bluuebunny/crossref_metadata_2025_split huggingface dataset:
Agonist derived molecular probes for A2 adenosine receptors
Authors: Jacobson Kenneth A. , Pannell Lewis K. , Ji Xiao‐Duo , Jarvis Michael F. , Williams Michael , Hutchison Alan J. , Barrington William W. , Stiles Gary L. 
Published: May 2005

AbstractThe adenosine agonist 2‐(4‐(2‐carboxyethyl)phenylethylamino)‐5′‐N‐ethylcarboxamidoadenosine (CGS21680) was recently reported to be selective for the A2 adenosine receptor subtype, which mediates its hypotensive action. To investigate structure/activity relationships at a distal site, CGS21680 was derivatized using a functionalized congener approach. The carboxylic group of CGS21680 has been esterified to form a methyl ester, which was then treated with ethylenediamine to produce an amine congener. The amine congener was an intermediate for acylation reactions, in which the reactive acyl species contained a reported group, or the precursor for such. For radioiodination, derivatives of p‐hydroxyphenylpropionic, 2‐thiophenylacetic, and p‐aminophenylacetic acids were prepared. The latter derivative (PAPA‐APEC) was iodinated electrophilically using [125I]iodide resulting in a radioligand which was used for studies of competition of binding to striatal A2 adenosine receptors in bovine brain. A biotin conjugate and an aryl sulfonate were at least 350‐fold selective for A2 receptors. For spectroscopic detection, a derivative of the stable free radical tetramethyl‐1‐piperidinyloxy (TEMPO) was prepared. For irreversible inhibition of receptors, meta‐ and para‐phenylenediisothiocyanate groups were incorporated in the analogs. We have demonstrated that binding at A2 receptors is relatively insensitive to distal structural changes at the 2‐position, and we report high affinity molecular probes for receptor characterization by radioactive, spectroscopic and affinity lebelling methodology.
Avidin column as a highly efficient and stable alternative for immobilization of ligands for affinity chromatography
Authors: Bayer Edward A. , Wilchek Meir 
Published: May 2005

AbstractThe avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin‐coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti‐transferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBr‐activated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.
Interaction between immobilized and soluble protein subunits. Analysis and applications
Authors: Chiancone Emilia , Gattoni Maurizio 
Published: May 2005

AbstractA distinctive property of oligomeric and self‐associating proteins is the high specificity of the subunit recognition process. Protein subunits immobilized covalently on a solid matrix maintain this characteristic and are able to bind soluble subunits of the same or a closely related protein under conditions that allow the establishment of a finite association/dissociation equilibrium. The basic theory for studying the immobilized‐soluble subunit interaction is presented together with the methodology for a proper protein immobilization. Specific examples are discussed to illustrate on the one hand benefits and caveats of using immobilized protein subunits to measure interaction constants, and on the other preparative applications of subunit affinity columns.
Enzymatic semisynthesis of human insulin: An update
Authors: Morihara Kazuyuki 
Published: May 2005

AbstractPeptide bond formation can be enzymatically catalysed by the reverse reaction of proteases. Application is seen in the industrial production of human insulin. Human insulin derivative can be enzymatically prepared using either porcine insulin or the single chain B(1–29)‐A(1–21) insulin precursor as the starting material. This is accomplished by either (1) digesting the starting material at Lys29 with Achromobacter lyticus protease I (Ach) and then coupling with Thr‐X (X = blocking residue) (two‐step reaction) or (2) subjecting Ala‐B30 of procine insulin or Gly‐A1 of the single chain insulin precursor to transpeptidation with Thr‐X (one‐step reaction). Trypsin and Ach can be used for either type of reaction and, in the immobilized form, for the two‐step reaction. Since the single chain insulin precursor can be produced by gene technology (yeast), use of immobilized trypsin or Ach and the two‐step reaction using the single chain insulin precursor as the starting material ensures the continuous production of human insulin making it a feasible method for industrial manufacture.
Purification and characterization of fab fragments from anti‐mouse NGF polyclonal antibodies
Authors: Callegaro L. , Skaper S. D. , Vantini G. , Benvegnù D. , Di Martino A. , Schiavo N. , Triban C. , Minozzi C. , Leon A. 
Published: May 2005

