Docking and DFT Studies to explore the Topoisomerase II ATP Pocket employing 3-Substituted 2,6-Piperazindiones for drug design

Topoisomerases (Topos) are very important protein targets for drug design in cancer treatment. Human Topo type IIα (hTopo IIα) has been widely studied experimentally and theoretically. Here, we performed protein rigid/flexible side-chain docking to study a set of thirty-nine 3-substituted-2,6-piperazindiones (labelled 1a, (R)-[(2–20)a] and (S)-[(2–20)b]) derived from α-amino acids. To explain the ligand–protein complexes at the electronic level [using the highest occupied and the lowest unoccupied molecular orbitals (HOMO and LUMO) energies], density functional theory calculations were carried out. Finally, to show adenosine triphosphate (ATP) binding-site constituents, the Q-SiteFinder program was used. The docking results showed that all of the test compounds bind to the ATP-binding site on hTopo IIα. Recognition is mediated by the formation of several hydrogen bond acceptors or donators. This site was the largest (631 Å³) according to the Q-SiteFinder program. When using the protein rigid docking protocol, compound 13a derived from (R)-Lys showed the highest affinity. However, when a flexible side-chain docking protocol was used, the compound with the highest affinity was 16a, derived from (R)-Trp. Frontier molecular orbital studies showed that the HOMO of the ligand interacts with the LUMO located at side-chain residues from the protein-binding site. The HOMO of the binding site interacts with the LUMO of the ligand. We conclude that some ligand properties including the hindrance effect, hydrogen bonds, π–π interactions and stereogenic centres are important for the ligand to be recognised by the ATP-binding site of hTopo IIα.     

[3H]5,7-Dichlorokynurenic Acid Recognizes two Binding Sites in Rat Cerebral Cortex Membranes

Abstract

Binding of [³H]5,7-dichlorokynurenic acid ([³H]DCKA), a competitive antagonist of the strychnine-insensitive glycine site of the N-methyl-D-aspartate (NMDA) receptor channel complex, was characterized in synaptic plasma membranes from rat cerebral cortex. Non linear curve fitting of [³H]DCKA saturation and homologous displacement isotherms indicated the existence of two binding sites: a specific, saturable, high affinity site, with a pK_D value of 7.24 (K_D = 57.5 nmol/1) and a maximum binding value (B_max) of 6.9 pmol/mg of protein and a second site, with micromolar affinity. The pharmacological profile of both binding components was determined by studying the effect on [³H]DCKA and [³H]glycine binding of a series of compounds known to interact with different excitatory and inhibitory amino acid receptors. These studies confirmed the identity of the high affinity site of [³H]DCKA binding with the strychnine-insensitive glycine site of the NMDA receptor channel complex. 3-[2-(Phenylaminocarbonyl)ethenyl]-4,6-dichloroindole-2-carboxylic acid sodium salt (GV 150526A), a new, high affinity, selective glycine site antagonist (1), was the most potent inhibitor of this component of binding (pK_i = 8.24, K_i = 5.6 nmol/1). The low affinity component of [³H]DCKA binding was insensitive to the agonists glycine and D-serine and the partial agonist (±)-3-amino-1-hydroxy-2-pyrrolidone (HA 966), though recognised by glycine site antagonists. The precise nature of this second, low affinity [³H]DCKA binding site remains to be elucidated.     

