bluuebunny/crossref_metadata_2025 huggingface dataset:
Zymogen activation: Effect of peptides sequentially related to the bovine β‐trypsin N‐terminus on kazal inhibitor and benzamidine binding to bovine trypsinogen
Authors: Ascenzi Paolo , Coletta Massimo , Amicoi Gino , Bolognesi Martino , Guarneri Mario , Menegatti Enea
Published: May 2005

The activating effect of peptides sequentially related to the Ile 16‐Vall7‐Gly 18 N‐terminus of bovine β‐trypsin (nemely Ile‐Val‐Gly, Ile‐Val, Ile‐Leu Ile‐Ala, Val‐Val, Leu‐Val, and Val‐Leu) on the thermodynamic parameters for the binding of the porcine pancreatic secretory trypsin inhibitor (Kazal inhibtor) and benzamidine to bovine trypsinogen was investigated at pH 5.5 (Bis tris‐HCI buffer, I=0.1 M) and T=21 ± 0.5°C. Thermodynamic parameters for Kazal inhibitor and benzamidine association to the binary peptide/zymogen adducts are more favorable than those observed for ligand binding to the proenzyme alone, although never as much as those reported for the formation of bovine β‐trypsin/Kazal inhibitor and bovine β‐trypsin/benzamidine adducts. Analogously, the affinity of activating peptides for the binary proenzume/Kazal inhibitor and binary proenzyme/benzamidine complexes is higher than that observed for peptide binding to free bovine trysinogen. Differences in affinity for ligand binding to free bovine trypsinogen. Differences in affinity for ligand binding to free bovine trypsinogen, to its binary adducts and to bovine β‐trypsin suggest the presence of different activation levels of the proenzyme, none of which structurally coincide with that achieved in bovine β‐trypsin. The existence of different discrete states suggests that the zymogen‐to‐active enzyme transition should not be considered as a two‐state process but as a multistep event.
Hidden self‐association of proteins
Authors: Muramatsu Nobuhiro , Minton Allen P.
Published: May 2005

Sedimentation equilibrium measurements were carried out on solution bovine serum albumin, aldolase, and ovalbumin in phosphte‐buffered saline, pH7.2, at 10°C. The data obtain for each protein were analyzed to yield the dependence of apparent weight‐average molecular weight upon protein concentration, over a concentration range of ca 1–200 g/L. Using the approximate theory of Chatelier and Minton ((1987) Biopolyymers 26, 507–524), models are formulated for the dependence of apparent weight‐average molecular weight upon cocentration in non‐ideal solutions containing proteins which may self‐associate accroding to a monomer/n‐mer or a monomwer/dimer/tetramer scheme. The concentration dependence data for serum albumin may be accounted for, assuming either no self‐asociation or weak monomer/dimer/association. The data for aldolase may be accounted for assuming either weak monomer/dimer or weak monomer/timer association. The data for ovalbumin may be accounted for assuming eiether weak monomer/trimer or weak monomer/dimer/tetramer association. The association do not approach saturation at the hightest concentrations studied, and the standard‐state free energy change accompanying self‐association amount to less than 4 kcal/mol off intermolecular contacts, suggesting that non‐specific clustering of protein molecules at high concentration rather than the formation of specific complexes is being observed.
Differences between sites of binding to DNA and strand cleavage for complexes of bleomycin with iron of cobalt
Authors: McLean Michael J. , Dar Ahsana , Waring Michael J.
Published: May 2005

The sequence specificity of bleomycin A5 and of its light‐activated cobalt complex were compared by examining the relative cleavage of each strand of two DNA fragments by either species. Significant differences between the two metallobleomycins were observed. The iron–bleomycin (Fe–BLM) complex cleaved the DNA molecules preferentially at dinucleotides GpT and GpC, whereas the light‐activated cobalt‐bleomycin complex (Co–BLM) showed a preference for cutting at the dinucleotide Gp A in addition to cleavage at every GpT dinucleotide. Further, new sites of preferential cleavage were noted for Co–BLM in regions of the DNA where enhanced reaction with DNAaseI can be observed in the presence of the antibiotic. No differences in the cutting behaviour of the Fe–BLM were evident upon irradiation of the reaction mixture. A reduction in the relative efficiency of cutting at GpC sequences by Co–BLM is responsible for the previously observed diminution of double‐strand breaks under condition of photoactivated cleavage. The results are discussed in terms of the likely production of highly reactive, diffusible cutting elements in the light activated reaction, which cause cleavage of the DNA in regions where the antibiotic is not bound.
pH regulation of calcium recognition by an amino acid containing acyclic ionophore
Authors: Spisni A. , Franzoni L. , Corradini R. , Marchelli R. , Dossena A.
Published: May 2005