AbstractA functional role for Nerve Growth Factor (NGF) in the peripheral nervous system is well‐documented, but a similar case for NGF in the central nervous system remains to be established. One approach to answering this question would be the availability of high‐affinity monospecific Fab fragments obtained against NGF. In the present studies we describe the preparation and characterization of such Fab fragments from anti‐mouse NGF polyclonal antibodies. Following their purification by the use of a NGF Sepharose‐coupled affinity column, the Fab fragments were examined for biological competence in several ways. In vitro, the anti‐Fab fragments blcoked the neuronotrophic activity of NGF, as measured by the survival of chicken embryonic day 8 dorsal root ganglion neurons. In vivo, these Fab fragments, when administered systemically to neonatal rats, produced a decrease of noradrenaline levels in two sympathetically innervated organs, the heart and the spleen. These findings suggest that affinity purified Fab fragments of anti‐NGF antibodies can be a useful tool for studying the physiological function of NGF in the nervous system.
Binding of the soybean Bowman‐Birk proteinase inhibitor and of its chymotrypsin and trypsin inhibiting fragments to bovine α‐chymotrypsin and bovine β‐trypsin. A thermodynamic study
Authors: Ascenzi Paolo , Amiconi Gino , Bolognesi Martino , Menegatti Enea , Guarneri Mario 
Published: May 2005

AbstractThe effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the soybean Bowman‐Birk proteinase inhibitor (BBI) and of its chymotrypsin and trypsin inhibiting fragments (F‐C(p), F‐T(p) and F‐T(t), respectively) to bovine α‐chymotrypsin (α‐chymotrypsin) and bovine β‐trypsin (β‐trypsin) has been investigated. On the basis of Ka values, the proteinase inhibitor affinity can be arranged as follows: β‐chymotrypsin: BBI ≈ β‐trypsin:BBI ≈ β‐trypsin:F‐T(t) ≈ β‐trypsin:F‐T(p) ≫ α‐chymotrypsin:F‐C(p), F‐C(p), F‐T(p) and F‐T(t) do not inhibit β‐trypsin and α‐chymotrypsin action, respectively. On lowering the pH from 9.5 to 4.5, values of Ka for BBI, F‐C(p), F‐T(p) and/or F‐T(t) binding to α‐chymotrypsin and β‐trypsin decrease, thus reflecting the acid‐pK shift of the invariant His57 catalytic residue from 7.0, in the free enzymes, to 5.2, in the proteinase:inhibitor complexes. Considering the known molecular models, the observed binding behaviour of BBI, F‐C(p), F‐T(p) and F‐T(t) was related to the inferred stereochemistry of the proteinase:inhibitor contact regions.
Effect of modification of enkephalin C‐terminal functions on affinity selection of opioid receptors
Authors: Kodama Hiroaki , Uchida Hiroaki , Yasunaga Teruo , Kondo Michio , Costa Tommaso , Shimohigashi Yasuyuki 
Published: May 2005

AbstractEnkephalin analogs with various C‐terminal modifications have been synthesized to evaluate the corresponding structural elements in the opioid receptors. The carboxyl group of the C‐terminal leucine5 or glycine5 was converted into the mercaptomethyl (–CH2SH) and hydroxymethyl (–CH2OH) groups, starting from leucinthiol or leucinol for Leu5‐derivatives and from cysteamine or ethanolamine for Gly5‐derivatives. Interactions of synthetic peptides with the opioid receptors were examined by the radioligand receptor binding assays using rat brain and tritiated enkephalin analogs. The data suggest that the C‐terminal carboxyl group in enkephalins is important, but not electrostatically, for interaction with delta‐opioid receptors. With leucinthiol–enkephalin in biological assays which examine its inhibitory activity for electrically stimulated contractions of isolated smooth muscle, it was found that the reactive thiol group exists in the mu receptors present in the guinea pig ileum. Leucinthiol‐enkephalin became bound covalently to this receptor‐thiol group via disulfide formation after prolonged incubation.
Proposed loci of interaction between bombesin and its receptor
Authors: Tritsch G. L. 
Published: May 2005