嗜热丁烷氧化古菌Candidatus Syntrophoarchaeum烷基转移酶MtaA催化丁基辅酶M的分子机制研究

【背景】嗜热古菌Candidatus Syntrophoarchaeum可以与硫酸盐还原细菌共生,通过逆转产甲烷途径进行正丁烷的氧化,但在该过程中负责催化丁基辅酶M氧化的酶尚未确定。【目的】利用分子动力学模拟证明Ca.Syntrophoarchaeum中mta A基因编码的蛋白可以特异性催化丁基辅酶M中丁基的转移,并非转移甲基。【方法】使用Methanosarcina mazei辅酶M甲基转移酶Mta A的晶体结构(PDB ID:4ay8)作为模板,对Mta A_1 (Gen Bank登录号OFV65993.1)和Mta A_2 (Gen Bank登录号OFV65678.1)进行同源建模。使用分子对接得到两者分别结合CH_3-Co M和C_4H_9-Co M时的结构,并用AMBER18进行分子动力学模拟。【结果】当Mta A_1和Mta A_2分别结合C_4H_9-Co M时,表现出与4ay8晶体结构类似的TIM-Barrel折叠三维结构,但在活性中心形状、Zn~(2+)与底物距离以及活性位点附近氨基酸配位方式等方面存在差异,这可能是导致Ca.Syntrophoarchaeum中mta A基因编码的蛋白催化丁基辅酶M氧化的原因。其中Mta A_2与4ay8结构更相似,活性中心氨基酸配位更完整,暗示其更可能具备催化活性。然而当Mta A_1和Mta A_2分别结合CH_3-Co M时,整体结构不合实际,活性中心Zn~(2+)与底物距离过远,表明底物几乎不可能与酶结合。【结论】Ca.Syntrophoarchaeum中的Mta A_1和Mta A_2很可能是特异性的丁基转移酶,而非催化甲基的转移,其中Mta A_2具备活性的可能性更高。     

Effects of nitrogen supply and irradiance on growth and nitrogen status in the moss Dicranum majus from differently polluted areas

Abstract

A growth experiment was undertaken to study the effects of nitrogen supply and irradiance on growth and nitrogen status in the moss Dicranum majus Sm. from two areas receiving different amounts of atmospheric nitrogen deposition. Intact samples of D. majus carpets were taken from two Picea abies forests, one located in southern Norway (high-N site) and the other in central Norway (low-N site). The moss carpets were grown for 120 days at three irradiance levels (PPFD: 20,40 or 80 μmol m^?2 s^?l) and sprayed daily with equal amounts of a nutrient solution containing 30, 180 or 330 μM nitrogen as NO₃ - and NH₄ +. Concentrations and total amounts of nitrogen, soluble proteins and chlorophyll were highest in moss plants from the high-N site, both at the start and the end of the experiment. The elongation growth was highest at the lowest irradiance level. As total biomass production did not differ between nitrogen and light treatments, moss growth was presumably limited by other factors, even at the lowest supply rates. Concentrations and total amounts of nitrogen increased with increasing nitrogen supply in moss plants from both sites. Accumulated nitrogen was partly stored as protein and chlorophyll. Recycling of nitrogen from old to young tissues is discussed as a possible explanation for the rather low nitrogen demand in D. majus and the persistently higher nitrogen contents in moss plants from the high-N site.     

Identification,characterization and purification of a 160 kD bumetanide-binding glycoprotein from the rabbit parotid

We demonstrate the presence of a 160 kD protein in rabbit parotid basolateral membranes that can be labeled with the irreversible sulfhydryl reagent [¹⁴C]-N-ethylmaleimide in a bumetanide-protectable fashion. The specificity of this labeling, and our previous evidence for the existence of an essential sulfhydryl group closely associated with the bumetanide-binding site on the parotid Na⁺−K⁺−Cl⁻ cotransporter (J. Membrane Biol. 112:51–58, 1989), provide strong evidence that this protein is a part or all of the parotid bumetanide-biding site. When this protein is treated with endoglycosidase F/N-glycosidase F to remove N-linked oligosaccharides, its apparent molecular weight decreases to 135 kD. The pI of this deglycosylated protein is ≈6.4. The bumetanide-binding protein was purified using two preparative electrophoresis steps. First, a Triton X-100 extract enriched in this protein was run on preparative electrophoresis to obtain fractions containing proteins in the 160 kD range. These were then deglycosylated with endoglycosidase F/N-glycosidase F and selected fractions were pooled and rerun on preparative electrophoresis to obtain a final 135 kD fraction. The enrichment of the bumetanide-binding protein in this final 135 kD fraction estimated from [¹⁴C]-N-ethylmaleimide labeling was approximately 48 times relative to the starting membrane extract. Since the bumetanide-binding site represents approximately 2% of the total protein in this starting extract, this enrichment indicates a high degree of purity of this protein in the 135 kD fraction.     

