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I would like to congratulate the Journal of Biochemistry (JB) on its 100th anniversary. I served as the Editor-in-Chief for four years starting in 2010 and succeeded Dr Naoyuki Taniguchi. [ABSTRACT FROM AUTHOR]

I J. Biochem. i 2021. doi:10.1093/jb/mvz016 This is a retraction to: K F Houslay, B A Fertig, F Christian, A J Tibbo, J Ling, J E Findlay, M D Houslay, G S Baillie. Phosphorylation of PDE4A5 by MAPKAPK2 attenuates fibrin degradation via p75 signalling, The Journal of Biochemistry, Volume 166, Issue 1, July 2019, Pages 97-106, https://doi.org/10.1093/jb/mvz016 The corresponding author is retracting this article following an institutional investigation into the authenticity of the figures in the paper. [Extracted from the article]

Isothermal and Chimeric primer-initiated Amplification of Nucleic acids (ICAN) allows the amplification of target DNA under isothermal conditions at around 55°C using only a pair of 5′-DNA-RNA-3′ chimeric primers, thermostable RNaseH and a DNA polymerase with strand-displacing activity (H. Mukai et al. J. Biochemistry, in the preceding paper in this issue). Here we elucidated the mechanism of ICAN by analysing the nicking site of RNaseH, behaviour of chimeric primers and extension products. We found that the ICAN reaction was composed of two unique mechanisms, multi-priming and template-switching, that were responsible for the highly efficient amplifying capability of ICAN. The simultaneous occurrence of two types of reactions, one based on multi-priming and the other based on template-switching, is likely to drive the DNA amplification in ICAN. [ABSTRACT FROM PUBLISHER]

Tamio Yamakawa isolated a glycosphingolipid from horse erythrocyte membrane, named it hematoside, and reported the results in Journal of Biochemistry. This was the first paper to report that glycosphingolipids are located in the cell membrane. He also isolated a glycosphingolipid, globoside, from human erythrocytes and demonstrated for the first time that ABO blood group antigens are glycosphingolipids in the erythrocytes. He reported the correct chemical structure of sulfatide, and found seminolipid, which is unexpectedly a glyceroglycolipid, as the major glycolipid of mammalian testis and spermatozoa. He started and developed the research of glycolipid biochemistry and established the basis for the further development of glycobiology. He published most of his original work in Journal of Biochemistry and made great efforts to improve review process of the journal as an editor-in-chief. [ABSTRACT FROM PUBLISHER]

Lectin-based structural glycomics requires a search for useful lectins and their biochemical characterization to profile complex features of glycans. In this paper, two GlcNAc-binding lectins are reported with their detailed oligosaccharide specificity. One is a classic plant lectin, Griffonia simplicifolia lectin-II (GSL-II), and the other is a novel fungal lectin, Boletopsis leucomelas lectin (BLL). Their sugar-binding specificity was analyzed by frontal affinity chromatography using 146 glycans (125 pyridylaminated and 21 p-nitrophenyl saccharides). As a result, it was found that both GSL-II and BLL showed significant affinity toward complex-type N-glycans, which are either partially or completely agalactosylated. However, their branch-specific features differed significantly: GSL-II strongly bound to agalacto-type, tri- or tetra-antennary N-glycans with its primary recognition of a GlcNAc residue transferred by GlcNAc-transferase IV, while BLL preferred N-glycans with fewer branches. In fact, the presence of a GlcNAc residue transferred by GlcNAc-transferase V abolishes the binding of BLL. Thus, GSL-II and BLL forms a pair of complementally probes to profile a series of agalacto-type N-glycans. [ABSTRACT FROM PUBLISHER]

This paper describes a new HPLC method for the sugar analysis of algal galactans containing 3,6-anhydrogalactose, which is readily destroyed during usual acid hydrolysis and methanolysis. By anhydrous mercaptolysis of galactans at 60 degreesC for 6 h with the newly developed solvent system, 0.5 N HCl/[ethanethiol:methanol (2:1, v/v)], component sugars, including 3,6-anhydrogalactose, were liberated quantitatively as their diethyl dithioacetals. The resultant sugar diethyl dithioacetals were found to have strong UV-absorption (absorption maximum of 3,6-anhydrogalactose diethyl dithioacetal in water, 191-192 nm; molar extinction coefficient, 4,400). The sugar diethyl dithioacetals were then resolved simultaneously within 15 min by reversed phase HPLC with 30% acetonitrile as the eluent and detected by UV-absorption at 215 nm. Amounts of sugar diethyl dithioacetals less than 50 pmol can be determined without further derivatization.

N-Acetylgalactosamine 4-sulfotransferase (GalNAc4ST) transfers sulfate to position 4 of nonreducing terminal GalNAc residues. We previously cloned human GalNAc4ST-1 cDNA. In this paper, we report the cloning, characterization and chromosomal mapping of mouse GalNAc4ST-1 and GalNAc4ST-2. Mouse GalNAc4ST-1 and GalNAc4ST-2 contain single open reading frames that predict type II transmembrane proteins composed of 417 and 413 amino acid residues, respectively. The amino acid sequence identity between the two isoforms is 49%. When the cDNA was transfected to COS-7 cells, sulfotransferase activities toward carbonic anhydrase VI and GalNAcβ1-4GlcNAcβ1-ctyl were overexpressed, but the sulfotransferase activity toward chondroitin showed no increase over the control level. Northern blot analysis showed that the 2.4 kb messages of GalNAc4ST-1 and GalNAc4ST-2 were strongly expressed in the kidney, where both of the human isoforms were hardly expressed. Reverse transcription-PCR analysis showed that, unlike human GalNAc4ST-1, the expression of mouse GalNAc4ST-1 in the pituitary gland was only marginal, while that of GalNAc4ST-2 in the pituitary gland was as high as that in the kidney. These results suggest that the functions of the two GalNAc4ST isoforms may differ between human and mouse. By fluorescence in situ hybridization, the GalNAc4ST-1 and GalNAc4ST-2 genes were localized to mouse chromosome 7B3 distal--B5 proximal and chromosome 18A2 distal--1 proximal, respectively. [ABSTRACT FROM AUTHOR]