Calcium ion recognition by a dicarboxylic ionophore containing two (S)‐phenylalanine residues joined via an amide bond by a flexible tri‐oxa‐undecanoyl bridge (Phe‐3‐O) has been investigated in a wide pH range (from pH 2 to 12). Experiments were performed in methanol and chloroform by 1H and 13C NMR, relaxation times and 2D NMR (COSY, NOESY and J‐resolved experiments). Recognition is shown to be regulated by pH, as it occurs in at least three different coordination modes according to the experimental conditions. Even at low pH (pH 2) the ion is already complexed in the compartment created by the ethereal oxygens and the amide carbonyls. At higher pH, it becomes fully encapsulated in a pseudo‐cyclic structure and at very basic pH it is localized between the amide carbonyls and the carboxylates. For these peculiar properties Phe‐3‐O appears to be a very promising ionophore in a transmembrane pH gradient system.
Recognition in cell adhesion. A comparative study of the conformations of RGD‐containing peptides by Monte Carlo and NMR methods
Authors: Cachau Raul E. , Serpersu Engin H. , Mildvan Albert S. , August J. Thomas , Amzel L. Mario
Published: May 2005

Many of the proteins that mediate cell adhesion processes–fibronectin, fibrinogen, vitronectin, von Willebrand factor, osteopontin, laminin and various collagens – contain the amino acid sequence Arg‐Gly‐Asp. Short peptides that include this sequence have been shown to inhibit the interactions of cell adhesion proteins with their receptors and to have dramatic effects on developmental processes involving cellular recognition. In order to determine which conformations are accessible to Arg‐Gly‐Asp containing peptides, we analyzed tri‐, tetra‐ and pentapeptide using molecular mechanics and Monte Carlo methods, and studied the solution conformations of the pentapeptide Gly‐Arg‐Gly‐Asp‐Ser using nuclear magnectic resonance techniques. The Monte Carlo method was used to: (a) identify the low energy conformations of the peptides and (b) evaluate their thermodynamic properties. In the case of the pentapeptide Gly‐Arg‐Gly‐Asp‐Gly, the four stable conformations include three with reverse turns and one open structure. The conformations found in this analysis are compatible with the nuclear magnetic resonance (nuclear Overhauser effect) data.
Interaction of the peptide CF3‐Leu‐Ala‐NH‐C6 H4‐CF3 (TFLA) with porcine pancreatic elastase. X‐ray studies at 1.8 Å
Authors: de la Sierra Ines Li , Papamichael Emmanuel , Sakarellos Constantinos , Dimicoli Jean‐Luc , Prangé Thierry
Published: May 2005

The peptide trifluoroacetyl‐Leu‐Ala‐(p‐trifluoromethylanilide), is a reversible inhibitor of pancreatic porcine elastase and is characterized by a Km of 2.5 × 10−8 M. Co‐crystals of the 1:1 complex were obtained in an acetate buffer + dimethylformamide solution at pH 5.7. Diffraction data were recorded on films at the LURE synchrotron facility. The inhibitor was localized on difference Fourier maps, and the refinement of the structure was performed by simulated annealing (XPLOR). The current agreement factor is R = 19% (for 13224 observed structure factors and 1.8 Å effective resolution). The RMS deviations from ideality of bond distances and angles are 0.02 Å and 2°, respectively. The inhibitor molecule was found in the active site, bent around the side chain of Phe‐215 in a geometry that resembles the previously reported structure of the CF3‐Lys‐Ala complex at 2.5 Å, in a parallel β‐sheet association with the loop 214–216. The analysis of the close contacts (less than 3.5 Å) indicates that the trifluoromethylamide bond interacts with the active site and not the Leu‐Ala or Ala‐anilide bonds. The two fluorinated groups of the inhibitor exhibit different specificities: the trifluoroacetyl group (N terminus) is tightly stacked between the two chain loops 191–195 and 213–215, while the trifluoromethylanilide (C terminus) shows less specificity and only a single contact.
Ligand‐binding properties of estrogen receptor proteins after interaction with surface‐immobilized Zn(II) ions: Evidence for localized surface interactions and minimal conformational changes
Authors: Hutchens T. William , Li Chee Ming
Published: May 2005

