bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of specific interactions of coenzymes, regulatory nucleotides and cibacron blue with nucleotide binding domains of enzymes by analytical affinity chromatography
Authors: Thresher Wayne C. , Swaisgood Harold E. 
Published: May 2005

AbstractThe dissociation constant for the complex of rhodanese and Cibacron Blue, determined by analytical affinity chromatography using rhodanese immobilized on controlled‐pore glass (CPG) beads (200 nm pore diameter) and aminohexyl‐Cibacron Blue, was 44 μM which agreed well with the kinetic inhibition constant, suggesting that the dye binds at or near the active site of this enzyme. Formation of a binary complex of the dye and lactate dehydrogenase (LDH) was also characterized by direct chromatography of LDH on CPG/immobilized Cibacron Blue (KD = 0.29 μM). The binary complex formed between LDH and NADH was characterized by analytical affinity chromatography using both CPG/immobilized LDH and immobilized Cibacron Blue. Since the dye competes with NADH in binding to the active site of LDH, competitive elution chromatography using the immobilized dye allows determination of the dissociation constant of the soluble LDH · NADH complex. Agreement between the dissociation constants determined by direct chromatography of NADH on immobilized LDH (KD = 1.4 μM) and that determined for the soluble complex (KD = 2.4 μM) indicates that immobilization of LDH did not affect the interaction. Formation of various binary, ternary and quaternary complexes of bovine liver glutamate dehydrogenase (GDH) with glutamate, NADPH, NADH, and ADP was also investigated using immobilized GDH. This approach allows characterization of the enzyme/ligand interactions without the complicating effect of enzyme self‐association. The affinity for NADPH is considerably greater in the ternary complex (including glutamate) as compared to the binary complex (0.38 μM vs 22 μM); however, occupancy of the regulatory site by ADP greatly reduces the affinity in both complexes (6.4 μM and 43 μM, respectively). These results confirm the proposed mechanism of enhancement of the catalytic rate by ADP binding via a weakening of coenzyme binding in the active ternary complex. A method is also presented for evaluation of very weak interactions within ternary systems. This method yields dissociation constants for the GDH · glutamate complex of 52 mM and 2.8 mM with and without the regulatory site occupied by ADP, respectively. Hence the analytical affinity chromatography allows determination of affinity constants that range over 5 orders of magnitude.
Cytokine conformations: Predictive studies
Authors: Parry David A. D. , Minasian Elizabeth , Leach Sydney J. 
Published: May 2005

AbstractThe amino acid sequences of human and murine haemopoietins have been analysed using algorithms predictive for secondary structure. The results for 19 of these proteins (human and murine Interleukins 2, 3, 4, 5, 6, 7 and granulocyte, macrophage and granulocyte macrophage‐colony stimulating factors as well as human erythropoietin) suggest that they each contain a 4‐α‐helical bundle, ca 25 Å long, as a common conformational feature. The most important predictive indicator was considered to be occurrence of quasi‐repeating sequences of seven amino acids of the form (a‐b‐c‐d‐e‐f‐g)n with a polar side chains (usually leucine) lying alternately three and four residues apart in the a and d positions. As with other proteins of known secondary structure this periodicity favors the formation of α‐helical elements, each with an a polar external strip, which interdigitate closely with one another when tested appropriately. Molecular models based on these putative 4‐α‐helical bundles are presented – with special references to human granulocyte macrophage‐colony stimulating factor. The extent to which such models are consistent with experiments designed to delineate receptor binding sites is discussed.
Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to serine (Pro)enzymes: A comparative thermodynamic study
Authors: Ascenzi Paolo , Aducci Patrizia , Amiconi Gino , Ballio Alessandro , Guaragna Alberto , Menegatti Enea , Schnebli Hans Peter , Bolognesi Martino 
Published: May 2005