Protein Data Bank (PDB), jointly founded in 1971 by Brookhaven National Laboratory, USA, and the Cambridge Crystallographic Data Centre, UK, is the single global archive of experimentally determined biological macromolecular structures. PDB deposition is mandatory for publication in most scientific journals, which means 'no PDB deposition, no structural publication'. The current PDB archive contains more than 180,000 entries and includes many structures from Asian institutions. The first protein structure from Japan was that of cytochrome c determined by Prof Masao Kakudo's group at the Institute for Protein Research, Osaka University, in 1971 at a resolution of 4 Å, and a subsequent atomic structure at 2.3 Å resolution was deposited to PDB in 1976 as the 1st Asian and 21st entry of the entire PDB archive. Since then, 317 protein structures whose primary citation was the Journal of Biochemistry (J. Biochem.) have been deposited to PDB. Based on this long history between PDB and J. Biochem. a statistical analysis of all structural reports in J. Biochem. has been carried out using the relational database system at PDBj (https://pdbj.org) and reviewed the yearly distribution, resolution, quality of structure, type of target protein, number of citations and comparison against other major journals. [ABSTRACT FROM AUTHOR]

In this review, we present an overview of the recent advances in chemical toolboxes that are used to provide insights into 'live' protein functions in living systems. Protein functions are mediated by various factors inside of cells, such as protein−protein interactions, posttranslational modifications, and they are also subject to environmental factors such as pH, redox states and crowding conditions. Obtaining a true understanding of protein functions in living systems is therefore a considerably difficult task. Recent advances in research tools have allowed us to consider 'live' biochemistry as a valid approach to precisely understand how proteins function in a live cell context. [ABSTRACT FROM AUTHOR]

GTPase domains from members of the HflX protein family have their catalytic glutamine residue of the DxxGQ motif substituted by phenylalanine, while they are still able to hydrolyse GTP. This appears to challenge the traditional view of GTP hydrolysis mechanism of Ras-like GTPases. SsGBP from the hyperthermophilic archaeon Sulfolobus solfataricus provided the first crystal structure of the HflX family. Here, we report structure-based mutagenesis analyses on SsGBP. Six-point mutations were individually introduced in the Ras-like GTPase domain including regions of P-loop, switches I and II. Intrinsic GTPase activities and thermal stabilities of these variants together with the wild-type full-length SsGBP and its isolated GTPase domain were analysed. Both functional and structural analyses of G235P and G235S mutants, which showed total and partial loss of the GTP hydrolyzing activity, respectively, support our hypothesis that the role of aligning a nucleophilic water molecule by the Ras Gln60 residue is replaced by the backbone amide group of Gly235 in SsGBP. Together with functional studies of other mutants, we conclude that the classical view of GTP hydrolysis mechanism likely remains the same in the HflX family with a twist in the entity of the nucleophilic alignment. [ABSTRACT FROM PUBLISHER]

Histamine dehydrogenase from Nocardioides simplex (HmDH) which catalyzes the oxidative deamination of histamine is an iron–sulphur–containing flavoprotein. For our further understanding on the intramolecular electron transfer process, the reductive half reaction of HmDH with histamine has been studied by stopped flow spectrophotometry at pH 7.5 and 10. The reaction at pH 7.5 is found to be analysed on a kinetic model composed of three sequential first-order reactions. The first fast phase, of which the rate constant shows a hyperbolic dependence on the histamine concentration, is assigned to a direct two-electron reduction of the oxidized flavin (CFMNO) by histamine with no involvement of the semiquinone form of the flavin (CFMNS). The second moderate process is the substrate-independent intramolecular single-electron transfer from the reduced flavin to the oxidized iron–sulphur cluster. The third slow process is considered to reflect the second binding of histamine to CFMNS, which is responsible for the substrate inhibition. At pH 10, the reaction is analysed with one pseudo-first-order reaction phase which is substrate-dependent two-electron reduction of CFMNO coupled with the subsequent fast intersubunit single-electron transfer. The UV–vis spectroscopy of HmDH suggests the deprotonation of Tyr residues, which seems to cause the switching of the electron transfer property. [ABSTRACT FROM PUBLISHER]

We have been interested in partially folded proteins with marginal stability and activity, because they have a potential to be mature proteins by artificial evolution. A module is defined as a contiguous peptide chain forming a compact region in a globular protein. Modules may be used as building blocks to create partially folded proteins. Barnase, a ribonuclease consisting of 110 amino acids, has been divided into six modules (M1–M6), four peptide fragments, M12 (1–52), M123 (1–73), M1234 (1–88) and M12345 (1–98), have been constructed by progressive elongation of the modules from the N-terminus. Only M12345 (1–98) had a partially folded conformation, but it lacked detectable RNase activity. A mixture of M12345 (1–98) with M56 (89–110) showed weak but distinct RNase activity. Unfolded M12345 (1–96) was constructed by removal of two residues from the C-terminus of M12345 (1–98). The mixture of M12345 (1–96) with M56 (89–110) also showed RNase activity. Further, the interaction endowed M12345 (1–96) with conformational stability. We propose that N- and C-terminal fragments obtained by successive elongation of modules would interact to be a complex with marginal stability and activity, which would be used for creating a mature complex by artificial evolution. [ABSTRACT FROM PUBLISHER]

Using an organelle proteomics approach, we previously studied the rat peroxisome in order to characterize the proteins participating in its biogenesis. A peroxisome-specific isoform of Lon (pLon) protein was accordingly identified. However, the precise role of pLon in peroxisomes remains to be elucidated. Here, we demonstrate that pLon plays a role in processing and activating a specific regulatory protein belonging to the peroxisome targeting signal (PTS) 1-containing proteins. Proteomic analysis of proteins co-immunoprecipitated with Lon suggested that Lon interacts with PMP70 and several enzymes involved in β-oxidation, including acyl-CoA oxidase (AOX). The processing of AOX for its activation in peroxisomes was strongly inhibited in cells expressing a dominant negative form of pLon. Furthermore, a catalase possessing a modified PTS1 sequence was misdistributed in this cell line. pLon exhibits little, if any, in vitro AOX processing activity, and does not process PTS2-containing 3-ketoacyl-coenzyme A thiolase (PTL). Therefore, pLon may specifically control, sort and process PTS1 proteins. Based on the relationship between pLon and the β-oxidation enzymes that regulate peroxisomal morphology, the observation of enlarged peroxisomes in cells expressing recombinant pLon suggests that pLon is a critical factor determining peroxisome morphology. [ABSTRACT FROM PUBLISHER]