The site‐ or domain‐specific immobilization of steroid receptor proteins with preserved structure and function would facilitate the identification and purification of receptor‐associated regulatory components and nucleic acids. We have demonstrated previously that restricted surface regions of the estrogen receptor protein contain high affinity binding sites for immobilized Zn(II) ions. Possible conformational changes in receptor at the stationary phase immobilized metal ion interface were evaluated by monitoring alterations in the equilibrium dissociation constant (Kd) for [3H]estradiol. Soluble estrogen receptor proteins (unliganded) present in immature calf uterine cytosol were immobilized via surface‐exposed Zn(II)‐binding sites to beads of agarose derivatized with iminodiacetate (IDA)‐Zn(II) ions. The IDA‐Zn(II) bound receptor was incubated with increasing concentrations of [3H]estradiol (0.01–20 nM) in the presence and absence of unlabeled competitor (diethylstilbestrol) to determine the level of specific hormone binding. Steroid‐binding experiments were performed in parallel with identical aliquots of soluble receptor. Analyses of the equilibrium binding data revealed the presence of a single class of high‐affinity (Kd = 2.44 ± 1.5 nM, n = 10) steroid‐binding sites which were only marginally affected by receptor immobilization via surface‐exposed Zn(II) bindings sites (Kd = 2.58 ± 0.56 nM, n = 4). These data are consistent with the location of surface accessible Zn(II) binding site(s) on the receptor at or near the DNA binding domain which, upon occupancy, do not influence the steroid binding domain. The high affinity of receptor for immobilized metal ions and lack of evident conformational changes in the immobilized receptor suggest that stationary phases of immobilized metal ions loaded with receptor proteins may be useful as affinity tools for the analysis and purification of receptor‐associated macromolecules.
Specific isolation by anhydrotrypsin‐agarose chromatography of a recombinant protein tagged with an affinity tail arginine at the C‐terminus
Authors: Hirabayashi Jun , Kasai Ken‐ichi
Published: May 2005

A characteristic property of anhydrotrypsin, i.e., its ability to strongly bind C‐terminal arginine, proved to be useful as a tool for specific enrichment of a recombinant protein. An arginine tail was introduced at the C‐terminus, for example, of a human β‐galactoside‐binding lectin by site‐directed mutagenesis. The resulting mutant recombinant lectin, which retained sugar‐binding activity as high as the wild‐type lectin, became recognizable by anhydrotrypsin. It was adsorbed on an anhydrotrypsin‐agarose column and eluted with benzoylglycylarginine. The added arginine tail could be specifically removed by carboxypeptidase B. When E. coli lyzate containing the mutant lectin was applied to the column more than 10‐fold enrichment of the mutant lectin was attained. This procedure should be generally applicable and may be advantageous because the addition of a single arginine residue may have minimal effect on the structure and function of the target protein.
Detection of antigen—antibody interactions by surface plasmon resonance. Application to Epitope Mapping
Authors: Fägerstam Lars G. , Frostell Åsa , Karlsson Robert , Kullman Mari , Larsson Anita , Malmqvist Magnus , Butt Helena
Published: May 2005

Surface plasmon resonance (SPR) detection requires no labelling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosapler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two‐site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti‐mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, and ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi‐site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV‐1 core protein p24 were used in all experiments.
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

The 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

The 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

The 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

Studies 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

We 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

Novel 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

The 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

Endo 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

Values 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

This 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

A 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

An 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

The 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

A 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

Protein 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

The 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.
Analysis of affinity supports by 13C CP/MAS NMR spectroscopy: Application to carbonyldiimidazole‐ and novel tresyl chloride‐synthesized agarose and silica gels
Authors: Zumbrink Thomas , Demiroglou Anastasios , Jennissen Herbert P.
Published: May 2005

