bluuebunny/crossref_metadata_2025 huggingface dataset:
Tris (2,2′‐bipyridyl)ruthenium (III) as a chemiluminescent reagent for detection in capillary electrophresis
Authors: Dickson James A. , Ferris Matt M. , Milofsky Robert E.
Published: May 2005

A post‐ column chemiluminescent technique for thedetection of compounds that are poor chromoshores using electorogenerated chemiluminescence following separation by capillartgy electrophoresis is described. The luminrescent signal is generated followintg the reaction of anlyres (e.g. amines) with Ru(bpy)33+, which isx electrochemically generated post‐columan from Ru(bpy)32+. Tripropylamine and proline are used as two model compounds to demostrate the feasibility of the method. Detection limits for the prototype system were in the micromolar rage, suggesting that this technnique offers an alternative to indirect detection of compounds that are poor chromophores with an added selectivity advangage. The system includes the use of a conductive joint to isolate the separation field from the potential necessary to drive the elctrogenerated chemiluminescent reactiion. Addition of the chemiluminescent reagent Ru(bpy)32+ post‐column did not decrease the efficiency of the separation. The design and favrication of the novel cell is discussed.
Molecular recognition: Model studies with convergent functional groups
Authors: Rebek Julius
Published: May 2005

Model studies of molecular recognition are reviewed with emphasis on contributions from macrocyclic chemistry. Recent developments concerning new molecular shapes are discussed. The advantages of a molecular cleft are presented. In these structures functional groups converge to create a microenvironment complementary to substrates. Specifically, di‐, tri‐ and tetracarboxylic acids of varying sizes are shown to recognize smaller molecules of complementary shape. The new receptors function by a combination of hydrogen bonding and aryl stacking interactions. Substrates include amines, acids, amino acids, metal ions and nucleotide components. The relationship between functional group orientation and catalysis is explored and two systems capable of concerted acid/base catalysis are introduced.
Monosized, magnetic polymer particles: Their use in separation of cells and subcellular components, and in the study of lymphocyte functionin vitro
Authors: Lea T. , Vartdal F. , Nustad K. , Funderud S. , Berge A. , Ellingsen T. , Schmid R. , Stenstad P. , Ugelstad J.
Published: May 2005

By employing the principles of “activated swelling”, monosized, superparamagnetic polymer particles have been prepared ranging in size from 1–100 μm. Both during and after the swelling process, the particles can be modified to meet a series of specific demands making them potentially very interesting for many separation and assay purposes.

Using monoclonal antibodies to direct the magnetic beads to their targets, immunomagnetic separation has turned out to be one of the most specific, reliable and, above all, the fastest technique available today to isolate particulate material for further studies. So far, most efforts have been concentrated on methodology for fractionation of cells in suspension, such as removal of tumour cells from bone marrow or isolation of lymphoid cells from peripheral blood. These studies have both established the parameters necessary for optimal performance and at the same time laid the groundwork for future developments making immunomagnetic separation an exciting new tool in many research areas.

High speed and specificity are the most conspicuous features of immunomagnetic cell separation. These properties have been exploited in the successful development of a new technique for tissue typing of cells directly from peripheral blood specimens. Both higher sensitivity and specificity have been obtained. The same principles can be used for fast and safe quantification of cell populations and subpopulations in blood and cell suspensions.

The functions of, and interactions between, peripheral blood cell populations or subpopulations in the immune response have also been studied with high precision. The significance of direct cell contact on the one hand, and soluble factors on the other, can now be established in detail. Immunomagnetic beads have also been used to study the interaction between various T lymphocyte membrane molecules in the early phases of the activation process.