The cDNA for an isotype of clytin, a calcium-binding photoprotein from the luminous jellyfish Clytia gregarium, was identified and named clytin-II. The histidine-tagged apoprotein of clytin-II expressed into the periplasmic space of Escherichia coli cells was isolated by nickel chelate affinity chromatography. Recombinant clytin-II regenerated from apoprotein by incubation with coelenterazine was purified. The yield of purified clytin-II was 13 mg from 2 l of cultured cells with purity >95%. The luminescence properties of clytin-II were characterized by comparison with clytin-I and aequorin, and semi-synthetic clytin-II was also prepared. The initial luminescence intensity of clytin-II triggered by Ca2+ was 4.5 times higher than that of clytin-I and aequorin, but the luminescence capacity was close to clytin-I and aequorin. Thus, clytin-II is a useful protein, showing high sensitivity in the signal-to-noise ratio of luminescence intensity. [ABSTRACT FROM PUBLISHER]

Recently, the existence of a ‘histone code’ has been proposed to explain the link between the covalent chemical modification of histone proteins and the epigenetic regulation of gene activity. Although the role of the four ‘core’ histones has been extensively studied, little is known about the involvement of the linker histone, histone H1 and its variants, in this code. For many years, few sites of chemical modification had been mapped in linker histones, but this has changed recently with the use of functional proteomic techniques, principally mass spectrometry, to characterize these modifications. The functionality of many of these sites, however, remains to be determined. [ABSTRACT FROM PUBLISHER]

A novel bacterium, Photobacterium sp. JT-ISH-224, that produces α-/β-galactoside α2,3-sialyltransferase and β-galactoside α2,6-sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding α-/β-galactoside α2,3-sialyltransferase from P. phosphoreum and β-galactoside α2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an α2,3-sialyltransferase and an α2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The α2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum α2,3-sialyltransferase, whereas the α2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae α2,6-sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The α2,3-sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, α-methyl-galactopyranoside and β-methyl-galactopyranoside with low apparent Km and the α2,6-sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent Km. [ABSTRACT FROM PUBLISHER]

Drosophila calpains, Calpain A and Calpain B, show typical calpain domain structures similar to mammalian calpains. However, the small subunit of mammalian calpains, shown to be essential in both genetic and biochemical aspects, is absent in Drosophila calpains and is not required for enzymatic activity. How they compensate for the lack of small subunit is mostly unknown. Here we conducted experiments using recombinant Drosophila Calpain B for further characterization of the enzyme with particular focuses on two issues: possibility of homodimerization and mode of autolysis. The native molecular weight of Calpain B indicates that the active enzyme is primarily monomeric. Co-expression of two recombinant Calpain B proteins each with a unique affinity tag and a subsequent single round of affinity tag purification resulted in isolation of only one recombinant calpain type, suggesting there is no homodimeric interaction. Also the C-termini of Drosophila calpains lack many of the key hydrophobic residues considered to be important in the dimerization of mammalian calpains. Further, initial autolysis of Calpain B seems to occur intramolecularly, which supports the monomeric nature of Drosophila calpains. These results strongly suggest that dimerization is not an essential requirement for Drosophila calpains. [ABSTRACT FROM PUBLISHER]

Three α-elicitins, named hibernalin1, hibernalin2 and hibernalin3 (hib1, hib2 and hib3, respectively), were isolated by reverse phase-low-pressure liquid chromatography from culture filtrates of Phytophthora hibernalis Carne 1925, the causal agent of citrus lemon brown rot. Hib1 proved to be identical to syringicin previously isolated from culture filtrates of Phytophthora syringae. Hib2 and hib3 shared the same primary structure with hib1, but contained, at position 50, Met sulphoxide or sulphone, respectively. By SDS–PAGE, the three proteins showed the same electrophoretic mobility, corresponding to about 10 kDa. Exact Mr values were obtained by MALDI-TOF-MS (10,194.82 for hib1, 10,209.33 for hib2 and 10,223.80 for hib3), while by ESI-MS an Mr value of 10,194.90 was found for hib1 and no results for hib2 and hib3. The hibernalin forms showed a high propensity to self-association, after exposure to acetonitrile. Hib1 showed to be active in both the hypersensitivity response and electrolytes leakage assays; the sample containing hib1 and hib2 was only weakly active in the first assay and inactive in the second assay, while the sample containing all three hibernalin forms proved to be inactive in both tests. It is proposed that the different activities of the three hibernalin samples could be very likely attributed to both Met50 oxidation and aggregation. [ABSTRACT FROM PUBLISHER]

Cystathionine γ-synthase (CGS) catalyses the first step of the transsulfuration pathway that converts l-cysteine to l-homocysteine in bacteria, whereas this pathway is absent in human. In this report, we identified a new metB gene from Helicobacter pylori strain SS1, and the recombinant H. pylori Cystathionine γ-synthase (HpCGS) was successfully cloned, expressed and purified in Escherichia coli system. Enzymatic study of HpCGS indicated that the Km and kcat/Km values against the substrate O-succinyl-l-homoserine (l-OSHS) were 3.02 mM and 98.7 M−1s−1, respectively. Moreover, four natural products (α-lapachone, 9-hydroxy-α-lapachone, Paulownin and Yangambin, Fig. 1) were discovered to demonstrate inhibitory activities against HpCGS with IC50 values of 11 ± 3, 9 ± 1, 19 ± 2 and 27 ± 6 μM, respectively. All these four inhibitors prevent the binding of l-OSHS to HpCGS in a non-competitive fashion. In vitro antibacterial assays further indicated that these four discovered compounds could highly inhibit the growth of H. pylori and exhibited strong inhibitory specificity against H. pylori related to E. coli. [ABSTRACT FROM PUBLISHER]