A major problem in affinity technology is the analysis of the synthesized solid supports, since liquid phase methodology can generally not be applied. Recently we reported a novel reaction sequence for the 2,2,2‐trifluoroethanesulfonyl chloride (tresyl chloride) coupling of nucleophiles [Demiroglou et al. (1994) Angew. Chem. Int. Ed. Engl. 33, 120–123] to agarose and concluded that previously proposed structures could not be correct. However it was not possible for us to conclusively define the new reaction products because the agarose derivatives could not be solubilized for customary liquid state 13C NMR analysis. Therefore in this paper solid state 13C CP/MAS NMR spectroscopy is applied for the first time to the polysacharide agarose. Using alkyl agarose derivatives prepared by the carbonyldiimidazol method as control we found that reliable spectra in agreement with the published reaction mechanism could be obtained. The method was then applied to the novel reaction products of the tresyl chloride reaction. From the solid state 13C NMR spectra and other quantitative data it is concluded that a β‐sulfonyl carboxylic acid is generated during alkaline hydrolysis of tresyl agarose and that alkyl amines are coupled by a β‐sulfonyl amide bond in an elimination–addition reaction in the absence of SO‐and SC‐scission of the tresyl group. In the case of alkane thiol coupling the absence of SC‐scission cannot be demonstrated indicating either a different reaction mechanism or possibly a mixture of reaction products.
Cholesterol‐dependent modulation of type 1 cannabinoid receptors in nerve cells
Authors: Bari Monica , Paradisi Andrea , Pasquariello Nicoletta , Maccarrone Mauro
Published: May 2005

Type 1 cannabinoid receptors (CB1R) are G‐protein‐coupled receptors that mediate several actions of the endocannabinoid anandamide (N‐arachidonoylethanolamine; AEA) in the central nervous system. Here we show that cholesterol enrichment of rat C6 glioma cell membranes reduces by approximately twofold the binding efficiency (i.e., the ratio between maximum binding and dissociation constant) of CB1R and that activation of CB1R by AEA leads to approximately twofold lower [35S]GTPγS binding in cholesterol‐treated cells than in controls. In addition, we show that CB1R‐dependent signaling via adenylate cyclase and p42/p44 mitogen‐activated protein kinase is almost halved by cholesterol enrichment. Unlike CB1R, the other AEA‐binding receptor TRPV1, the AEA synthetase NAPE‐PLD, and the AEA hydrolase FAAH are not modulated by cholesterol, whereas the catalytic efficiency (i.e., the ratio between maximal velocity and Michaelis‐Menten constant) of the AEA membrane transporter AMT is almost doubled compared with control cells. These data demonstrate that, among the proteins of the “endocannabinoid system,” only CB1R and AMT critically depend on membrane cholesterol content. This observation may have important implications for the role of CB1R in protecting nerve cells against (endo)cannabinoid‐induced apoptosis. © 2005 Wiley‐Liss, Inc.
Übersicht über Verfahren und Vorschriften zur Kurzbeanspruchung im Rahmen der Klimaprüfung
Authors: Kutzelnigg Artur
Published: May 2005

In Form von tabellarischen Übersichten werden die in der deutschen und ausländischen Literatur, insbesondere der Normenliteratur, beschriebenen Kurzbeanspruchungsverfahren im Rahmen der Klimaprüfung zusammenfassend dargestellt. Der Stoff ist nach folgenden Gesichtspunkten gegliedert:

Klimabedingungen vor der Prüfung

Konstantklimate (Kälte, trockene Wärme, hohe Luftfeuchtigkeit.)

Sonderbeanspruchungen (Wasserlagerung, Salzsprühnebel, Schimmel usw.)

Wechselklima (Temperaturwechsel, Betauung usw.)

Kombinierte Prüfzyklen (Wechselbetauung in einer Atmosphäre, die Schwefeldioxyd enthält, Licht mit zusätzlichen Einflüssen usw)

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Zum Problem der interkristallinen‐ und der Spannungskorrosion an den homogenen Kupfer‐Gold und Kupfer‐Zink und an ausscheidungsfähigen Aluminium‐Zink‐Magnesium‐Mischkristallen
Authors: Klatte Heinrich
Published: May 2005