麦积山石窟赋存环境中空气细菌的时空分布特征

【目的】空气微生物沉降及污染与文化遗产的微生物退化密切相关,本文对世界文化遗产地麦积山石窟赋存环境空气中细菌浓度和群落结构的季节性变化特征进行了系统研究,为石窟环境监测预警和文物预防性保护提供依据。【方法】利用生物气溶胶采样器,在2016年春、夏、秋和冬季分别采集空气样品;基于传统培养方法获得空气中细菌浓度及纯培养菌株;通过提取基因组DNA、扩增细菌16S rRNA、测序和系统发生树等分子技术研究细菌群落时空动态变化规律;结合环境监测数据,分析影响遗产地空气细菌变化的主要因素。【结果】监测期内,空气细菌浓度在(281.20–1409.20)CFU/m3之间,最高浓度出现在MJ4处的夏季,最低浓度出现在MJO处的春季;具有明显季节性变化特征,在空间层位分布上有所差异,但不显著(P0.05)。培养的细菌菌株经鉴定属于4个门11个属;芽孢杆菌属(Bacillus)、Paenarthrobacter、节杆菌属(Arthrobacter)、薄层菌属(Hymenobacter)和考克氏菌属(Kocuria)等为优势属。【结论】麦积山石窟空气细菌群落结构具有明显的季节性和空间分布动态变化特征;在石窟不同层位,空气中细菌群落分布与相对湿度、温度与降雨量相关;部分细菌种属如芽孢杆菌属、微球菌属(Micrococcus),为壁画及彩塑生物腐蚀的潜在病害菌;麦积山石窟及周边环境空气细菌的监测可为石窟保护和旅游开放管理提供重要参考。     

Molecular Characterization of the Glycated Plasma Membrane Calcium Pump

We have previously demonstrated (Diabetes 39:707–711, 1990) that in vitro glycation of the red cell Ca²⁺ pump diminishes the Ca²⁺-ATPase activity of the enzyme up to 50%. Such effect is due to the reaction of glucose with lysine residues of the Ca²⁺ pump (Biochem. J. 293:369–375, 1993). The aim of this work was to determine whether the effect of glucose is due to a full inactivation of a fraction of the total population of Ca²⁺ pump, or to a partial inactivation of all the molecules. Glycation decreased the V _max for the ATPase activity leaving unaffected the apparent affinities for Ca²⁺, calmodulin or ATP. The apparent turnover was identical in both, the glycated and the native enzyme. Glycation decreased the V _max for the ATP-dependent but not for the calmodulin-activated phosphatase activities. Concomitantly with the inhibition, up to 6.5% of the lysine residues were randomly glycated. The probabilistic analysis of the relation between the enzyme activity and the fraction of nonmodified residues indicates that only one Lys residue is responsible for the inhibition. We suggest that glucose decreases the Ca²⁺-ATPase activity by reacting with one essential Lys residue probably located in the vicinity of the catalytic site, which results in the full inactivation of the enzyme. Thus, Ca²⁺-ATPase activity measured in erythrocyte membranes or purified enzyme preparations preincubated with glucose depends on the remaining enzyme molecules in which the essential Lys residue stays unglycated. Received: 9 March 1999/Revised: 11 May 1999     

Modeling Study on the Cleavage Step of the Self-Splicing Reaction in Group I Introns

Abstract

A three-dimensional model of the Tetrahymena thermophila group I intron is used to further explore the catalytic mechanism of the transphosphorylation reaction of the cleavage step. Based on the coordinates of the catalytic core model proposed by Michel and Westhof (Michel, F., Westhof, E.J. Mol. Biol. 216, 585–610 (1990)), we first converted their ligation step model into a model of the cleavage step by the substitution of several bases and the removal of helix P9. Next an attempt to place a trigonal bipyramidal transition state model in the active site revealed that this modified model for the cleavage step could not accommodate the transition state due to insufficient space. A lowering of PI helix relative to surrounding helices provided the additional space required. Simultaneously, it provided a better starting geometry to model the molecular contacts proposed by Pyle et al. (Pyle, A M., Murphy, F. L., Cech, T. R. Nature 358, 123–128. (1992)), based on mutational studies involving the J8/7 segment Two hydrated Mg²⁺ complexes were placed in the active site of the ribozyme model, using the crystal structure of the functionally similar Klenow fragment (Beese, L.S., Steitz, T.A. EMBO J. 10, 25–33 (1991)) as a guide. Thepresence of two metal ions in the active site of the intron differs from previous models, which incorporate one metal ion in the catalytic site to fulfill the postulated roles of Mg²⁺ in catalysis. The reaction profile is simulated based on a trigonal bipyramidal transition state, and the role of the hydrated Mg²⁺ complexes in catalysis is further explored using molecular orbital calculations.     