Divalent glycosides carrying N-acetyl-d-glucosamine (GlcNAc) and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. First, hexan-1,6-diyl bis-(2-acetamido-2-deoxy-β-d-glucopyranoside) (GlcNAc-Hx-GlcNAc) and 3,6-dioxaoct-1,8-diyl bis-(2-acetamido-2-deoxy-β-d-glucopyranoside) (GlcNAc-Doo-GlcNAc) were enzymatically synthesized by transglycosylation of an N,N′N″,N′″-tetraacetylchitotetraose [(GlcNAc)4] donor with a primary diol acceptor, utilizing a chitinolytic enzyme from Amycolatopsis orientalis. The resulting divalent glycosides were further converted to the respective hexan-1,6-diyl bis-[β-d-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranoside] (LacNAc-Hx-LacNAc) and 6-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-hexyl β-d-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranoside (LacNAc-Hx-GlcNAc), and respective 3,6-dioxaoct-1,8-diyl bis-[β-d-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranoside] (LacNAc-Doo-LacNAc) and 8-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-3,6-dioxaoctyl β-d-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-d-glucopyranoside (LacNAc-Doo-GlcNAc) by galactosyltransferase. The interaction of wheat germ agglutinin (WGA) with a series of divalent glycosides and related compounds were studied using a biosensor based on surface plasmon resonance (SPR) and by precipitation analysis. Our results demonstrated that divalent glycosides carrying GlcNAc on both sides and GlcNAc and LacNAc on each side are capable of precipitating WGA as divalent ligands, but that the corresponding monovalent controls and divalent glycosides carrying LacNAc on both sides are unable to precipitate the lectin and bind as univalent ligands. [ABSTRACT FROM PUBLISHER]

Nattokinase (Subtilisin NAT, NK) is a bacterial serine protease with high fibrinolytic activity. To probe their roles on protease activity and substrate specificity, three residues of S3 site (Gly100, Ser101 and Leu126) were mutated by site-directed mutagenesis. Kinetics parameters of 20 mutants were measured using tetrapeptides as substrates, and their fibrinolytic activities were determined by fibrin plate method. Results of mutation analysis showed that Gly100 and Ser101 had reverse steric and electrostatic effects. Residues with bulky or positively charged side chains at position 100 decreased the substrate binding and catalytic activity drastically, while residues with the same characters at position 101 could obviously enhance protease and fibrinolytic activity of NK. Mutation of Leu126 might impair the structure of the active cleft and drastically decreased the activity of NK. Kinetics studies of the mutants showed that S3 residues were crucial to keep protease activity while they moderately affected substrate specificity of NK. The present study provided some original insight into the P3–S3 interaction in NK and other subtilisins, as well as showed successful protein engineering cases to improve NK as a potential therapeutic agent. [ABSTRACT FROM PUBLISHER]

Bark lectins from the elderberry plants belonging to the genus Sambucus specifically bind to Neu5Acα2,6Gal/GalNAc sequence and have long been used for the analysis of sialoglycoconjugates that play important roles in many biological phenomena. However, molecular basis of such a unique carbohydrate binding specificity has not been understood. To answer these questions, we tried to identify the amino-acid residues in the Japanese elderberry bark lectin, Sambucus sieboldiana agglutinin that enabled the lectin to recognize sialic acid by using in silico docking simulation and site-directed mutagenesis. These studies showed that three amino-acid residues, S197, A233 and Q234, in the C-terminal subdomain of SSA-B chain are critical for the binding to the sialic acid in Neu5Acα2,6Gal/GalNAc sequence. Replacement of one of these residues to the one in the corresponding position of ricin B-chain completely abolished the binding to a sialoglycoprotein, fetuin. Conserved presence of these amino acid residues in the corresponding sequences of two other elderberry lectins with similar binding specificity further supported the conclusion. These findings indicated that the replacement of the corresponding amino-acid residues in a putative Gal/GalNAc-specific ancestral lectin to these amino-acid residues generated the unique Neu5Acα2,6Gal/GalNAc-specific elderberry lectins in the course of molecular evolution. [ABSTRACT FROM PUBLISHER]

We developed an efficient method of isothermally amplifying DNA termed ICAN, Isothermal and Chimeric primer-initiated Amplification of Nucleic acids. This method allows the amplification of target DNA under isothermal conditions at around 55°C using only a pair of 5′-DNA-RNA-3′ chimeric primers, a thermostable RNaseH and a DNA polymerase with strong strand-displacing activity. ICAN is capable of amplifying DNA at least several times greater than the amount produced with PCR by increasing primer concentration. This method would be applicable for on-site DNA detection including gene diagnosis, and would also be suitable for ‘real time’ detection when combined with a cycling probe. [ABSTRACT FROM PUBLISHER]

Cellular transformation, the conversion of normal cells into tumorigenic cells in vitro, is characterized by immortalization, anchorage- and serum-independent growth and tumour formation in the nude mouse. Among these, anchorage-independent growth is one of the defining characteristics of transformed cells and tumour cells. Without attachment to the extracellular substrate, most normal cells cannot grow or survive, but tumour cells can proliferate. Many oncogenes and tumour suppressors are involved in regulating this process, among which is Abl tyrosine kinases. Previous work showed that v-Abl, an oncogenic variant of c-Abl kinase, induces anchorage-independent growth in the context of p53 deficiency, and a recent study by our group showed that loss of c-Abl kinase also facilitates anchorage-independent growth. The cellular context, such as a deficiency in both p53 and RB, is critical to induce anchorage independence by loss of c-Abl kinase. In this review, we discuss the mechanisms of cellular transformation by oncogenic and normal Abl kinases. [ABSTRACT FROM PUBLISHER]

The dinuclear μ-okso vanadium (III) complex compound H4V2OCl4(Ad)2 synthesized in our laboratory was investigated as a potential cytotoxic agent against yeast cells. The results of these studies could be helpful in the explanation of the mechanism governing the V (III) compound action on yeast as a simple model of eukaryotic cells. The important factors influencing the toxicity of this complex compound are: the stage of the yeast life cycle, the rate of growth and the pH of reaction mixture. The lethal effect was distinctly stronger when the reaction mixture was slightly acidic (pH = 4). In such solutions V(III) mononuclear species with adenine was relatively stable, and during the time of experiment possibly only a slow oxidation process to V(IV) occurred. In the solutions with pH = 7, several hydrolytic, perhaps mixed—valence, polynuclear species were present and their action on the yeast cells was rather weak. The increased lethal activity of this compound in acidic solutions may be useful in specific treatment against cancer cells whose cytoplasm and/or closest surrounding has lower pH value. The next important result was an observation that the killing activity of this compound was enhanced for yeast cells being in log phase. Also these which had a slower rate of growth (possessing some auxotrophic mutations) were more resistant than those growing faster. The extent of yeast mutagenesis caused by the complex compound is negligible, as the number of mutants found in experiments was within the limit of experimental error. These results are promising and the investigated complex can be considered as a potential anti cancer agent. [ABSTRACT FROM PUBLISHER]

