excluding any exchange of hydrazones with each other. In the range of concentrations of biological interest, e.g., 2–40 nM for MDA and 0.5–10 nM for 4-HNE, the derivatization reactions of MDA and 4-HNE with DNPH and d3-DNPH showed overlapping kinetics and comparable yields. The MS response of both DNPH and d3-DNPH hydrazones was similar. The precision of AIDA, calculated as %RSD, was within 3.2–8% for MDA and 4.5–11% for 4-HNE. Accuracy was tested by analyzing a spiked EBC pool sample and acceptable results (accuracy within 98–108% for MDA and 93–114% for 4-HNE) were obtained by AIDA after subtraction of the blank, which was not negligible. The results of quantitative analysis of MDA and 4-HNE in EBC samples obtained by AIDA assay with four analyses per sample were in good agreement with those obtained by external calibration method on the same samples. 相似文献
9.
The binding of the myristoylated alanine-rich C kinase substrate (MARCKS) to mixed, fluid, phospholipid membranes is modeled with a recently developed Monte Carlo simulation scheme. The central domain of MARCKS is both basic (ζ = +13) and hydrophobic (five Phe residues), and is flanked with two long chains, one ending with the myristoylated N-terminus. This natively unfolded protein is modeled as a flexible chain of “beads” representing the amino acid residues. The membranes contain neutral (ζ = 0), monovalent (ζ = −1), and tetravalent (ζ = −4) lipids, all of which are laterally mobile. MARCKS-membrane interaction is modeled by Debye-Hückel electrostatic potentials and semiempirical hydrophobic energies. In agreement with experiment, we find that membrane binding is mediated by electrostatic attraction of the basic domain to acidic lipids and membrane penetration of its hydrophobic moieties. The binding is opposed by configurational entropy losses and electrostatic membrane repulsion of the two long chains, and by lipid demixing upon adsorption. The simulations provide a physical model for how membrane-adsorbed MARCKS attracts several PIP2 lipids (ζ = −4) to its vicinity, and how phosphorylation of the central domain (ζ = +13 to ζ = +7) triggers an “electrostatic switch”, which weakens both the membrane interaction and PIP2 sequestration. This scheme captures the essence of “discreteness of charge” at membrane surfaces and can examine the formation of membrane-mediated multicomponent macromolecular complexes that function in many cellular processes. 相似文献
10.
To simplify the method of ACE-inhibitory peptide production, defatted canola meal was subjected to enzymatic proteolysis. Alcalase 2.4L and protease M “Amano” were found to be the most efficient enzymes in releasing ACE-inhibitory peptides from canola proteins among 13 tested enzymes. The IC50 values of canola protein hydrolysates ranged from 18.1 to 82.5 μg protein/mL. Differences in ACE-inhibitory activities of various protein hydrolysates reflected varied enzyme specificities. A positive correlation was determined between ACE-inhibitory activity and the degree of hydrolysis (r = 0.5916, p < 0.001). Ion-exchange chromatography of canola protein hydrolysate increased the protein content greater than 95% without loss of ACE-inhibitory activity. This fraction was resistant to the degradation of gastrointestinal enzyme and ACE during in vitro incubation and may be a useful ingredient in the formulation of hypotensive functional food products. 相似文献
11.
S. Khodadadi A. MalkovskiyA. Kisliuk A.P. Sokolov 《Biochimica et Biophysica Acta - Proteins and Proteomics》2010,1804(1):15-19
We performed Raman and Brillouin scattering measurements to estimate glass transition temperature, Tg, of hydrated protein. The measurements reveal very broad glass transition in hydrated lysozyme with approximate Tg ∼ 180 ± 15 K. This result agrees with a broad range of Tg ∼ 160–200 K reported in literature for hydrated globular proteins and stresses the difference between behavior of hydrated biomolecules and simple glass-forming systems. Moreover, the main structural relaxation of the hydrated protein system that freezes at Tg ∼ 180 K remains unknown. We emphasize the difference between the “dynamic transition”, known as a sharp rise in mean-squared atomic displacement <r2> at temperatures around TD ∼ 200–230 K, and the glass transition. They have different physical origin and should not be confused. 相似文献
12.