Kinetic studies on the reactions of Fusarium galactose oxidase with five different substrates in the presence of dioxygen

 Reactions (25  °C) of galactose oxidase, GOase_ox from Fusarium NRRL 2903 with five different primary-alcohol-containing substrates RCH₂OH:- D-galactose (I) and 2-deoxy-d-galactose (II) (monosaccharides); methyl-β-d-galactopyranoside (III) (glycoside);d-raffinose (IV) (trisaccharide); and dihydroxyacetone (V) have been studied in the presence of O₂. The GOase_ox state has a tyrosyl radical coordinated at a square-pyramidal Cu^II active site, and is a two-equivalent oxidant. Reactant concentrations were [GOase_ox] (0.8–10 μM), RCH₂OH (1.0–6.0 mM), and O₂ (0.14–0.29 mM), with I=0.100 M (NaCl). The reactions, monitored at 450 nm by stopped-flow spectrophotometry, terminated with depletion of the O₂. Each trace was fitted to the competing reactions GOase_ox+RCH₂ OH → GOase_redH₂+RCHO (k ₁), and GOase_redH₂+O₂→ GOase_ox+H₂O₂ (k ₂), with GOase_redH₂ written as the doubly protonated two-electron-reduced Cu^I product. It was necessary to avoid auto-redox interconversion of GOase_ox and GOase_semi . Information obtained at pH 7.5 indicates a 5 : 95 (ox : semi) "native" mix equilibration complete in ∼3 h. At pH >7.5, rate constants 10^–4 k ₁ / M^–1 s^–1 for the reactions of GOase_ox with (I) (1.19), (II) (1.07), (III) (1.29), (IV) (1.81), (V) (2.94) were determined. On decreasing the pH to 5.5, k ₁ values decreased by factors of up to a half, and acid dissociation pK _as in the range 6.6–6.9 were obtained. UV-Vis spectrophotometric studies on GOase_ox gave an independently determined pK _a of 6.7. No corresponding reactions of the Tyr495Phe variant were observed, and there are no similar UV-Vis absorbance changes for this variant. The pK _a is therefore assigned to protonation of Tyr-495 which is a ligand to the Cu. The rate constant k ₂ (1.01×10⁷ M^–1 s^–1) is independent of pH in the range 5.5–9.0 investigated, suggesting that H⁺ (or H-atoms) for the O₂ → H₂O₂ change are provided by the active site of GOase_red . The Cu^I of GOase_red is less extensively complexed, and a coordination number of three is likely. Received: 4 February 1997 / Accepted: 16 May 1997     

基于双层BiGRU网络的哺乳动物组织m6A甲基化位点预测

目的 N⁶-甲基化腺苷（N⁶-methyladenosine，m⁶A）是RNA中最常见、最丰富的化学修饰，在很多生物过程中发挥着重要作用。目前已经发展了一些预测m⁶A甲基化位点的计算方法。然而，这些方法在针对不同物种或不同组织时，缺乏稳健性。为了提升对不同组织中m⁶A甲基化位点预测的稳健性，本文提出一种能结合序列反向信息来提取数据更高级特征的双层双向门控循环单元（bidirectional gated recurrent unit，BiGRU）网络模型。方法 本文选取具有代表性的哺乳动物组织m⁶A甲基化位点数据集作为训练数据，通过对模型网络、网络结构、层数和优化器等进行搭配，构建双层BiGRU网络。结果 将模型应用于人类、小鼠和大鼠共11个组织的m⁶A甲基化位点预测上，并与其他方法在这11个组织上的预测能力进行了全面的比较。结果表明，本文构建的模型平均预测接受者操作特征曲线下面积（area under the receiver operating characteristic curve，AUC）达到93.72%，与目前最好的预测方法持平，而预测准确率（accuracy，ACC）、敏感性（sensitivity，SN）、特异性（specificity，SP）和马修斯相关系数（Matthews correlation coefficient，MCC）分别为90.07%、90.30%、89.84%和80.17%，均高于目前的m⁶A甲基化位点预测方法。结论 和已有研究方法相比，本文方法对11个哺乳动物组织的m⁶A甲基化位点的预测准确性均达到最高，说明本文方法具有较好的泛化能力。     