AbstractThe non‐coding strand of the bombesin receptor gene, when ‘translated’ 5′ 3′, contains and interrupted sequence of the 10 amino acids of bombesin. This finding forms the basis for proposing the points of contact between bombesin and its receptor as well as a partial conformation of the binding region of the receptor as well as partial conformation of the binding region of the receptor.
Peculiarities of recognition of CCA/TGG sequences in DNA by restriction endonucleases MvaI and EcoRII
Authors: Gromova Elizabeth S. , Kubareva Elena A. , Vinogradova Marina N. , Oretskaya Tatjana S. , Shabarova Zoe A. 
Published: May 2005

AbstractTo elucidate the mechanism of action of restriction endonucleases MvaI and EcoRII a study was made of their interaction with a set of synthetic substrates in which the heterocyclic bases or the sugar–phosphate backbone had been modified; individual nucleotide residues had been removed or replaced with hydrocarbon bridges, and mismatched base pairs had been introduced. The groups of atoms in the heterocyclic bases and the phosphates in the recognition site that produce the most significant influence on the functioning of endonucleases MvaI and EcoRII were discerned. Profound differences were found in the functioning of the MvaI and EcoRII neo‐schizomers. The catalytic activity of EcoRII is significantly affected by any alteration in the recognition site structure and conformation, with a modification in one strand of the substrate causing the same decrease in the hydrolysis rate of both strands. Endonuclease MvaI is tolerant to a number of structural abnormalities; the latter sometimes affect only hydrolysis of one strand of the recognition site. The enzyme can preferentially cleave one of the substrate strands. Mismatched base pairs retard and sometimes block the hydrolysis. The effect depends on the particular enzyme, mismatch and its location.
Evaluation of peptide/metal ion interactions by UV laser desorption time‐of‐flight mass spectrometry
Authors: Hutchens T. William , Nelson Randall W. , Yip Tai‐Tung 
Published: May 2005

AbstractA relatively recent method developed to determine the molecular weights of intact peptides and proteins, matrix‐assisted UV laser desorption time‐of‐flight mass spectrometry (LDTOF‐MS), has been evaluated as a new means to investigate the metal ion‐binding properties of model synthetic peptides. A contiguous sequence of 25 residues on the surface of the 74 kDA human plasma metal‐binding transport protein histidine‐rich glycoprotein (HRG) has been identified as a bioactive metal‐binding domain. The peptide, (GHHPH)5G, was synthesized and evaluated by LDTOF‐MS before and after the addition of Cu(II) in solution with 2,5‐dihydroxybenzoic acid as the matrix. In the absence of added Cu(II), the major protonated molecular ion (M + H)+ was observed to have a mass equal to its calculated mass (2904.0 Da). In the presence of Cu(II), however, five additional peaks were observed at mass increments of approximately 63.9 Da. The maximum Cu(II)‐binding capacity observed for the 26‐residue peptide (5 g‐atoms/mol) suggest that up 1 Cu(II) may be bound per 5‐residue internal repeat unit (GHHPH) within this peptide: several other monovalent and divalent metal cations were not bound under identical conditions of analysis. The Cu(II)‐binding stoichiometry was verified by spectrophotometric titration and by frontal analyses of the immobilized peptide with a solution of Cu(II) ions. These results demonstrate the ability to verify directly the solution‐phase binding capacity of metal‐binding peptides by LDTOC‐MS.
Design of novel affinity adsorbents for the purification of trypsin‐like proteases
Authors: Burton Nicolas P. , Lowe Christopher R. 
Published: May 2005