Um die Bedingungen, unter denen Spannungskorrosion an homogenen Mischkristallen auftritt, zu ermitteln und einen Mechanismus der Rißbildung zu entwickeln, wird von Gold‐Kupfer‐Mischkristallproben mit 10 bis 90 At.‐% Gold und Kupfer‐Zink‐Mischkristallproben mit 63 bis 90 Gew.‐% Kupfer, nachdem sie verschieden lange unter Quecksilber gelagert worden sind, die Zugfestigkeit bestimmt. Erfolgt dabei der Zugversuch unter Anwesenheit des Quecksilbers, so beobachtet man einen hohen Festigkeits‐abfall, der von der Mischkristallkonzentration abhängt und bei 50 At.‐% am größten ist. Der gleiche Effekt wird auch von chemischen Agenzien ausgelöst, doch beschränkt er sich hier auf die unedle Seite des Mischkristallsystems. Da an reinen Metallen dieser Festigkeitsabfall nicht auftritt, wird auf eine erhöhte Reaktionsfähigkeit der Mischkristallkorngrenze geschlossen, die als Ursache der Spannungskorrosionsempfindlichkeit anzusehen ist. Die bei der Spannungskorrosion auftretende Rißbildung ist dabei darauf zurückzuführen, daß das Korrosionsmittel stets mit einem sich plastisch verformenden Material reagiert, dessen Reaktionsfähigkeit während des Verformungsvorganges stark erhöht ist.
Korrosionsschutz‐ und Werkstoff‐Probleme des modernen Säurebaues
Authors: Schücking Günter
Published: May 2005

Es wird auf die Aufgaben und auf die Industrialisierung des modernen Säurebaues hingewiesen. Der Säurebau befaßt sich mit Säure‐ und Korrosionsschutz‐Problemen sowie mit der Planung und Erstellung säurefester Anlagen und Industriebauten. Eine Zusammenstellung der charakteristischsten Arbeitsmethoden gibt einleitend eine Übersicht, getrennt für Beton und Metall. Es werden zahlreiche Roh‐, Hilfs‐ und Werkstoffe aufgezählt und ihre wesentlichsten physikalischen Konstanten tabellarisch zusammengestellt. Dabei wird auf die Bedeutung der Schutzschicht hingewiesen und auf Fehlerquellen aufmerksam gemacht. Zwei neue Hilfsstoffe, Korroplast N und H, finden im modernen Säurebau in steigendem Maße Verwendung. Einige säurefeste Auskleidungen nach neuesten Erkenntnissen werden beschrieben und eine Übersicht über die gängigsten Kitt‐Typen gegeben. Im Zusammenhang mit den zwangsläufig erforderlichen eigenen Entwicklungsarbeiten im Rahmen des Säurebaues werden einige Beispiele angeführt. Auch an den industriellen Abwasser‐ und Abluft‐Problemen ist die Säurebau‐Industrie maßgeblich beteiligt, und sie werden in ihren wesentlichsten Grundzügen skizziert. Eine ausführliche Beständigkeitsliste wird beigefügt.
Über den kathodischen Korrosionsschutz im Boden auf galvanischem Wege II
Authors: Markovic T. , Dugi Z. , Sevdic D.
Published: May 2005

Nachdem über die Ergebnisse der mit kathodisch geschützten Eisenproben in belüfteten und unbelüfteten Böden durchgeführten Korrosionsversuche bereits berichtet wurde (Mitteilung I), wird in der vorliegenden Arbeit über weitere Korrosionsversuche berichtet, die mit kathodisch geschütztem Eisen im Erdreich zur Klärung des Einflusses des W‐Bd‐Verhältnisses durchgeführt wurden. Wie zu erwarten war, wurde der Angriff der geschützten Probe verringert, wenn der Boden die Sättigungsgrenze an Wasser erreicht hatte. Auf Grund der durchgeführten Versuche können quantitative, zuverlässige Aussagen über die Wirkung des kathodischen Schutzes auf den Korrosionsablauf des geschützten Metalls in Abhängigkeit vom W‐Bd‐Verhältniswert, der für die Aggressivität des Bodens maßgebend ist, gemacht und zur Aufstellung allgemeiner Korrosionsschemen für einen kathodisch zu schützenden Werkstoff herangezogen werden.
Chemische Reaktionen des Zinns und ihre Auswirkungen auf verzinnte Konservendosen
Authors: Heintze K. , Braun Fr.
Published: May 2005