Disulfide Linkages and a Three-Dimensional Structure Model of the Extracellular Ligand-binding Domain of Guanylyl Cyclase C

Guanylyl cyclase C (GC-C) is a single-transmembrane receptor that is specifically activated by endogenous ligands, including guanylin, and the exogenous ligand, heat-stable enterotoxin. Using combined HPLC separation and MS analysis techniques the positions of the disulfide linkages in the extracellular ligand-binding domain (ECD) of GC-C were determined to be between Cys⁷–Cys⁹⁴, Cys⁷²–Cys⁷⁷, Cys¹⁰¹–Cys¹²⁸ and Cys¹⁷⁹–Cys²²⁶. Furthermore, a three-dimensional structural model of the ECD was constructed by homology modeling, using the structure of the ECD of GC-A as a template (van den Akker et al., 2000, Nature, 406: 101–104) and the information of the disulfide linkages. Although the GC-C model was similar to the known structure of GC-A, importantly its ligand-binding site appears to be located on the quite different region from that in GC-A.     

Antioxidative Activities of Galloyl Glucopyranosides from the Stem-Bark of Juglans mandshurica

Two phenolics, 1,2,6-trigalloylglucose (1) and 1,2,3,6-tetragalloylglucose (2), isolated from the stem-bark of Juglans mandshurica were evaluated for their antioxidative activities. The results showed that compounds 1 and 2 exhibited strong scavenging activities against 1,1′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis-(3-ethylbenzenthiazoline-6-sulphonic) acid (ABTS^?+), and superoxide radicals (O₂ ^??), and also had a significant inhibitory effect on lipid peroxidation and low-density lipoprotein (LDL) oxidation. The strong superoxide radical scavenging of 1 and 2 resulted from the potential competitive inhibition with xanthine at the active site of xanthine oxidase (OX). In addition, compounds 1 and 2 displayed significant lipoxygenase inhibitory activity, the mode of inhibition also being identified as competitive. In comparison, the antioxidative activities of compounds 1 and 2, together with gallic acid, indicated that the number of galloyl moieties could play an important role in the antioxidative activity.     

Inhibition of Acetylcholinesterase by three New Pyridinium Compounds and their Effect on Phosphonylation of the Enzyme

Abstract

Three new mono-pyridinium compounds were prepared: 1-phenacyl-2-methylpyridinium chloride (1), 1-benzoylethylpyridinium chloride (2) and 1-benzoylethylpyridinium-4-aldoxime chloride (3) and assayed in vitro for their inhibitory effect on human blood acetylcholinesterase (EC 3.1.1.7, AChE). All the three compounds inhibited AChE reversibly; their binding affinity for the enzyme was compared with their protective effect (PI) on AChE phosphonylation by soman and VX. Compound 1 was found to bind to both the catalytic and the allosteric (substrate inhibition) sites of the enzyme with estimated dissociation constants of 6.9 μM (K_cat) and 27 μM (K_all), respectively. Compound 2 bound to the catalytic site with K_cat= 59 μM and compound 3 only to the allosteric site with K_all = 328 μM. PI was evaluated from phosphonylation measured in the absence and in presence of the compounds applied in a concentration corresponding to their K_cat or K_all value, and was also calculated from theoretical equations deduced from the reversible inhibition of the enzyme. Compounds 1 and 3 protected the enzyme from phosphonylation by soman and VX, whereas no protection was observed in the presence of compound 2 under the same conditions. Irrespective of the binding sites to AChE, PI for compounds 1 and 3 evaluated from phosphonylation agreed with PI calculated from reversible inhibition. Compound 3 was found to be a weak reactivator of methylphosphonylated AChE with k_r = 1.1 × 10²Lmol^-1 min^-1.     