AbstractThe binding of the recombinant proteinase inhibitor eglin c from the leech Hirudo medicinalis to serine (pro)enzymes belonging to the chymotrypsin and subtilisin families has been investigated from the thermodynamic viewpoint, between pH 4.5 and 9.5 and from 10°C to 40°C. The affinity of eglin c for the serine (pro)enzymes considered shows the following trend: Leu‐proteinase [the leucine specific serine proteinase from spinach (Spinacia oleracea L.) leaves] > human leucocyte elastase ⋍ human cathepsin G ⋍ subtilisin Carlsberg ⋍ bovine α‐chymotrypsin > bovine α‐chymotrypsinogen A ⋍ porcine pancreatic elastase ⋍ bovine β‐trypsin. The serine (pro)enzyme–inhibitor complex formation is an entropy‐driven process. On increasing the pH from 4.5 to 9.5, the affinity of eglin c for the serine (pro)enzymes considered increases thus reflecting the acid pK shift to the invariant hystidyl catalytic residue from ≈︁ 6.9 in the free serine proteinases and bovine α‐chymotrypsinogen A to ⋍ 5.1 in the serine (pro)enzyme–inhibitor complexes. Considering the known molecular models, the observed binding behaviour of eglin c was related to the inferred stereochemistry of the serine(pro)enzyme–inhibitor contact regions.
Identity elements of Escherichia coli tRNAAla
Authors: Tamura Koji , Asahara Haruichi , Himeno Hyouta , Hasegawa Tsunemi , Shimizu Mikio 
Published: May 2005

AbstractStudies using the T7 transcription system revealed that the discriminator base A73 and the G20 in the variable pocket play important roles in the Escherichia coli alanine tRNA identity. The C60 in the T‐loop, which is unique to alanine tRNA, was not found to be crucial for alanine identity. Anticodon replacement into the valine anticodon UAC did not decrease alanine charging activity, and no alanine charging activity was detected in the mutant valine tRNA possessing the alanine anticodon UGC.
Immunomagnetic separation and analysis of non‐malignant variants and parental malignant mouse lymphoma cells
Authors: Lazarovici Philip , Bergel Michael , Hasson Tsury , Rahamim Ezra , Hochman Jacob 
Published: May 2005

AbstractWe have devised conditions whereby non‐tumorigenic, immunogenic cell variants of S49 mouse lymphoma were analyzed and separated from parental tumorigenic lymphoma cells. This was carried out using polyclonal antibodies (raised against the immunogenic variants) and immunomagnetic beads. The efficacy of the procedure depended on the amount of polyclonal antiserum, the immunobead to cell ratio, incubation time and the number of repetitions of the procedure. Experiments with mixed tumorigenic and non‐tumorigenic cells have resulted in an enrichment of up to 200‐fold of the non‐tumorigenic, immunogenic cells in the population. These findings indicate the potential use of this procedure (in conjunction with other approaches) to isolate from a population of tumorigenic cells those variant cells that might be used to immunize against the parental tumor.
Chiral separation using molecularly imprinted heteroaromatic polymers
Authors: Kempe Maria , Mosbach Klaus , Fischer Lutz 
Published: May 2005

AbstractNovel molecularly imprinted polymer systems utilizing 4‐vinylpyridine and 1‐vinylimidazole as functional monomers have been developed for enantioselective recognition of carboxylic and N‐protected amino acids. Non‐covalent interactions between the functional monomers and the template molecules were the source of the subsequent recognition sites in the resultant polymers. The capacity of the polymers for molecular recognition was investigated by using them as stationary phases in the HPLC mode. Polymers prepared with 4‐vinylpyridine were found to be more efficient in racemic resolution than those prepared with 1‐vinylimidazole. When applying a racemic mixture of the template molecule, the polymers showed highest affinity for the enantiomer used as template. Imprints of a racemic template molecule, as expected, did not exhibit enantioselectivity. The optimal molar ratio of 4‐vinylpyridine to the template Cbz‐L‐Asp‐OH in the polymerization mixture was determined to be 12:1. In addition to enantioselectivity, the investigated polymers demonstrated ‘ligand selectivity’ e.g., a Cbz‐L‐Asp‐OH‐imprinted polymer was able to separate Cbz‐D,L‐Asp‐OH, but was unable to separate Cbz‐D,L‐Glu‐OH.
The binding of phosphorylase kinase to immobilized calmodulin
Authors: Jennissen H. P. , Botzet G. 
Published: May 2005