d-Aspartate oxidase (DDO, EC 1.4.3.1) catalyzes dehydrogenation of d-aspartate to iminoaspartate and the subsequent re-oxidation of reduced FAD with O2 to produce hydrogen peroxide. In the mammalian neuroendocrine system, d-aspartate, a natural substrate, plays important roles in the regulation of the synthesis and secretion of hormones. To elucidate the kinetic and structural properties of native DDO, we purified DDO from porcine kidney to homogeneity, cloned the cDNA, and overexpressed the enzyme in Escherichia coli. The purified DDO was a homotetramer with tightly-bound FAD. The enzyme consisted of 341 amino acids and had GAGVMG as the dinucleotide binding motif and a C-terminal SKL peroxisomal-targeting signal sequence. Porcine DDO showed a strong affinity for meso-tartrate (Kd = 118 μM). The oxidase exhibited pronounced substrate activation at d-aspartate and d-glutamate concentrations, [S], higher than 0.2 and 4 mM, respectively, and the [S]/v versus [S] plot showed marked downward curvature (v, the initial velocity), whereas substrate inhibition occurred with N-methyl-d-aspartate. These kinetic properties of DDO suggested that at high substrate concentrations, the FAD-reduced form of the enzyme also catalyzes the reaction: the oxidative half-reaction precedes the reductive one. The present direct approach to the analysis of non-Michaelis kinetics is indispensable for understanding the functional properties of DDO. [ABSTRACT FROM PUBLISHER]

An efficient method for site-selective modification of proteins using an unnatural amino acid, 3-azidotyrosine has been developed. This method utilizes the yeast amber suppressor tRNATyr/mutated tyrosyl-tRNA synthetase pair as a carrier of 3-azidotyrosine in an Escherichia coli cell-free translation system, and triarylphosphine derivatives for specific modification of the azido group. Using rat calmodulin (CaM) as a model protein, we prepared several unnatural CaM molecules, each carrying an azidotyrosine at predetermined positions 72, 78, 80 or 100, respectively. Post-translational modification of these proteins with a conjugate compound of triarylphosphine and biotin produced site-selectively biotinylated CaM molecules. Reaction efficiency was similar among these proteins irrespective of the position of introduction, and site-specificity of biotinylation was confirmed using mass spectrometry. In addition, CBP-binding activity of the biotinylated CaMs was confirmed to be similar to that of wild-type CaM. This method is intrinsically versatile in that it should be easily applicable to introducing any other desirable compounds (e.g., probes and cross-linkers) into selected sites of proteins as far as appropriate derivative compounds of triarylphosphine could be chemically synthesized. Elucidation of molecular mechanisms of protein functions and protein-to-protein networks will be greatly facilitated by making use of these site-selectively modified proteins. [ABSTRACT FROM PUBLISHER]

We have developed two experimental methods for observing Escherichia coli RecA-DNA filament under a fluorescence microscope. First, RecA-DNA filaments were visualized by immunofluorescence staining with anti-RecA monoclonal antibody. Although the detailed filament structures below submicron scale were unable to be measured accurately due to optical resolution limit, this method has an advantage to analyse a large number of RecA-DNA filaments in a single experiment. Thus, it provides a reliable statistical distribution of the filament morphology. Moreover, not only RecA filament, but also naked DNA region was visualized separately in combination with immunofluorescence staining using anti-DNA monoclonal antibody. Second, by using cysteine derivative RecA protein, RecA-DNA filament was directly labelled by fluorescent reagent, and was able to observe directly under a fluorescence microscope with its enzymatic activity maintained. We showed that the RecA-DNA filament disassembled in the direction from 5′ to 3′ of ssDNA as dATP hydrolysis proceeded. [ABSTRACT FROM PUBLISHER]

Virus-like particles (VLPs), a promising next-generation drug delivery vehicle, can be formed in vitro using a recombinant viral capsid protein VP1 from SV40. Seventy-two VP1 pentamers interconnect to form the T = 7d lattice of SV40 capsids, through three types of C-terminal interactions, α-α′-α″, β-β′ and γ-γ. These appear to require VP1 conformational switch, which involve in particular the region from amino acids 301–312 (herein Region I). Here we show that progressive deletions from the C-terminus of VP1, up to 34 amino acids, cause size and shape variations in the resulting VLPs, including tubular formation, whereas deletions beyond 34 amino acids simply blocked VP1 self-assembly. Mutants carrying in Region I point mutations predicted to disrupt α-α′-α″-type and/or β-β′-type interactions formed small VLPs resembling T = 1 symmetry. Chimeric VP1, in which Region I of SV40 VP1 was substituted with the homologous region from VP1 of other polyomaviruses, assembled only into small VLPs. Together, our results show the importance of the integrity of VP1 C-terminal region and the specific amino acid sequences within Region I in the assembly of normal VLPs. By understanding how to alter VLP sizes and shapes contributes to the development of drug delivery systems using VLPs. [ABSTRACT FROM PUBLISHER]

The lux operon is an uncommon gene cluster. To find the pathway through which the operon has been transferred, we sequenced the operon and both flanking regions in four typical luminous species. In Vibrio cholerae NCIMB 41, a five-gene cluster, most genes of which were highly similar to orthologues present in Gram-positive bacteria, along with the lux operon, is inserted between VC1560 and VC1563, on chromosome 1. Because this entire five-gene cluster is present in Photorhabdus luminescens TT01, about 1.5 Mbp upstream of the operon, we deduced that the operon and the gene cluster were transferred from V. cholerae to an ancestor of Pr. luminescens. Because in both V. fischeri and Shewanella hanedai, luxR and luxI were found just upstream of the operon, we concluded that the operon was transferred from either species to the other. Because most of the genes flanking the operon were highly similar to orthologues present on chromosome 2 of vibrios, we speculated that the operon of most species is located on this chromosome. The undigested genomic DNAs of five luminous species were analysed by pulsed-field gel electrophoresis and Southern hybridization. In all the species except V. cholerae, the operons are located on chromosome 2. [ABSTRACT FROM PUBLISHER]