Midvale Community Lead Study

Abstract

The primary objective of this study was to ascertain whether children living in close proximity to mill tailings and a former lead smelter site were currently exhibiting elevated blood lead (PbB) concentrations. To address this issue, the mean PbB for community children and the relationship between PbB and the proximity of the child's residence to the site was estimated. A secondary objective was to identify and quantify accessible lead (Pb) and arsenic (As) in the environment (e.g. Pb in soil, dust, paint and water or As in soil and dust). A third objective was to test for association between specific sources of environmental Pb and PbB and to estimate the relative contribution of these proximate sources of lead to the children's PbB. The data analytic methods allowed estimation of both direct and indirect impact of environmentally accessible Pb. The average PbB level of all children screened in Midvale was 5.2 μg dL^?1. Three percent exceeded 15 μg dL^?1; 12.7% exceeded 10 μg dL^?1. Pb-based house paint and Pb contaminated soil were identified as principal contributors to PbB. PbB was found to increase 1.25 μg dL^?1 per 1,000 ppm increase in lead in soil. Proximity of residence to the mill and smelter site was found to be a strong predictor of Pb in soil, and therefore indirectly related to increases in PbB.     

Kinetics of the course of reactivation of aminoacylase reconstituted using Mn2+ ions

The kinetic theory of the substrate reaction during irreversible change of enzyme activity previously described by Tsou (Tsou (1988),Adv. Enzymol. Relat. Areas Mol. Biol.61, 381–436] has been applied to a study of the kinetics of the course of reactivation during reconstitution of apo-aminoacylase using Mn²⁺ or Zn²⁺. The kinetic parameters for Mn²⁺-and Zn²⁺-reconstituted enzymes and the microscopic rate constants for reactivation during reconstitution were determined. The kinetic analysis suggests the presence of a second Mn²⁺ binding site in Mn²⁺-reconstituted aminoacylase.     

Polyphasic analysis in the description of Sulfitobacter salinus sp. nov., a marine alphaproteobacterium isolated from seawater

Purpose

A polyphasic analysis was performed on a novel bacterium, designated strain KMU-143^T, which was isolated from seawater collected in the Republic of Korea.

Methods

A novel marine bacterium KMU-143^T was analyzed and described using a polyphasic taxonomic method including 16S rRNA gene sequence analysis, DNA–DNA hybridization, and physiological, biochemical, and chemotaxonomic analyses.

Results

Strain KMU-143^T was Gram-stain-negative, strictly aerobic, oval-shaped, non-motile, and chemoorganoheterotrophic. Phylogenetic analysis based on the 16S rRNA gene sequence demonstrated that the novel marine bacterium belongs to the family Rhodobacteraceae, of the class Alphaproteobacteria, and that it possessed the highest (97.1%) sequence similarity with Sulfitobacter pontiacus ChLG 10^T and Sulfitobacter undariae W-BA2^T. DNA–DNA relatedness values between strains KMU-143^T, S. pontiacus JCM 21789^T, and S. undariae KCTC 42200^T were less than 70%. The major isoprenoid quinone of the novel isolate was ubiquinone-10 (Q-10) and the major (> 10%) cellular fatty acids were C16:0 and C18:1 ω7c. The genomic DNA G+C content of strain KMU-143^T was 56.1 mol%. The polar lipid profile of the strain KMU-143^T was found to consist of phosphatidylglycerol, diphosphatidylglycerol, phosphatidylcholine, an unidentified aminolipid, and two unidentified lipids.

Conclusion

Based on the discriminative phylogenetic position and combination of genotypic and phenotypic properties, the strain is considered to represent a new species of the genus Sulfitobacter for which the name Sulfitobacter salinus sp. nov. is proposed. The type strain of S. salinus sp. nov. is KMU-143^T (= KCCM 90322^T = NBRC 113459^T).