AbstractA number of ligands for the selective purification by affinity chromatography of the trypsin‐like protease, porcine pancreatic kallikrein, were designedde novoby computer‐aided molecular design. The ligands were designed to mimic the side‐chains of a number of arginyl dipeptides and included a benzamidine moiety substituted on a triazine ring. The ligands displayed inhibitory activities against pancreatic kallikrein which mirrored the specificity constants of the dipeptides they were designed to mimic. The ligand with the highest affinity for the enzyme, and analogue of a Phe‐Arg dipeptide, when immobilized to Sepharose CL‐4B via a hexamethylene spacer arm, purified pancreatic kallikrein 110‐fold in one step from a crude pancreatic acetone extract.
Complex carbohydrate–lectin interaction at the interface: a model for cellular adhesion. II. Reactivity of both the oligosaccharide chain and sugar‐binding domain of a glycoprotein lectin
Authors: Chakrabarti Abhijit , Podder Sunil K. 
Published: May 2005

AbstractWe describe studies of a new model cell adhesion system involving liposomes bearing lectins and the glycosphingolipid, asialomonosialoganglioside (asialoGM1). The model provides a simple analysis of experimental data to elucidate the mechanism of heterophilic cell–cell adhesion mediated by multiple protein–carbohydrate interactions. Phospholipid vesicles bearing the fatty acid conjugate of a glycoprotein lectin from Ricinus communis (RCAI vesicle) are shown to react with vesicles bearing the fatty acid conjugate of Concanavalin A (Con A) and asialoGM1 (Con A vesicle). The kinetics of aggregation and monosaccharide‐induced disaggregation of the two types of vesicles were followed by monitoring the time‐dependent change in turbidity. Depending on the surface density of the asialoGM1, 40–60% of the resulting precipitin complex was dissociable only in the presence of both RCAI‐specific galactose and Con A‐specific α‐methyl‐D‐mannoside. Results indicate simultaneous participation of both the saccharide‐binding domain and carbohydrage sequence of RCAI, a model cell adhesion molecule, to stabilize the encounter complex by two types of interactions. These findings support the possibility of stable cell–cell adhesion in vivo occurring via interactions between cell adhesion molecules on apposing cell‐surface membranes.
Chymotrypsin inhibitory conformation of dipeptides constructed by side chain–side chain hydrophobic interactions
Authors: Sakamoto Hiroshi , Shimohigashi Yasuyuki , Maeda Iori , Nose Takeru , Nakashima Kin‐ichi , Nakamura Ichiro , Ogawa Tomoshisa , Ohno Motonori , Kawano Keiichi 
Published: May 2005

AbstractA complete series of configurationally isomers (L‐L, L‐D, D‐L AND D‐D) of a dipeptide Leu‐Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz 1H NMR, the L‐D and D‐L isomers, but not hte L‐L and D‐D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu‐βCH2 and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe‐βCH2, the NOE enhancement observed between Leu‐δCH3 and Phe‐phenyl groups revealed that these groups are in close proximity. These data indicated that L‐D and D‐L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac‐Try‐OEt as a substrate, the L‐L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D‐L isomer was strongest with Ki of 2.2 × 10−5 M. It was found that the D‐L isomer was only slowly hydrolysed but the L(or D)‐D isomer was not. H‐D‐Phe‐L‐Leu‐OBzl with the inverse sequence of H‐D‐Leu‐L‐Pre‐OBzl inhibited chymotrypsin more strongly (Ki = 6.3 × 10−6 M). Since the free acid analogue of the D‐L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester‐benzyl group fits the S1 site of chymotrypsin, while the side chain‐side chain complexing hydrophobic core fits the S2 site.
PLIM: A protein–ligand interaction modeller
Authors: Harris Mark R. , Kihlen Mats , Bywater Robert P. 
Published: May 2005

AbstractA Computer program is presented that models the binding of selected chemical groups to a protein surface. The groups are successively incorporated at energetically favourable positions to build up a pharmacophore pattern that may be used as the basis for a database search for possible ligands. The ability to predict known binding points in a trypsin–inhibitor complex is demonstrated, and the results from a run on dihydrofolate reductase are shown to be usable as a pharmacophore pattern for a database search.
Influence of fractal kinetics on molecular recognition
Authors: Savageau Michael A. 
Published: May 2005