Polymorphisms in the endothelial nitric oxide synthase (eNOS) gene may influence the risk of ischemic stroke (IS), but the results are still debatable. A meta-analysis was performed to investigate the association between the eNOS gene polymorphisms in IS risk. Case–control studies on the association between the G894T, T-786C, and 4b/a polymorphisms and IS were searched up to July 2012, and the genotype frequencies in the control group were found to be consistent with the Hardy–Weinberg equilibrium (HWE). The effect summary odds ratio (OR) and 95% confidence intervals (CIs) were obtained. Meta-regression was used to explore the potential sources of heterogeneity. Funnel plots and Egger's test was used to estimate small study biases, and heterogeneity was assessed by chi-square-based Q-test and I2 test. There were total 6537/6475 cases/controls for G894T, 3459/3951 cases/controls for 4b/a, and 2125/2673 cases/controls for T-786C polymorphism. For G894T and 4b/a, a significant association of 894 T allele and 4a allele with increased risk of IS was found in Asians (TT + GT vs. GG: p < 0.00001, OR = 1.60, 95% CI = 1.38–1.79, Pheterogeneity = 0.11; aa + ba vs. bb: P < 0.00001, OR = 1.60, 95% CI = 1.30–1.97, Pheterogeneity = 0.02), but not in Caucasians (TT + GT vs. GG: P = 0.60, OR = 0.94, 95% CI = 0.75–1.19, Pheterogeneity = 0.002; aa + ba vs. bb: P = 0.13, OR = 0.81, 95% CI = 0.62–1.06, Pheterogeneity = 0.63). For T-786C polymorphism, there were no significant differences in genotype distribution between IS and control in Asians (CC + TC vs. TT: P = 0.15, OR = 1.14, 95% CI = 0.95–1.37, Pheterogeneity = 0.94) and in Caucasians (CC + TC vs. TT: P = 0.72, OR = 0.96, 95% CI = 0.75–1.22, Pheterogeneity = 0.53). This analysis provides strong evidence that the eNOS T-786C gene polymorphism is not associated with IS, the G894T and 4b/a polymorphisms might be associated with IS, at least in Asians. 相似文献
13.
The resting metabolic rate (RMR) of seasonally-acclimated Mabuya brevicollis of various body masses was determined at 20, 25, 30, 35 and 40 °C, using open-flow respirometry. RMR (ml g−1 h−1) decreased with increasing mass at each temperature. RMRs increaProd. Type: FTPsed as temperature increased. The highest and lowest Q10 values were obtained for the temperature ranges 20–25 °C and 30–35 °C for the summer-acclimated lizards. The exponent of mass “b” in the metabolism-body mass relation ranged from 0.41 to 0.61. b values were lower in the autumn and winter-acclimated lizards than in spring and summer-acclimated lizards. Seasonal acclimation effects were evident at all temperatures (20–40 °C) for M. brevicollis. Winter-acclimated skinks had the lowest metabolic rates at different temperatures. The pattern of acclimation exhibited by M. brevicollis may represent a useful adaptation for lizards inhabiting subtropical deserts to promote activity during their active seasons. 相似文献
14.
Null genotypes of GSTM1 and GSTT1 contribute to increased risk of diabetes mellitus: A meta-analysis
Diabetes mellitus (DM) is a common disease which results from various causes including genetic and environmental factors. Glutathione S-Transferase M1 (GSTM1) and Glutathione S-Transferase T1 (GSTT1) genes are polymorphic in human and the null genotypes lead to the absence of enzyme function. Many studies assessed the associations between GSTM1/GSTT1 null genotypes and DM risk but reported conflicting results. In order to get a more precise estimate of the associations of GSTM1/GSTT1 null genotypes with DM risk, we performed this meta-analysis. Published literature from PubMed, Embase and China Biology Medicine (CBM) databases was searched for eligible studies. Pooled odds ratios (OR) and corresponding 95% confidence intervals (95%CI) were calculated using a fixed- or random-effects model. 11 publications (a total of 2577 cases and 4572 controls) were finally included into this meta-analysis. Meta-analyses indicated that null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 were all associated with increased risk of DM (GSTM1: OR random-effects = 1.60, 95%CI 1.10–2.34, POR = 0.014; GSTT1: OR random-effects = 1.47, 95%CI 1.12–1.92, POR = 0.005; GSTM1–GSTT1: OR fixed-effects = 1.83, 95%CI 1.30–2.59, POR = 0.001). Subgroup by ethnicity suggested significant associations between null genotypes of GSTM1 and GSTT1 and DM risk among Asians (GSTM1: OR random-effects = 1.77, 95%CI 1.24–2.53, POR = 0.002; GSTT1: OR random-effects = 1.58, 95%CI 1.09–2.27, POR = 0.015). This meta-analysis suggests null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 are all associated with increased risk of DM, and null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 are potential biomarkers of DM. 相似文献
15.