Finally, the usefulness of specially developed particles for the fractionation of subcellular components is described.
Substance P synthesis by enzymatic fragment condensation using product–directed antibodies as molecular traps
Authors: Nyberg Fred
Published: May 2005

This paper describes the enzyme catalyzed synthesis of the undecapeptide substance P from its non‐associated fragments (1–7) and (8–11) or (1–8) and (9–11). The fragment condensation was mediated by the use of product specific antibodies as molecular traps. As catalyst a previously purified endopeptidase was used which specifically hydrolyzes substance P at the Phe7–Phe8 and Phe8–Gly9 bonds. The synthesis was performed in analytical scale and product formation was guided by reversed phase HPLC combined with radioimmunoassay. It appeared that the substance P fragments (1–8) and (9–11) were condensed to a larger extent than (1–7) and (8–11). This observation may well result from the higher affinity of the antibodies observed for substance P (8–11) as compared to that found for the other fragments. Increased concentration of the antibodies also seemed to result in enhanced resynthesis of substance P.
Estrogen receptor interaction with immobilized metals: Differential molecular recognition of Zn2+, Cu2+ and Ni2+ and separation of receptor isoforms
Authors: Hutchens T. William , Li Chee Ming
Published: May 2005

We have utilized iminodiacetate (IDA) gels with immobilized Zn2+, Cu2+ and Ni2+ ions to evaluate the metal binding properties of uterine estrogen receptor proteins. Soluble (cytosol) receptors labeled with [3H]estradiol were analyzed by immobilized metal affinity chromatography (IMAC) before as well as after (1) 3 M urea‐induced transformation to the DNA‐binding form, and (2) limited trypsin digestion to separate the steroid‐ and DNA‐binding domains. Imidazole (2–200 mM) affinity elution and pH‐dependent (pH 7–3.6) elution techniques were both evaluated and found to resolve several receptor isoforms differentially in both the presence and absence of 3 M urea. Individual receptor forms exhibited various affinities for immobilized Zn2+, Cu2+ and Ni2+ ions, but all intact receptor forms were strongly adsorbed to each of the immobilized metals (Ni2+ > Cu2+ » Zn2+) at neutral pH. Generally, similar results were obtained with IDA–Cu2+ and IDA–Ni2+ in the absence of urea. Receptors were tightly bound and not eluted before 100 mM imidazole or pH 3.6. Different results were obtained using IDA–Zn2+; at least four receptor isoforms were resolved on IDA–Zn2+. Receptor–metal interaction heterogeneity and affinity for IDA–Zn2+ and IDA–Cu2+, but not IDA–Ni2+, were substantially decreased in the presence of 3 M urea. The receptor isoforms identified and separated by IDA–Zn2+ chromatography were not separable using high‐performance size‐exclusion chromatography, density gradient centrifugation, chromatofocusing or DNA‐affinity chromatography. The affinity of trypsin‐generated (mero) receptor forms for each of the immobilized metals was decreased relative to that of intact receptor. High‐affinity metal‐binding sites were mapped to the DNA‐binding domain, but at least one of the metal‐binding sites is located on the steroidbinding domain. Recovery of all receptor forms from the immobilized metal ion columns was routinely above 90%. These results demonstrate the differential utility of various immobilized metals to characterize and separate individual receptor isoforms and domain structures. Receptor‐metal interactions warrant further investigation to establish their effects on receptor structure/function relationships. In addition to the biological implications, recognition of estrogen receptor proteins as metal‐binding proteins suggests new and potentially powerful receptor immobilization and purification regimes previously unexplored by those in this field.
Conformational and receptor binding properties of human EGF and TGF‐α second loop fragments
Authors: Han Kyou‐Hoon , Ferretti James A. , Niu Chien‐Hua , Lokeshwar Vinatu , Clarke Robert , Katz Deborah
Published: May 2005