Behandelt wird das Verhalten von Zinn und Weißblech unter atmosphärischen Einflüssen. Daran anknüpfend wird das Problem der Außenkorrosion von Weißblechdosen erörtert und Ratschläge zu ihrer Verhinderung gegeben. Die Wirkung von Säuren sowie von sauren Konservierungsgütern auf Zinn und Weißblech wird eingebend untersucht. Lochfraß, Wasserstoffbombage und Umkehr der Polarität des Zinn‐Eisensystems durch Zinnkomplexbildner werden behandelt. Desgleichen werden die verschiedenen Korrosionserscheinungen durch alkalisch wirkende Substanzen dargestellt. Sulfidverfärbungen durch sulfide enthaltende oder Sulfid abspaltende Stoffe werden unter den verschiedensten Bedingungen besprochen; desgleichen eine Reihe von neutralen Stoffen, die Weißblech angreifen können. Abschließend werden einige Hinweise über Stablblechqualität, Verzinnungsart und Lackierung von Weißblechdosen in Bezug auf Korrosionserscheinungen gegeben.
Die Korrosion in Hochtemperaturkesseln
Authors: Werner M.
Published: May 2005

Die Erfahrungen über das Korrosionsverhalten der hochwarmfesten Stähle in Hochtemperaturkesseln werden in Deutschland an Hand der im Betriebe befindliche 600°‐Kessel nach rd. 40 000‐stündiger Betriebszeit besprochen. Die Dampfzunderung in reinem Dampf ist gering. Gefahren treten erst auf, sobald der Dampf merkbar verunreinigt ist. dieses läß sich bei dem heutigen hohen Stande der Wasseraufbereitung vermeiden, so daß Korrosionsschäden von der Wasserseite her als ungewöhnlich zu betrachten sind.

Schäden von der Rauchgasseite her sind bekannt geworden, sobald in der Flugasche Alkalisulfate vorkommen. Der Schadensbereich liegt in erster Linie dort, wo ein Eutektikum der Sulfate zu schmelzen vermag. Es werden Wege angegeben, wie diese Korrosion zu vermeiden ist.

Vanadinhaltige Stäle scheiden für Temperaturen oberhalb 600° aus, da dann dieselbe Vanadinzunderung auftreten kann, welche von der Verfeuerung vanadinhaltiger Öle her bekannt ist.
Korrosionsschutz bei Seeversand im Tropenklima
Authors: Determann Hermann
Published: May 2005

Die beim Überseeversand gebräuchlichen Korrosionsschutzmethoden werden als bekannt vorausgesetzt. Hier wird über spezielle Beobachtungen bein praktischen Versuchsfahrten berichtet. Kieselgel also Trocknungsmittel wirkt immer korrosionhemmend, wenn die Menge ausreicht, um die Luftfeuchtigkeit auf dem ganzen Reiseweg unterkritisch zu halten. Bei kleineren Kieselgelmengen kann je nach Dichtigkeit des Verschlusses verstärkte oder verminderte Korrosion auftreten. Bakterien fördern stark die Korrosion. Bananen bilden für solche Bakterien einen guten Nährboden. Korrosionsschutzfett oder Wachs bieten trotzdem ausreichenden Schutz. VPI also Korrosionsschutzgas für Stahl hat sich beim Überseeversand gut bewährt.
Aufbau, Arbeitsweise und Einsatz eines neuen Universal‐Biegeprüfers
Authors: Köhler Walther
Published: May 2005

Einleitend wird auf die technische Bedeutung des Biegeversuchs für die Werkstoffprüfung hingewiesen. Anschließend werden Aufbau, Arbeitsweise, Eichung und Einsatz eines neuen Universal‐Biegeprüfers in allen Einzelheiten behandelt. Ein ausführlicher wiedergegebenes, an E‐ und NE‐Metallen sowie organischen Werkstoffen (Kunststoffe, Holz) erhaltenes Versuchsmaterial belegt sinnfällig die universelle Anwendung (Aufnahme von Biegespannungs‐Durchbiegungs‐Diagrammen, E‐Modul‐ und Trägheitsmoment‐Bestimmungen) des beschriebenen Biegeprüfers.
Neue silikatische Schutzschichten auf Metalloberflächen
Authors: Loebich Otto
Published: May 2005

Verfasser beschreibt eine neue Silikaschicht (Inverron‐Schicht) für Metalloberflächen, die einfacher aufzubringen ist als eine Emaillierung, die gegenüber Lacken und Anstrichen den Vorteil der Unbrennbarkeit und Temperaturbeständigkeit bis 400°C hat, sich durch gute chemische Beständigkeit auszeichnet und den Metallglanz der Oberfläche nicht beeinträchtigt.