AbstractThe binding of phosphorylase kinase to calmodulin‐Sepharose 4B was studied by column and batch methods. It was found that the Ca2+ dependence of the interaction strongly depended strongly depended on the degree of substitution of agarose with calmodulin. Equilibrium adsorption isotherms (i.e., bulk ligand binding functions and lattice site binding functions) of phosphorylase kinase were measured on calmodulin‐Sepharose. Sigmoidal bulk ligand binding functions (bulk adsorption coefficients: 1.5–5.8) were found which indicate intermolecular attraction during binding. Hyperbolic lattice site binding functions (lattice adsorption coefficients: 1.0) were obtained thus excluding the existence of a critical surface concentration of immobilized calmodulin and indicating single independent binding sites on the gel surface and on phosphorylase kinase. These findings were combined to optimize the adsorption of phosphorylase kinase on calmodulin‐Sepharose, for purification procedures at low Ca2+ concentrations (5–10 μM) minimizing proteolysis by calpains. With this novel method phosphorylase kinase from rabbit and frog skeletal muscle could be purified ca 100‐ and 200‐fold, respectively, in two steps.
Endo β‐N‐acetylglucosaminidase F cleavage specificity with peptide free oligosaccharides
Authors: Anumula Kalyan Rao 
Published: May 2005

AbstractEndo β‐N‐acetylglucosaminidase activities were determined based on conversion of oligosaccharides containing two N‐acetylglucosamines to the oligosaccharides with a single N‐acetylglucosamine at the reducing terminal and following their separation on a carbohydrate analyzer. The oligosaccharides eluted from the high performance anion exchange column in the order of fucosyl‐N,N′ ‐diacetylchitobiose, N,N′ ‐diacetylchitobiose and N‐acetylglucosamine containing reducing terminals. Using this assay, differences in cleavage specificity of the glycoproteins was determined. The commercial Endo F‐peptide N‐glycosidase/glycanyl amidase (PNGase)mixture readily leaved high mannose and complex oligosaccharides (neutral and sialyated) with common core α1–6 linked fucose found in porcine thyroglobulin including the trimannosyl‐chitobiose core structure. However, the same Endo F mixture did not cleave the non‐fucosylated complex oligosaccharides found in human transferrin and also the common core structure. Glycopeptide counterparts with and without fucose were good substrates for the endoglycosidases. These results show that the specificity of these enzymes is such that they can recognize the conformational differences between free oligosaccharides and glycopeptides with and without the common core α1–6 linked fucose. In contrast, highly purified Endo F cleaved only the high mannose type oligosaccharides and was unable to cleave ovalbumin hybrid type oligosaccharides. However, it was similar to Endo H when reduced ovalbumin oligosaccharides were used as substrates, consistent with the recently isolated Endo F subfraction F1 being similar to Endo H [Trimble, R. B. and Tarentino, a. L. (1991). J. Biol. Chem. 266, 1646]. Results obtained in this study suggest that the complex oligosaccharides cleaving enzymes F2 and F3 show high specificity towards peptide free oligosaccharides with the core α1‐6 linked fucose, unlike the glycopeptide substrates. Therefore PNGase free Endo F1, F2 and F3 mixtures should be useful in the functional evaluation of the oligosaccharides in glycoproteins.
Binding of native and [homoserine lactone‐52]‐52,53‐seco‐bovine basic pancreatic trypsin inhibitor (kunitz inhibitor) to porcine pancreatic β‐kallikrein‐B and bovine α‐chymtorpsin: Thermodynaic study
Authors: Oddone Rolando , Barra Donatella , Amiconi Gino , Ascenzi Paolo , Tarricone Cataldo , Bolognesi Martino , Bortolotti Fabrizio , Menegatti Enea 
Published: May 2005

AbstractValues of the association equilibrium constant (Ka) for the binding of the native and of the cyanogen bromide‐cleaved bovine basic pancreatic trypsin inhibitor (native BPTI and [Hse lactone‐52]‐52,53‐seco‐BPTI, respectively) to neuraminidase‐treated porcine pancreatic β‐Kallikrein‐B (kallikrein) and bovine α‐chymotrypsin (chymotrypsin) have been determined between pH4.0 and 9.0, and 20.0°C. Over the whole pH range explored, native BPTI and [Hse lactone‐52]‐52,53‐seco‐BPTI show the same affinity for kallikrein. On the other hand, the affinity of [se lactone‐52]‐52,53‐seco‐BPTI for chymotrypsin is high4er, around neutrality, than that found for native BPTI by about one order of magnitude, coverging in the acidic pH limb. The simplest mechanism accounting for the observed data implies that, on lowering the pH from 9.0 to 4.0 (i) the decrease in affinity for the binding of native BPTI to kalikrein and chymotrypsin, as well as for the association of [Hse lactone‐52]‐52,53‐seco‐BPTI to kalikrein, reflects the acidic pK shift, upon inhibitor association, of a single inozing group; and (ii) the decrease of Ka values for [Hse lactone‐52]‐52,53‐seco‐BPTI binding to chymotrypsin appears to be modulated by the acidic pK shift, upon inhibitor association, of two non‐equivalent proton‐binding residues. On the basis of the stereochemistry of the serine proteinase/inhibitor contact region(s), these data indicate that long‐rang structural changes in [Hse lactone‐52]‐52,53‐seco‐BPTI are energetically linked to the chymotrypsin: inhibitor complex formation. This observation represents an important aspect for the mechanism of molecular recognition and regulation in BPTI.
Quantitative footprinting analysis
Authors: Shubsda Michael , Kishikawa Hiroko , Goodisman Jerry , Dabrowiak James 
Published: May 2005