The solution conformation of the second loop fragment of human EGF, [Ala20]EGF(14–31), was determined using two‐dimensional NMR homonuclear Hartmann–Hahn and rotating frame nuclear Overhauser enhancement spectroscopy. The results are compared with the conformation of the second loop fragment of human TGF‐α, [Ala21] TGF‐α(16–32), and with that of the second loop of intact EGF. Comparison of the two experimentally determined structures of the second loop fragments shows significant differences in the turn regions of each peptide. For the EGF fragment, hydrophobic side chain groups protrude away from the ring, whereas for the TGF‐α fragment hydrophilic groups are directed away from the ring. Although these turn regions represent the putative receptor binding sites, neither second loop fragment binds to the EGF receptor. The biological activity is discussed in terms of the conformational differences found for the two second loop fragments.
Chemical probes reveal no evidence of Hoogsteen base pairing in complexes formed between echinomycin and DNA in solution
Authors: McLean Michael J. , Waring Michael J.
Published: May 2005

Five different DNA fragments have been treated with a range of conformationally sensitive reagents in an effort to probe structural chages in DNA associated with binding of the bis‐intercalating antibiotic echinomycin. For each probe, the intensity and pattern of its reactivity with DNA have been analyzed in order to elucidate the effect of antibiotic binding on the accessibility of a specific site of sites to chemical attack. It was found that in one of the DNA fragments, pTyr2DNA, several purine residues exhibit enhanced reactivity to diethyl pyrocarbonate (DEPC) in the absence of bound antibiotic, and that this strongly sequence specific reaction is enhanced in the presence of quite low echinomycin concentrations. The echinomycin‐dependent reactivities towards DEPC of three homologous DNA fragments, chosen for their subtly different antibiotic binding characteristics, were also investigated. It was found that small changes in base sequence generate striking changes in susceptibility to modification by DEPC. The abolition of one antibiotic binding site leads to the creation of an new, intense DEPC‐reactive site. In the presence of moderate concentrations of echinomycin, specific thymidine residues exhibit enhanced reactivity towards osmium tetroxide. No differences in the reactivities of the DNA fragments towards bromoacetaldehyde, S1 nuclease, dimethyl sulphate of potassium tetrachloropalladinate were observed in the presence of the antibiotic. DEPC reactions were performed on tubercidin (7‐deaza‐adenosine) to determine the DEPC reactive positions in situations where N‐7 is inaccessible. Tubercidin was found to be generally resistant to attack by DEPC followed by treatment with base. We conclude that the bulk of structural changes induced by the binding of echinomycin to DNA do not involve Hoogsteen base pairing, but rather are due to sequence‐specific unwinding of the helix in a manner which is strongly dependent on the nature of surrounding nucleotide sequences.
Molecular recognition between oligopeptides and nucleic acids. Sequence specific binding of (4S)‐(+)‐ and (4R)‐(−)‐dihydrokikumycin B to DNA deduced form 1H NMR, footprinting studies and thermodynamic data
Authors: Lee Moses , Shea Regan G. , Hartley John A. , Lown J. Willliam , Kissinger Koren , Dabrowiak James C. , Vesnaver Gorazd , Breslauer Kenneth J. , Pon Richard T.
Published: May 2005

The sequence specific binding of the antibiotic (4S)‐(+)‐dihydrokikumycin B and its (4R)‐(−)enantiomer, [(S)‐I and (R)‐I, respectively] to DNA were characterized by DNase I and MPE footprinting, calorimetry, UV, spectroscopy, circular dichroism, and 1H NMR studies. Footprinting analyses showed that both enantiomers [(S)‐I and (R)‐I] bind to AT‐rich regions of DNA. 1H NMR studies (ligand induced chemical shift changes and NOE differences) of the dihydrokikumycins with d‐[CGCAATTGCG]2 show unambiguously that the N to C termini of the ligands are bound to 5′‐A5T6T7‐3′ reading from left to right. From quantitative 1D‐NOE studies, the AH2(5)‐ligand H7 distance of complex A [(S)‐I plus decamer (which is bound more strongely)] and complex B[(R)‐I and decamer] are estimated to be 3.8 ± 0.3 Å and 4.9 ± 0.4 Å, respectively. This difference in binding properties is reflected in the thermodynamic profiles of the two enantiomeric ligands determined by a combination of spectroscopic and calorimetric techniques. The binding freee energies (ΔG°) of (S)‐I and (R)‐I to poly d(AT)·poly d(AT) at 25°C are −31.8 and −29.3 kJ mol−1, respectively while the corresponding binding enthalpies (ΔH°) are −11.3 and −0.8 kJ mol−1. These data permit the construction of models for the binding of the enantiomeric dihydrokikumycins to DNA and account for the more efficient binding of the natural (S) isomer to DNA.
Evidence for the presence of disulfide bridges in opioid receptors essential for ligand binding. Possible role in receptor activation
Authors: Gioannini T. L. , Liu Y. F. , Park Y‐H. , Hiller J. M. , Simon E. J.
Published: May 2005