Jan Vacek Martina Zatloukalová Thomas Desmier Veronika Nezhodová Jan Hrbáč Martin Kubala Vladimír Křen Jitka Ulrichová Patrick Trouillas 《Chemico-biological interactions》2013
Besides the well-known chemoprotective effects of polyphenols, their prooxidant activities via interactions with biomacromolecules as DNA and proteins are of the utmost importance. Current research focuses not only on natural polyphenols but also on synthetically prepared analogs with promising biological activities. In the present study, the antioxidant and prooxidant properties of a semi-synthetic flavonolignan 7-O-galloylsilybin (7-GSB) are described. The presence of the galloyl moiety significantly enhances the antioxidant capacity of 7-GSB compared to that of silybin (SB). These findings were supported by electrochemistry, DPPH (2,2-diphenyl-1-picrylhydrazyl) scavenging activity, total antioxidant capacity (CL-TAC) and DFT (density functional theory) calculations. A three-step oxidation mechanism of 7-GSB is proposed at pH 7.4, in which the galloyl moiety is first oxidized at Ep,1 = +0.20 V (vs. Ag/AgCl3M KCl) followed by oxidation of the 20-OH (Ep,2 = +0.55 V) and most probably 5-OH (Ep,3 = +0.95 V) group of SB moiety. The molecular orbital analysis and the calculation of O–H bond dissociation enthalpies (BDE) fully rationalize the electrooxidation processes. The metal (Cu2+) complexation of 7-GSB was studied, which appeared to involve both the galloyl moiety and the 5-OH group. The prooxidant effects of the metal-complexes were then studied according to their capacity to oxidatively induce DNA modification and cleavage. These results paved the way towards the conclusion that 7-O-galloyl substitution to SB concomitantly (i) enhances antioxidant (ROS scavenging) capacity and (ii) decreases prooxidant effect/DNA damage after Cu complexation. This multidisciplinary approach provides a comprehensive mechanistic picture of the antioxidant vs. metal-induced prooxidant effects of flavonolignans at the molecular level, under ex vivo conditions. 相似文献
16.
Bennett L. Ibey Andrei G. Pakhomov Betsy W. Gregory Vera A. Khorokhorina Caleb C. Roth Mikhail A. Rassokhin Joshua A. Bernhard Gerald J. Wilmink Olga N. Pakhomova 《Biochimica et Biophysica Acta (BBA)/General Subjects》2010
Background
Nanosecond electric pulses (EP) disrupt cell membrane and organelles and cause cell death in a manner different from the conventional irreversible electroporation. We explored the cytotoxic effect of 10-ns EP (quantitation, mechanisms, efficiency, and specificity) in comparison with 300-ns, 1.8- and 9-μs EP.Methods
Effects in Jurkat and U937 cells were characterized by survival assays, DNA electrophoresis and flow cytometry.Results
10-ns EP caused apoptotic or necrotic death within 2–20 h. Survival (S, %) followed the absorbed dose (D, J/g) as: S = αD(−K), where coefficients K and α determined the slope and the “shoulder” of the survival curve. K was similar in all groups, whereas α was cell type- and pulse duration-dependent. Long pulses caused immediate propidium uptake and phosphatidylserine (PS) externalization, whereas 10-ns pulses caused PS externalization only.Conclusions
1.8- and 9-μs EP cause cell death efficiently and indiscriminately (LD50 1–3 J/g in both cell lines); 10-ns EP are less efficient, but very selective (LD50 50–80 J/g for Jurkat and 400–500 J/g for U937); 300-ns EP show intermediate effects. Shorter EP open propidium-impermeable, small membrane pores (”nanopores”), triggering different cell death mechanisms.General significance
Nanosecond EP can selectively target certain cells in medical applications like tumor ablation. 相似文献17.