AbstractThis review outlines the steps for obtaining relative constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA sites. Slopes of plots outside the drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netrospin to a 139‐base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netrospin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.
Receptor interactions of the position 4 side chains of angiotensin II analogues: Importance of aromatic ring quadrupole
Authors: Moore Graham J. , Ganter Renee C. , Matsoukas John M. , Hondrelis John , Agelis George , Barlos Klemoenis , Wilkinson Scott , Sandall John , Fowler Patrick 
Published: May 2005

AbstractA triad of interacting group (TyrOHHis\documentclass{article}\pagestyle{empty}\begin{document}$ \underline\ominus$ \end{document}O2C) in angiotensin II (ANG II) has been postulated to create the tyrosinate anion pharmacophore (tyanophore) responsible for receptor activation/triggering (Biochim. Biophys. Acta 1991, 1065, 21). In the present study we investigated the effects on bioactivity of substituting the Tyr4 residue in [Sar1]ANG II with other anionic or electronegative amino acids, and with a number of aromatic amino acids lacking a hydroxyl group. [Sar1 Nva(δ‐OH)4]ANG II, [Sar1 Nva(δ‐OCH3)4]ANG II, [Sar1 Met4]ANG II, [Sar1 Gln4]ANG II, [Sar1 Glu4]ANG II and [Sar1 DL‐Alg4]ANG II had agonist activities in the rat isolated uterus assay of 4, 3, 19, 10, > 0.1 and > 0.1%, respectively, of that of ANG II. [Sar1 Nal4]ANG II, [Sar1 Pal4]ANG II, [Sar1 DL‐Phg(4′‐F)4]ANG II, [Sar1 Phe(4′‐F)4]ANG II, [Sar1 Phe(F5)4]ANG II and [Sar1 His4]ANG II had agonist activities of 4.5, 7, < 0.1, 0.2, 1 and 0.6%, respectively. All peptides investigated were devoid of measurable antagonist activity except [Sar1] Phe(4′‐F)4 ANG II (pA2 = 7.7). These findings illustrate that anionic or electronegative aliphatic side chains replacing tyrosinate at position 4 can partially activate the angiotension receptor. For ANG II analogues containing an aromatic amino acid other than Tyr at position 4, ligand binding and agonist activity are not dependent on the electronegativity or dipole moment of the aromatic ring, or on the ability of the 4′ ring substituent to accept a proton. Modelling based on ab initio calculations of aromatic ring multipoles illustrate that the apparent binding affinity (PA2) of ANG II analogues is associated with a perpendicular electrostatic interaction of the position 4 aromatic ring with a receptor‐based group. In addition, intramolecular interactions providing for the conformation of the ligand as it approaches its receptor appear to have a role in determining agonist vs antagonist activity.
An approach towards surface imprinting using the enzyme ribonuclease A
Authors: Kempe Maria , Glad Magnus , Mosbach Klaus 
Published: May 2005

AbstractAn Adsorbent showing enhanced selectivity for the enzyme RNase A was prepared by a surface imprinting procedure based on metal coordination. A metal chelating monomer, N‐(4‐vinyl)‐benzyl iminodiacetic acid, was polymerized onto methacrylate‐derivated silica particles in the presence of RNase A and metal ions. Lysozyme and RNase A were separated on the adsorbent used as stationary phase in high‐performance liquid chromatography.
Chaperone‐like effect of monoclonal antibodies on refolding of heat‐denatured carboxypeptidase A
Authors: Solomon Beka , Schwartz Fidi 
Published: May 2005