The mobility of purified μ opioid binding protein in SDS‐polyacrylamide gek electrophoresis is sensitive to the presence of reducing agents. In the presence of increasing concentrations of DTT the apparent molecular weight increases in a stepwise fashion from 53 kDa to 65 kDa. This reduction in mobility is attributed to the successive breakage of disulfide bridges, resulting in an increasingly asymmetric molecule. Treatment of cell membranes from various brain areas with reducing agents, such as DTT, produced a concentration‐dependent inhibition of opioid binding. Sensitivity to DTT inhibition varied between receptor types, μ > δ ≫ κ. For μ receptors, agonist binding was considerably more sensitive to DTT than antagonist binding. Inhibition by DTT is readily reversible and is unaffected by Na+ and/or Mg2+ ions. Reversibility may be partially prevented by the inclusion of a low concentration of a reducing reagent such as glutathione which does not inhibit binding but blocks reformation of disulfide bonds. Scatchard analysis of saturation data shows that DTT causes a pronounced decrease in binding affinity with little effect on receptor number. It is suggested that disulfide bonds are essential for ligand binding and that cleavage of one or more of these bonds may play a role in opioid receptor activation by agonists.
Regional ligand domain is involved in scavenger receptor‐mediated recognition of maleyl‐albumin by rat sinusoidal liver cells
Authors: Horiuchi Seikoh , Takata Kyoko , Araki Norie , Morino Yoshimasa
Published: May 2005

Scavenger receptor‐mediated endocytosis of maleyl‐albumin was studied with rat sinusoidal liver cells. Upon maleylation of > 28 mol lysine residues per protein, bovine serum albumin became an active ligand. Further modification of up to 37 mol lysine residues per protein resulted in a sharp increase in the ligand acitivity, reaching a maximum level thereafter. Removal of maleyl moieties from maleyl‐albumin (demaleylation) from 53 mol to 14 mol lysine residues per protein did not affect the ligand acitivity. However, further demaleylation to less than 5 mol lysine residues per protein led to complete loss of the ligand activity. Thus, the covalently incorporated maleyl moieties are needed for the ligand activity. The ligand activity was also generated when two peptides (Frag N and Frag C) from cyanogen bromide‐cleaved albumin were maleylated, indicating that the formation of an active ligand would not require a whole albumin molecule. Maleyl Frag C was further separated into three peptides; maleyl Frag C‐1 (261 amino acid residues), maleyl Frag C‐2 (102 residues) and maleyl Frag C‐3 (36 residues). The cellular binding and endocytic degradation of maleyl‐albumin or acetylated low density lipoprotein were effectively competed for by maleyl Frag C‐1 and maleyl Frag C‐2 but not by maleyl Frag C‐3. Thus, regional domains might be involved in the ligand recognition by the scavenger receptor.
Application of a degenerate consensus sequence to quantify recognition sites by vertebrate DNA topoisomerase II
Authors: Spitzner J. R. , Muller M. T.
Published: May 2005