To clarify the contribution of N-terminal region of eukaryotic initiation factor 4E (eIF4E) to the interaction with 4E-BP and to investigate the effect of 4E-BP phosphorylation on the interaction with eIF4E, the interaction profiles of the Ser65-unphosphorylated and phosphorylated peptides (Thr37–Thr70 fragment of 4E-BP1) with full-length and N-terminal 33 residues–deleted eIF4Es were investigated by fluorescence and SPR methods. The effect of N-terminal region of eIF4E on the interaction with 4E-BP1 peptides was shown to be dependent on the interaction state, that is, the steady-state fluorescence and kinetic-state SRP analyses showed the positive and negative contributions of the N-terminal region to the interaction with the peptide, respectively, despite its unphosphorylated or phosphorylated state. The comparison of the association constants of the peptide with those of full-length 4E-BP1 indicated the importance of N-terminal (1–36) and/or C-terminal (71–118) sequence of 4E-BP1 for the interaction, although the MD simulations suggested that the α-helical region (Arg56–Cys62) of 4E-BP1 peptide is sufficient for keeping the interaction. The MD simulations also indicated that a charge-dependent rigid hydration shell formed around the phosphate group makes the molecular conformation rigid, and single Ser65 phosphorylation is insufficient for releasing 4E-PB1 peptide from eIF4E. [ABSTRACT FROM PUBLISHER]

Lipids in dimeric photosystem II complexes prepared from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and two higher plants, spinach and rice, were analyzed to determine how many lipid molecules and what class of lipids are present in the photosystem II complexes. It was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the dimeric photosystem II complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively. In each of the organisms, the lipid composition of the photosystem II complexes was quite different from that of the thylakoid membranes used for preparation of the complexes. The content of phosphatidylglycerol in the photosystem II complexes of each organism was much higher than that in the thylakoid membranes. Phospholipase A2 treatment of the photosystem II complexes of Synechocystis that degraded phosphatidylglycerol resulted in impairment of QB-mediated but not QA-mediated electron transport. These findings suggest that phosphatidylglycerol plays important roles in the electron transport at the QB-binding site in photosystem II complexes. [ABSTRACT FROM PUBLISHER]

Proteolytic myosin subfragment 1 (S1) is known to be partially unfolded in its 50-kDa subdomain by mild heat treatment at 35°C [Burke et al. (1987) Biochemistry 26, 1492–1496]. Here, we report that this partial unfolding is accompanied by aggregation of S1 protein. Characteristics of the aggregate thus formed were: (i) formation of transparent sediment under centrifugation at 183,000 × g; (ii) amyloid-like, dye-binding properties such as Congo red–binding and Thioflavin T fluorescence enhancement; (iii) a uniformly sized spherical appearance in electron micrographs; and (iv) sensitivity to tryptic digestion. Gel filtration analysis of the aggregation process indicates that the spheroid was formed through an intermediate oligomeric stage. The aggregate inhibited spontaneous aggregation of an isolated 50 kDa fragment into a large amorphous mass. The remaining native regions in the partially unfolded S1 were probably responsible for this effect. These results show that, unlike the 50-kDa fragment, the partially unfolded S1 molecules do not form amorphous aggregates but assemble into spherical particles. The native regions in partially unfolded S1 may be a determinant of aggregate morphology. [ABSTRACT FROM PUBLISHER]

Membrane-associated guanylate kinase inverted (MAGI)-1 plays a role as a scaffold at cell junctions in non-neuronal cells, while S-SCAM, its neuronal isoform, is involved in the organization of synapses. A search for MAGI-1–interacting proteins by yeast two-hybrid screening of a kidney cDNA library yielded dendrin. As dendrin was originally reported as a brain-specific postsynaptic protein, we tested the interaction between dendrin and S-SCAM and revealed that dendrin binds to the WW domains of S-SCAM. Dendrin is known to be dendritically translated but its function is largely unknown. To gain insights into the physiological meaning of the interaction, we performed a second yeast two-hybrid screening using dendrin as a bait. We identified CIN85, an endocytic scaffold protein, as a putative dendrin-interactor. Immunocytochemistry and subcellular fractionation analysis supported the synaptic localization of CIN85. The first SH3 domain and the C-terminal region of CIN85 bind to the proline-rich region and the N-terminal region of dendrin, respectively. In vitro experiments suggest that dendrin forms a ternary complex with CIN85 and S-SCAM and that this complex formation facilitates the recruitment of dendrin and S-SCAM to vesicle-like structures where CIN85 is accumulated. [ABSTRACT FROM PUBLISHER]

When Saccharomyces cerevisiae cells are exposed to hyper-osmotic stress, the high-osmolarity glycerol response (HOG) pathway is activated to induce osmotic responses. The HOG pathway consists of two upstream osmosensing branches, the SLN1 and SHO1 branches, and a downstream MAP kinase cascade. Although the mechanisms by which these upstream branches transmit signals to the MAP kinase cascade are well understood, the mechanisms by which they sense and respond to osmotic changes are elusive. Here we show that the HOG pathway is activated in an SLN1 branch–dependent manner when cells are exposed to cold stress (0°C treatment). Dimethyl sulfoxide (DMSO) treatment, which rigidifies the cell membrane, also activates the HOG pathway in both SLN1 branch– and SHO1 branch–dependent manners. Moreover, cold stress, as well as hyper-osmotic stress, exhibits a synergistic effect with DMSO treatment on HOG pathway activation. On the other hand, ethanol treatment, which fluidizes the cell membrane, partially represses the cold stress–induced HOG pathway activation. Our results suggest that both osmosensing branches respond to the rigidification of the cell membrane to activate the HOG pathway. [ABSTRACT FROM PUBLISHER]