Monsur Alam Khan Mohammad Monirul Islam Yutaka Kuroda 《Biochimica et Biophysica Acta - Proteins and Proteomics》2013,1834(10):2107-2115
Understanding protein solubility, and consequently aggregation, is an important issue both from an academic and a biotechnological application viewpoints. Here we report the effects of 10 representative amino acids on the aggregation kinetics of proteins. The effects were determined by measuring the solubility of a simplified bovine pancreatic trypsin inhibitor (BPTI) variant, to which short artificial tags containing the amino acid of interest were added at its C-terminus. We determined the solubility of the tagged variants as a function of equilibration time (20 min to 48 h) and total protein concentration ranging from 0.10 mg/ml to 25.0 mg/ml. We observed, as anticipated, that proteins precipitated when the total protein concentration exceeded a critical value. However, when the total protein concentration was further increased, the apparent solubility reached a concentration above the critical value, and slowly decreased to a value under the critical concentration upon increasing the equilibration period. We rationalized these observations by identifying three different solubility values, the “transient solubility (TS)”, the “aggregation initiation concentration (AIC)” and the “long-term solubility (LS)”. AIC and LS are parameters determined essentially by the amino acid types composing the tags and could be considered as an amino acid's intrinsic property. On the other hand, TS is an apparent solubility that is measured after some (20 min in our case) equilibration time and is often considered as the “solubility” of the protein. Similar aggregation kinetic patterns were observed with natural proteins, indicating the generality of the observations made using our model protein. 相似文献
18.
Somnath Mukherjee 《Journal of molecular biology》2010,401(5):949-968
The dreaded pathogen Staphylococcus aureus is one of the causes of morbidity and mortality worldwide. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), one of the key glycolytic enzymes, is irreversibly oxidized under oxidative stress and is responsible for sustenance of the pathogen inside the host. With an aim to elucidate the catalytic mechanism and identification of intermediates involved, we describe in this study different crystal structures of GAPDH1 from methicillin-resistant S. aureus MRSA252 (SaGAPDH1) in apo and holo forms of wild type, thioacyl intermediate, and ternary complexes of active-site mutants with physiological substrate d-glyceraldehyde-3-phosphate (G3P) and coenzyme NAD+. A new phosphate recognition site, “new Pi” site, similar to that observed in GAPDH from Thermotoga maritima, is reported here, which is 3.40 Å away from the “classical Pi” site. Ternary complexes discussed are representatives of noncovalent Michaelis complexes in the ground state. d-G3P is bound to all the four subunits of C151S.NAD and C151G.NAD in more reactive hydrate (gem-di-ol) form. However, in C151S + H178N.NAD, the substrate is bound to two chains in aldehyde form and in gem-di-ol form to the other two. This work reports binding of d-G3P to the C151G mutant in an inverted manner for the very first time. The structure of the thiaocyl complex presented here is formed after the hydride transfer. The C3 phosphate of d-G3P is positioned at the “Ps” site in the ternary complexes but at the “new Pi” site in the thioacyl complex and C1-O1 bond points opposite to His178 disrupting the alignment between itself and NE2 of His178. A new conformation (Conformation I) of the 209-215 loop has also been identified, where the interaction between phosphate ion at the “new Pi” site and conserved Gly212 is lost. Altogether, inferences drawn from the kinetic analyses and crystal structures suggest the “flip-flop” model proposed for the enzyme mechanism. 相似文献
19.
Isolation and primary structures of seven serine proteinase inhibitors from Cyclanthera pedata seeds
Jolanta Kowalska Agnieszka ZabłockaTadeusz Wilusz 《Biochimica et Biophysica Acta (BBA)/General Subjects》2006
Seven new trypsin inhibitors, CyPTI I–VII, were purified from ripe seeds of Cyclanthera pedata by affinity chromatography on immobilized chymotrypsin in the presence of 5 M NaCl followed by preparative native PAGE at pH 8.9. The CyPTIs (Cyclanthera pedata trypsin inhibitors) belong to a well-known squash inhibitor family. They contain 28–30 amino acids and have molecular weights from 3031 to 3367 Da. All the isolated inhibitors strongly inhibit bovine β-trypsin (Ka > 1011 M− 1) and, more weakly, bovine α-chymotrypsin (Ka ≈ 104–106 M− 1). In the presence of 3 M NaCl the association constants of CyPTIs with α-chymotrypsin increased a few hundred fold. Taking advantage of this phenomenon, a high concentration of NaCl was used to isolate the inhibitors by affinity chromatography on immobilized chymotrypsin. It was found that although one of them, CyPTI IV, had split the Asn25–Gly26 peptide bond, its inhibitory activity remained unchanged. The hydrolyzed bond is located downstream of the reactive site. Presumably, the inhibitor is a naturally occurring, double-chain protein arising during posttranslational modifications. 相似文献
20.