A consensus sequence has been derived for vertebrate topoisomerase II cleavage of DNA (Spitzner, J. R. and Muller, M. T. (1988) Nucleic Acid. Res. 16, 5533–5556). An independent sample of 65 topoisomerase II sites (obtained in the absence of topoisomerase II inhibitors) was analyzed and found to match the consensus sequence as well as enzyme sites determined in the presence of the anti‐tumor drug 4′‐(9‐acridinyl‐amino)‐methanesulfon‐manisidide (m‐AMSA). As originally described, conventional application of the consensus sequence afforded accuracy in the prediction of the locations but not the frequencies of topoisomerase II cleavages. In the present report, we describe a new method which quantitatively discriminates sites from nonsites, called the ‘matrix mean’ method (the mean match of a site to the matrix of base proportions from the original consensus sequence derivation). Furthermore, we derived a second method, called the ‘unique score’ model, which predicts frequency of topoisomerase II activity at a cleavage site. In the unique score method both DNA strands of a site are examined to determine the total number of the consensus positions that match on at least one strand of a potential site. From the new data base of 65 topoisomerase II sites, cleavages were scored for relative cleavage strength. Linear regression analysis showed a significant (p < 0.01) correlation between the unique score and cleavage strength. The study was extended to show that the unique score model accurately and quantitatively predicts topoisomerase II sites either in the absence or presence of m‐AMSA using the same consensus sequence.
Compartmentation of deoxypyrimidine nucleotides for nuclear DNA replication in S phase mammalian cells
Authors: Reddy G. Prem Veer
Published: May 2005

DNA synthesis in S phase Chinese hamster embryo fibroblast cells in the presence of exogenous 3H‐dUrd shows incorporation of the labeled precursor with very little dilution by the large unlabeled intracellular precursor pools. Full mixing would predict a specific activity 10‐fold less than that measured. This coupled with the finding that 80% of the radioactivity from the exogenous 3H‐dUrd appears in the karyoplasts implies a compartmentation where 3H‐dUMP and 3H‐dTTP derived from exogenous 3H‐dUrd do not mix freely with endogenous cytoplasmic pools.
A heme‐ and metal‐binding hexampeptide from the sequence of rabbit plasma histidine‐rich glycoprotein
Authors: Morgan William T. , Deaciuc Victor , Riehm John P.
Published: May 2005

Rabbit histidine‐rich glycorprotein (HRG) binds low‐spin heme and metal tightly at several sites that contain histidine. As part of an on‐going effort to define and locate the binding sites for these and the other ligands of HRG, the sequence: NH2‐Gly‐His‐Phe‐Pro‐Phe‐His‐Trp‐… was found in a 16kDa heme‐binding peptide isolated from HRG. The spacing of the histidyl residues in this peptide, which contains the C‐terminal 79 residues of HRG, together with molecular modeling suggested that this sequence might constitute one heme binding site of HRG by accommodating heme in a bis‐histidyl linkage. Three peptides based on this sequence (I, HFPFHW; II, WHFPFH; and III, HFGFHW) were synthesized, and their ability to bind heme and metals examined. All three peptides bind heme as demonstrated by the changes produced in the absorbance of heme when mixed with the peptides. Substituting glycine for proline in the central position or moving the location of the tryptophan did not effect heme binding. The apparent Kd's of the mesoheme/peptide I, II and III complexes are 75 ± 25 μM, indicative of heme binding approximately 100 times less avid than the mesoheme/HRG complex (Kd ca. 1 μM), but nearly 1000 times tighter than that of the mesoheme/histidine complex (Kd ca. 60mM). The absorbance spectra of the mesoheme/peptide complexes, the loss of binding caused by modification of histidine residues, and the pH dependence of heme binding, all indicate that heme forms a low spin, bis‐histidyl type of complex with these peptides, like that formed with HRG itself. Copper, but not cadmium or nickel, was an effective inhibitor of heme binding by the peptides. The sequence of HRG congruent with the sequence of peptide I is proposed to be one heme‐ and metal‐binding site of rabbit HRG.
Binding of lanthanum and gadolinium ions to Concanavalin A studied calorimetrically at 25°C
Authors: Barone Guido , Castronuovo Giuseppina , Del Vecchio Pompea , Elia Vittorio , Guarrata Patrizia
Published: May 2005