The mitochondrial ADP/ATP carrier (AAC) transports substrate by interconversion of its conformation between m- and c-states. The 1st loop facing the matrix (LM1) is extruded into the matrix in the m-state and is suggested to intrude into the mitochondrial membrane on conversion to the c-state conformation [Hashimoto, M., Majima, E., Goto, S., Shinohara, Y., and Terada, H. (1999) Biochemistry 38, 1050–1056]. To elucidate the mechanism of the translocation of LM1, we examined the effects of site-directed mutagenesis of two adjoining residues, Cys56 and Asp55 in the bovine type 1 AAC and Cys73 and Asp72 in the yeast type 2 AAC, on the substrate transport activity. We found that (i) replacement of the Cys by bulky and hydrophilic residues was unfavorable for efficient transport activity, (ii) the carboxyl groups of the Asp residues of the bovine and yeast AACs were essential and strictly position-specific, and (iii) hence, the mutation to Glu showed transport activity comparable to that of the native AACs. Based on these results, we discussed the functional role of LM1 in the transport activity of AAC. [ABSTRACT FROM PUBLISHER]

d-Amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of d-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, d-serine is proposed to be a neuromodulator of the N-methyl-d-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular d-serine in glial cells. Here we show that after d-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of d-serine led to the production of hydrogen peroxide (H2O2) followed by apoptosis in C6/DAO cells. Among the amino acids tested, d-serine specifically exhibited a significant cell death–inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with d-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular d-serine can gain access to intracellular DAO, being metabolized to produce H2O2. These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, d-serine. [ABSTRACT FROM PUBLISHER]

The polypeptide α3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)3, and α3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of α helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed α helix like α3. However, their helix formation ability was weaker than that of α3 under some conditions. The Val-substituted polypeptides formed α helix only under restricted condition. The Ala-substituted polypeptides did not form α helix under any condition. Thus, it is clear that the order of the α helix formation ability is as follows: Leu ≥ Ile > Val > Ala. The formation of α helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the α helix structure correlates with the formation of a fibrous assembly. The amphipathic α helix structure would be stabilized by the formation of the fibrous assembly. [ABSTRACT FROM PUBLISHER]

Recently, we isolated CHO cells, termed SK32 cells, that express mutant Pex5p (G432R), and showed mislocalization of catalase in the cytosol, but peroxisomal localization of 3-ketoacyl-CoA thiolase (thiolase) in the mutant cells [Ito, R. et al. (2001) Biochem. Biophys. Res. Commun. 288, 321–327]. While analyzing the mutant cells, we found a novel Pex5p isoform (Pex5pM), which was shorter by seven amino acids than Pex5pL and longer by 30 amino acids than Pex5pS. Similar levels of mRNA syntheses for the PEX5 gene were observed in both the wild type and mutant cells, but the protein levels of Pex5p isoforms were markedly reduced in the mutant cells cultured at 37°C and only slightly discernible at 30°C, suggesting that they could be rapidly degraded. Furthermore, we characterized the peroxisomal localization of thiolase and acyl-CoA oxidase (Aox) in SK32 cells. The proteins in the organelle fraction were protected from proteinase K-digestion in the mutant cells, indicating that they were translocated inside peroxisomes. However, the conversion of Aox from component A to components B and C was completely prevented at both 30 and 37°C, and the precursor form of thiolase was partially processed to the mature one in a temperature-sensitive manner. Transformed SK32 cells stably expressing one of the wild type Pex5p isoforms were isolated, and then the maturation steps for thiolase and Aox were examined. Pex5pM and S restored the processing of the two enzymes, but Pex5pL did not. In addition, Pex5pL prevented the maturation of thiolase observed at 30°C. These results indicate that (i) the novel Pex5pM is functional and (ii) a seven amino acids-insertion, which is present in the L isoform but absent in the M isoform, plays some role in the process of maturation of thiolase and Aox. [ABSTRACT FROM PUBLISHER]

Vimelysin is a unique metalloproteinase from Vibrio sp. T1800 exhibiting high activity at low temperature and high stability in organic solvents such as ethanol. A 1,821 bp open reading frame of the vimelysin gene encoded 607 amino acid residues consisting of an N-terminal pro-region, a mature enzyme, and a C-terminal pro-region. The mature enzyme region showed 80%, 57% and 35% sequence identity with the mature forms of vibriolysin from V. vulnificus, pseudolysin from Pseudomonas aeruginosa, and thermolysin from Bacillus thermoproteolyticus, respectively. The catalytic residues and zinc-binding motifs of metalloproteinases are well conserved in vimelysin. The vimelysin gene was expressed in E. coli JM109 cells and the recombinant enzyme was purified as a 38-kDa mature form from cell-free extracts. The purified recombinant enzyme is indistinguishable from the enzyme purified directly from Vibrio. To obtain mutants exhibiting higher stability in organic solvents, random mutations were introduced by error-prone PCR and 600 transformants were screened. The N123D mutant exhibits two times higher stability in organic solvents than the wild-type enzyme. A plausible mechanism for the stability of the N123D mutant in organic solvents was discussed based on homology models of vimelysin and the N123D mutant. [ABSTRACT FROM PUBLISHER]

The crystal structure of phosphoribosyl anthranilate isomerase (PRAI) from Thermus thermophilus HB8 (TtPRAI) was solved at 2.0 Å resolution. The overall structure of TtPRAI with a dimeric structure was quite similar to that of PRAI from Thermotoga maritima (TmPRAI). In order to elucidate the stabilization mechanism of TtPRAI, its physicochemical properties were examined using DSC, CD, and analytical centrifugation at various pHs in relation to the association-dissociation of the subunits. Based on the experimental results for TtPRAI and the structural information on TtPRAI and TmPRAI, we found that: (i) the denaturation of TtPRAI at acidic pH is correlated with the dissociation of its dimeric form; (ii) the hydrophobic interaction of TtPRAI in the monomer structure is slightly greater than that of TmPRAI, but dimer interface of the TmPRAI is remarkably greater; (iii) the contributions of hydrogen bonds and ion bonds to the stability are similar to each other; and (iv) destabilization due to the presence of cavities in TtPRAI is greater than that of TmPRAI in both the monomer and dimer structures. [ABSTRACT FROM PUBLISHER]

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds. [ABSTRACT FROM PUBLISHER]