AbstractMolecular recognition is a central issue for nearly every biological mechanism. The analysis of molecular recognition to has been conducted within the framework of classical chemical kinetics, in which the kinetic orders of a reaction have positive integer values. However, recent theoretical and experimental advances have shown that the assumption inherent in this classical framework are invalid under a variety of conditions in shown that the assumptions inherent in this classical framework are invalid under a variety of condition in which the reaction environment may be considered nonideal. A good example is provided by reactions that are spatially constrainal and diffusion limited. Bio molecular reactions confined within two‐dimensional membranes, one‐dimensional channels or fractal surfaces in general exhibit kinetic orders that are noninteger. An appropriate framework for the study of these nonideal phenomena is provided by the Power‐Law formalism, which includes as special cases the Mass‐Action formalism of chemical kinetics and the Michaelis–Menten formalism of enzyme kinetics. The Power‐Law formalism is an appropriate representation not only for fractal kinetics per se, but also for other nonideal kinetic phenomena, provided the range of variation in concentration is not too large. After defining some elementary concepts of molecular recognition, and showing how these are manifested in classical kinetic terms, this paper contrasts the implications of classical and fractal kinetics in a few simple cases. The principal distinction lies in the ability of fractal kinetics to nonlinearly transform, rather than proportionally transmit, the input S/N ratio. As a consequence, fractal kinetics create a threshold for the input signal below which no recognition occurs and above which amplified recognition takes place. Thus, fractal kinetics implies an intimate relationship between design of the physiological mechanisms regulating the environment of the process and design of the molecular process itself. These results also suggest that recognition in the presence of a favorable input ration would emphasize rapid reactions, while recognition in the presence of an unfavorable input ratio would emphasize slow reactions.
Temporal analysis of metabolic systems and its application to metabolite channelling
Authors: Easterby John S. 
Published: May 2005

AbstractWhen a metabolic system undergoes a transition between steady states, the lag or transition time of the system is determined by the aggregated lifetimes of the metabolite pools. This allows the transition time, and hence the temporal responsiveness of the system, to be estimated from a knowledge of the starting and finishing steady states and obviates the need for dynamic measurements. The analysis of temporal response in metabolic systems may be integrated with the general field of metabolic control analysis by the definition of a temporal control coefficient (C) in terms of flux and concentration control coefficients. The temporal control coefficient exhibits summation and other properties analogous to the flux and concentration control coefficients. For systems in which static metabolite channels exits, the major kinetic advantage of channelling is a reduction in pool sizes and, as a result, a more rapid system response reflected in a reduced transition time. The extent of the channelling advantage may therefore be assessed from a knowledge of the system transition time. This reveals that no channelling advantage is achieved at high enzyme concentration (i.e., comparable to Km) or, in the case of ‘leaky’ channels, where rapid equilibrium kinetic mechanisms obtain. In the case of a perfect channel with no leakage and direct transfer of metabolite between adjacent enzyme active sites, the transition time is minimized and equal to the lifetime of the enzyme–substrate complex.
Immobilized enzymes as tools for the demonstration of metabolon formation. A short overview
Authors: Beeckmans Sonia , Van Driessche Edilbert , Kanarek Louis 
Published: May 2005

AbstractIn recent years it has become clear that a cell cannot be visualized as a ‘bag’ filled with enzymes dissolved in bulk water. The aqueous‐phase properties in the interior of a cell are, indeed, essentially different from those of an ordianry queous solutin. Large amounts of water are believed to be organized in layers at the surface of intracellular structural proteins and membranes. Such considerations prompt us to reconsider the operation and regulation of metabolic pathways. Enzymes of metabolic pathways are nowadays thought to be clustered and operate as ‘metabolons’. Very often interactions between enzymes of a pathway can exclusively be evidenced in Vitro in media which are known to reduce the water concentration in the vicinity of the proteins. Immobilized enzyme preparations have been shown to be excellent tools for this type of research. We describe here some recent studies where immobilized enzymes have been used in various applications to investigate associations among enzymes of a number of different metabolic pathways (glucolysis/gluconeogenesis, citric acid cycle and its connection to the electron transport chain, aspartate–malate shuttle, glyoxylate cycle). Advantages and disadvantages of the different techniques we also discussed.