The interaction between Concanavalin A (ConA) and the lanthanide ions La3+ and Gd3+ has been studied calorimetrically at 25°C. The measurements were carried out at a pH of 4.5, where the protein exists prevailingly as a dimer. Calorimetry allows the direct determination of the binding enthalpy and the evaluation of both the apparent association constant, and the apparent free energy and entropy. Three groups of data were collected. The first concerns the interaction of the ‘native’ protein, i.e., fully metallized with Mn2+ and Ca2+, with the lanthanides. The second concerns the interaction of the completely demetallized protein with La3+ and Gd3+. Finally, the affinity of each complex was tested for the specific sugar α‐methylmannopyranoside. The analysis of the thermodynamic parameters obtained, led to the following conclusions: (1) a specific site, named S3, exists on the protein for the lanthanides, distinct from the S1 site of the transition metal and from the S2 site, specific for calcium. There is only one S3 site per protomer when the protein has Mn2+ in S1 and Ca2+ in S2. Moreover, there is no appreciable competition for S1 and S2 from the lanthanides. The ‘native’ protein, metallized with La3+ or Gd3+, is a fully functional protein. (2) The demetallized protein (ApoCon A) has at least two sites per protomer for the lanthanides. The hypothesis is that, besides the S3 site, the lanthanides, in the absence of Mn2+, can also occupy the S1, but not the S2, site. The protein metallized only with gadolinium ions is completely inactive toward the interaction with the mannoside. The same happens when, along with gadolinium, only calcium or manganese is present. Hence, in the absence of the transition metal in S1 or of calcium in S2, the protein is not in the conformation suitable to interact with its specific substrate.
Molecular recognition between oligopeptides and nucleic acids. DNA sequence specificity and binding properties of an acridine‐linked netropsin hybrid ligand
Authors: Bailly Christian , Helbecque Nicole , Hénichart Jean‐Pierre , Colson Pierre , Houssier Claude , Rao K. Ekambareswara , Shea Regan G. , Lown J. William
Published: May 2005

The binding to DNA of a mixed function ligand (NETGA) is described, in which a potential intercalating group, an acridine moiety, is incorporated at the carboxyl terminus of the minor groove binding oligopeptide netropsin skeleton. Scatchard analysis of absorption data provided evidence of two modes of binding to DNA withK1= 9.1 × 105M−1at low r values (0.003–0.1), and a binding site sizen= 10, indicative of binding of both moeities. At high binding ratios (>0.1),K2= 0.9 × 105M−1andn= 5 corresponding to external binding. Complementary strand MPE footprinting on a pBR322 restriction fragment showed NETGA binds to 5′‐AAAT like netropsin. It causes enhanced cleavage by MPE, particularly at G‐C rich sequences and remote from the preferred binding sites. Viscometry measurements provided evidence for biphasic modes of the two binding portions of NETGA. Fluorescence polarization and linear dichroism measurements were in accord with distinct modes of interaction of the acridine (intercalation) and oligopeptide (minor groove binding) portions of NETGA. LD measurments of NETGA indicate that the oligopeptide moiety (netropsin‐like) has an orientation typical of minor groove binders, whereas the degree of intercalation of the acridine group is decreased by association of the oligopeptide moiety.
The formation of mispairs by 8‐oxyguanine as a pathway of mutations induced by irradiation and oxygen radicals
Authors: Poltev Valery I. , Shulyupina Nataly V. , Bruskov Vadim I.
Published: May 2005

Guanine conversion to 8‐oxyguanine (OG) was induced by irradiation and oxygen radicals. The pathways of mutations caused by such DNA damage have been considered. Minima of interaction energies of OG with nucleic acid bases are revealed via classical potential function calculations. The minima exist for OG*:G and OG*:A base pairs, with syn‐conformation of OG* and OG:T wobble base pairs. The mutual positions of glycosyl bonds in these base pairs are quite close to those for Watson‐Crick pairs, and energies of OG*:G, OG*:A and OG:T mispairs are close to the energy of A:T. The results calculated suggest that these mispairs could arise as intermediates in the transversions C:G to G:C and C:G to A:T, and the transition G:C to A:T.
Effects of monomethoxypoly(ethylene glycol) modification of ribonuclease on antibody recognition, substrate accessibility and conformational stability
Authors: Caliceti P. , Schiavon O. , Veronese F. M. , Chaiken I. M.
Published: May 2005