Temperature-dependent (25–80°C) infrared (IR) spectra were obtained for recombinant methionyl human granulocyte-colony stimulating factor (rmethuG-CSF) in aqueous solutions over the pD range of 5.5–2.1 to investigate its thermal stability at various pDs. Second derivative, Fourier self-deconvolution, and curve-fitting analyses were performed to analyze the obtained spectra. These spectral analyses demonstrated that in the thermal unfolding process the α-helix structure of rmethuG-CSF partially changes to an unordered structure and then the unordered structure forms aggregates. The temperature-dependent IR spectra revealed that the structure of rmethuG-CSF is the most stable at pD 2.5 in the pD range of 5.5–2.1. It has been suggested that the unordered structure formed before the marked structural change in the whole molecule is a perturbed form of the native structure of rmethuG-CSF and plays a role as a precursor for the aggregation. This alteration to the perturbed form is likely to be the first secondary structure change that occurs along the aggregation pathway. Of particular note is that the stability at pD 2.1 is slightly lower than that at pD 2.5, but that aggregates are formed at higher temperature at pD 2.1 than at pD 2.5, probably because the repulsive interaction between the unordered structure is stronger at pD 2.1. [ABSTRACT FROM PUBLISHER]

The effects of salts on the rate constants of inactivation by heat of yeast alcohol dehydrogenase (YADH) at 60.0°C were measured. Different effects were observed at low and high salt concentrations. At high concentrations, some salts had stabilizing effects, while others were destabilizing. The effects of salts in the high concentration range examined can be described as follows: (decreased thermal stability) NaClO4 < NaI = (C2H5)4NBr < NH4Br < NaBr = KBr = CsBr = (no addition) < (CH3)4NBr < KCl < KF < Na2SO4 (increased thermal stability). The decreasing effect of NaClO4 on YADH controlled the thermal stability of the enzyme absolutely and was not compensated by the addition of Na2SO4, a salt which stabilized the enzyme. However, Na2SO4 compensation did occur in response to the decrease in thermal stability caused by (C2H5)4NBr. The rate constants of inactivation by heat (k in) of the enzyme were measured at various temperatures. Effective values of the thermodynamic activation parameters of thermal inactivation, activation of free energy (ΔG ‡), activation enthalpy (ΔH ‡), and activation entropy (ΔS ‡), were determined. The thermal stability of YADH in 0.8 M Na2SO4 increased more than that of pyruvate kinase from Bacillus stearothermophilus, a moderate thermophile. The changes in the values of ΔH ‡ and ΔS ‡ were great and showed a general compensatory tendency, with the exception of in the case of NaClO4. The temperature for the general compensation effect (T c) was approximately 123°C. With Na2SO4, the thermal stability of YADH at a temperature below T c was greater than that in the absence of salt due to the higher values of ΔH ‡ and ΔS ‡, respectively, and thus was an example of low-temperature enzymatic stabilization. With (C2H5)4NBr, the thermal stability of YADH at a temperature below T c was lower than that in the absence of salt due to the lower values of ΔH ‡ and ΔS ‡, respectively, and thus was an example of low-temperature enzymatic destabilization. But with NaClO4, the changes in the values of ΔH ‡ and ΔS ‡ were small and the thermal stability of YADH was thus an example of high-temperature enzymatic destabilization. [ABSTRACT FROM PUBLISHER]

Group 2 major mite allergens Der f 2 and Der p 2 are classified into the recently identified group of MD-2-related lipid-recognition (ML) proteins, but the ligands and biological functions of these allergens are unknown. We have obtained a high-quality NMR structure for Der f 2, and found that it is more similar to the crystal structure of NPC2, a distant homologue, than to that of Der p 2, in terms of the separation and angle between the two major β-sheets. This made us propose that ML proteins undergo clamshell-like motions that change the sizes of ligand-binding spaces inside their immunoglobulin-fold β-sandwich to accommodate lipid molecules. This type of motion in lipopolysaccaride recognition of MD-2 is suggested to be likely as well by structural models. We also report the applicability of NMR differential exchange broadening experiments for complexes of intact monoclonal antibodies and antigens; using this technique, we have detected the conformational epitopes for monoclonal antibodies 15E11 and 13A4 as two separate surface patches. [ABSTRACT FROM PUBLISHER]

Adaptive strategies employed by the yeast Saccharomyces cerevisiae provide robustness and adaptability of its central metabolism. Since central metabolism in yeast has been well studied at the enzymatic and genetic levels, it represents an excellent system for evaluating the relative roles of duplicate genes and alternative metabolic pathways as possible mechanisms for the stability of central metabolism against null mutations. Yeast appears to employ a variety of mechanisms to ensure functional robustness of its central metabolism. Uninterrupted flow of energy and precursor metabolites through the pathways of central metabolism via glycolysis (EMP), pentose phosphate shunt (PPS), and the tricarboxylic acid (TCA) cycle are ensured by a variety of adaptive mechanisms. One of the most significant mechanisms appears to be gene duplication events that have produced a number of isozymes functioning under variable environmental and physiological conditions. Alternative pathways represent another important mechanism for increasing the robustness of the system. The robustness of the pathways of central metabolism is apparently higher than that of the other parts of metabolism, because of its exceptional importance to the organism’s vitality. The proportion of duplicated viable genes also is substantially larger in central metabolism than that in a pool of other metabolic genes. [ABSTRACT FROM PUBLISHER]

To address the effects of ligand binding on the structural fluctuations of Escherichia coli dihydrofolate reductase (DHFR), the hydrogen/deuterium (H/D) exchange kinetics of its binary and ternary complexes formed with various ligands (folate, dihydrofolate, tetrahydrofolate, NADPH, NADP+, and methotrexate) were examined using electrospray ionization mass spectrometry. The kinetic parameters of H/D exchange reactions, which consisted of two phases with fast and slow rates, were sensitively influenced by ligand binding, indicating that changes in the structural fluctuation of the DHFR molecule are associated with the alternating binding and release of the cofactor and substrate. No additivity was observed in the kinetic parameters between a ternary complex and its constitutive binary complexes, indicating that ligand binding cooperatively affects the structural fluctuation of the DHFR molecule via long-range interactions. The local H/D exchange profile of pepsin digestion fragments was determined by matrix-assisted laser desorption/ionization mass spectrometry, and the helix and loop regions that appear to participate in substrate binding, largely fluctuating in the apo-form, are dominantly influenced by ligand binding. These results demonstrate that the structural fluctuation of kinetic intermediates plays an important role in enzyme function, and that mass spectrometry on H/D exchange coupled with ligand binding and protease digestion provide new insight into the structure–fluctuation–function relationship of enzymes. [ABSTRACT FROM PUBLISHER]