AbstractThe availability of monoclonal antibodies which bind to a specific antigen at distinct and well‐defined sites has led to a better understanding of the effects of highly specific enzyme–antibody interactions on enzyme behaviour. By appropriate selection it has been possible to isolate those antibodies that are non‐inhibitory to biological activity of the enzyme and bind at strategic locations on the antigen molecule, resulting in a considerable stabilization effect on the enzyme conformation. Moreover, such monoclonal antibodies proved to have a chaperone activity leading to aconsiderable refolding effect on the enzyme which was already partially heat denatured. Renaturation of carboxypeptidase A after heat denaturation in the presence of selected monoclonal antibodies, was followed by recovery of its enzymatic activity. The refolding effect of anti‐CPA monoclonal antibodies, was followed by recovery of its enzymatic activity. The refolding effect of anit‐CPA monoclonal antibodies on heat‐denatured enzyme depends on the degree of denaturation of the enzyme and on the location of the antigenic site of each antibody. The additively effect of the pairs of monoclonal antibodies on the refolding process of CPA proved to be dependent on the localization of the antigenic sites of the monoclonal antibodies studied.
Chromatographic kinetic measurements of human serum albumin adsorption on monoclonal antibodies
Authors: Renard James , Vidal‐Madjar Claire , Sebille Bernard , Lapresle Claude 
Published: May 2005

AbstractA chromatographic method was employed to study the kinetics of human serum albumin (HSA) adsorbed on immobilized monoclonal antibodies. The antibodies of various specificities were covalently bound to a high performance liquid chromatography (HPLC) silica support. For very low desorption rates, successive amounts of the reacting protein were injected until column saturation. The analysis of the increase of the non‐retained fraction calculated from peak area measurements gives the capacity of the support and the rate of the biospecific adsorption process.The model is based on a second‐order Langmuir kinetic law and assumes a global mass transfer for the adsorption process. The use of a silica support of small pore size permits the reduction of the contribution for mass transfer in the stagnant fluid and the decrease in the column capacity: due to its large size, the reacting molecule is adsorbed on the external surface of the particle.The adsorption rate constants of HSA on five monoclonal anti_HSA antibodies of different specificities were determined. For all the immuno‐adsorbents studied, the adsorption rate constant is significantly lower than that found on immobilized polyclonal antibodies. Measurements at different flow rates reveal that the mass transfer due to the transport to the adsorbent surface is small and can be estimated.
Study of cell‐free protein synthesis in aqueous two‐phase systems
Authors: Marszal Ewa , Suchova Martina , Konecny Premysl , Scouten William H. 
Published: May 2005

AbstractProtein synthesis in an aqueous two‐phase system is reported here as a novel type of extractive bioconversion. Translation of BMV RNA was studied using either rabbit reticulocyte lysate or wheat‐germ extract in aqueous dextran–PEG systems. Both polymers inhibited protein synthesis and the translation system partitioned into both phases. In the rabbit reticulocyte two‐phase system, protein synthesis reached 30% of that in free solution, while in wheat in wheat‐germ extract it was 60% of that in free solution. Protein was found mainly in the dextran (lower) phase but its partitioning was related to the polymer concentration, and molecular weight, as well as the ionic strength of the system. Protein synthesis was highly influenced by PEG concentration, potassium chloride concentration, and dextran molecular weight.
Blocking of CD4 cell receptors for the human immunodeficiency virus type 1 (HIV‐1) by chemically modified bovine milk proteins: Potential for AIDS prophylaxis
Authors: Neurath A. Robert , Debnath Asim K. , Strick Nathan , Li Yun‐Yao , Lin Kang , Jiang Shibo 
Published: May 2005

AbstractThe chemical transformation of synthetic combinatorial libraries to increase the diversity of compounds of medicinal interest was reported recently. Chemical modification of natural products represents a complementary approach to accomplish this aim. Modification of lysines by aromatic acid anhydrides, preferentially by 3‐hydroxyphthalic and trimellitic anhydrides and trimellitic anhydride chloride, converted commonly available proteins (human and bovine serum albumin and casein) into potent inhibitors of (i) binding between the HIV‐1 gp120 envelope glycoprotein and the CD4 cell receptor, probably owing to their binding to CD4, and (ii) infection by HIV‐1. Modified bovind milk proteins are also potent HIV‐1 inhibitors and may have protential for anti‐Hiv‐1 prophylaxis.