     

Effects of vasopressin and aldosterone on the lateral mobility of epithelial Na+ channels in A6 renal epithelial cells

We have previously demonstrated that apical Na⁺ channels in A6 renal epithelial cells are associated with spectrin-based membrane cytoskeleton proteins and that the lateral mobility of these channels, as determined by fluorescence photobleach recovery (FPR) analysis, is severely restricted by this association (Smith et al., 1991. Proc. Natl. Acad. Sci. USA 88:6971–6975). Recent data indicate that the actin component of the cytoskeleton may play a role in modulating Na⁺ channel activity (Cantiello et al., 1991. Am. J. Physiol. 261:C882–C888); however, it is unknown if the Na⁺ channel's linkage to the spectrin-based membrane cytoskeleton is also involved in regulating channel activity. In this study, we have used FPR to examine if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for modulation of Na⁺ channel activity in filter grown A6 cells by vasopressin and aldosterone. We hypothesized that if the linkage of the Na⁺ channels to the membrane cytoskeleton is a site for regulation of Na⁺ channel activity by vasopressin and aldosterone, then hormone-mediated changes in either the membrane cytoskeleton or the affinity of the Na⁺ channel for the membrane cytoskeleton, should be reflected in changes in the lateral mobility and/or mobile fraction of Na⁺ channels on the cell surface. FPR revealed that although the rates of lateral mobility were not affected, there was a twofold increase in mobility fraction (f) of apical Na⁺ channels in aldosterone-treated (16 hr) monolayers (f = 32.31 ± 5.42%) when compared to control (unstimulated) (f = 14.2 ± 0.77%) and vasopressin-treated (20 min) (f = 12.7 ± 2.4%) monolayers. The twofold increase in mobile fraction of Na⁺ channels corresponds to the average increase in Na⁺ transport in response to aldosterone in A6 cells. The aldosterone-induced increase in Na⁺ transport and mobile fraction can be inhibited by the methylation inhibitor, 3-deazaadenosine, consistent with the hypothesis that a methylation event is involved in aldosterone induced upregulation of Na⁺ transport. We propose that the membrane cytoskeleton is involved in the aldosterone-mediated activation of epithelial Na⁺ channels.Supported by NIH grants DK37206 (DJB), NS26733 and NS28072 (KJA), DK46705 (PRS) and AHA New York Affiliate grant 91007G (LCS).     

Two-step concerted mechanism for alkane hydroxylation on the ferryl active site of methane monooxygenase

 A two-step concerted mechanism for the conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO) is discussed. We propose that the enzymatic reaction mechanism is essentially the same as that of the gas-phase methane-methanol conversion by the bare FeO⁺ complex. In the initial stage of our mechanism, the ferryl (Fe—O) "iron" active site of intermediate Q and substrate methane come into contact to form the initial Q (CH₄) complex with an OFe—CH₄ bond. The C—H bonds of methane are significantly weakened by the formation of a five-coordinate carbon species, through orbital interactions between a C _3v - or D _2d -distorted methane and the Fe—O active site. The important transition state for an H atom abstraction exhibits a four-centered structure. The generated intermediate involves an HO—Fe—CH₃ moiety, and it is then converted into the final product complex including methanol as a ligand through a methyl migration that occurs via a three-centered transition state. The two-step concerted mechanism is consistent with recent experiments on regioselectivity of enzyme-catalyzed alkane hydroxylations. Received: 15 September 1997 / Accepted: 20 December 1997     

Multi-spectroscopic and voltammetric evidences for binding,conformational changes of bovine serum albumin with thiamine

The interaction between thiamine hydrochloride (TA) and bovine serum albumin (BSA) was investigated by fluorescence, FTIR, UV–vis spectroscopic and cyclic voltammetric techniques under optimised physiological condition. The fluorescence intensity of BSA is gradually decreased upon addition of TA due to the formation of a BSA–TA complex. The binding parameters were evaluated and their behaviour at different temperatures was analysed. The quenching constants (K_sv) obtained were 2.6 × 10⁴, 2.2 × 10⁴ and 2.0 × 10⁴ L mol^?1 at 288, 298 and 308 K, respectively. The binding mechanism was static-type quenching. The values of ΔH° and ΔS° were found to be 26.87 kJ mol^?1 and 21.3 J K^?1 mol^?1, and indicated that electrostatic interaction was the principal intermolecular force. The changes in the secondary structure of BSA upon interaction with TA were confirmed by synchronous and 3-D spectral results. Site probe studies reveal that TA is located in site I of BSA. The effects of some common metal ions on binding of BSA–TA complex were also investigated.