The effects of modification of bovine pancreatic ribonuclease A by monomethoxypoly(ethylene glycol) (MPEG) were examined for changes in recognition by antiRNase antibodies, enzymatic activity against low and high molecular weight substrates and conformational stability to temperature elevation. Modified forms of RNase were prepared containing an average of 4, 9, and 11 mol of MPEG/mol protein, by amino group modification. These were analysed by binding to RNase antibodies crosslinked to solid phase‐immobilized protein A. The affinity column was incorporated into a high performance liquid chromatograph and the RNase species were studied by both zonal and frontal analytical affinity chromatography. An antibody dissociation constant of 7.6 × 10−8 M was found for unmodified RNase, as compared to values of 1.3 × 10−7 and 1.2 × 10−6 M for RNase with 4 and 9 covalently bound MPEG chains, respectively. Modification also led to progressive loss of enzymatic activity against RNA, down to 3% for the most highly modified enzyme. In contrast, enzymatic activity against cytidine‐2′,3′‐cyclic monophosphate was suppressed to a maximum of only 33% at the highest modification level, and the stability to temperature, as followed by circular dichroism, was reduced only partially, from 67°C for native protein to 57°C for RNase with 11 mol equivalents MPEG incorporated. The above differential effects on enzymatic activity, antibody binding and temperature effects are consistent with the view that MPEG modification has relatively small effects on conformational stability and small molecule accessibility, but more dramatic effects on large molecule (substrate as well as antibody) accessibility. The latter is likely a strong contribution to the reduced immunogenicity observed generally with MPEG modified proteins.
The specificity of the binding of platelet activating factor (PAF) to anti‐PAF antibodies
Authors: Smal Mary A. , Baldo Brian A. , Harle David G.
Published: May 2005

Quantitative hapten inhibition experiments employing sheep anti‐PAF antibodies and selected PAF analogues were undertaken with the aim of defining the antigenic determinant structures complementary to the antibody combining sites. The most important fine structural features for inhibition of antibody binding to PAF were shown to be an acetyl group at position 2 of the phospholipid glycerol backbone and an ether group at position 1. Of the naturally occurring compounds, C16‐ and C18:1‐PAF proved to be the most potent inhibitors and more active than C18‐PAF while phospholipids with a propionyl, butyryl or hexanoyl group at position 2 showed either weak or no inhibitory activity. The 1‐acyl, thioether and deoxy analogues proved inactive. Variations in the polar head group of PAF were found to be less critical with, for example, the dimethyl and ethanolamine derivatives retaining some activity. This antibody recognition pattern is very similar to that of the PAF receptor, although the antibodies appear to have a more specific requirement for an acyl linkage at position 2.
Citrate synthase 1 interacts with the citrate transporter of yeast mitochondria
Authors: Grigorenko Elena V. , Small W. Curtis , Persson Lars‐Olof , Srere Paul A.
Published: May 2005

We have previously shown that citrate synthase binds to an intrinsic protein of the mitochondrial inner membrane (D'Souza and Srere, 1983). In this paper we present evidence that this citrate synthase binding protein is the citrate transporter. We have used citrate synthase 1 mutants of Saccharomyces cerevesiae and transformants containing citrate synthase inactivated by site‐directed mutagenesis to study the effect of the CS1 protein upon mitochondrial function (Kispal and Srere). In the present study citrate uptake and oxidation were measured during state 3 conditions (presence of 200 μM ADP) in the mitochondria of several strains of Saccharomyces cerevesiae: a parental strain containing wild‐type mitochondrial citrate synthase (CS1) and strains derived from a CS1 deficient strain in which the CS1 gene was disrupted by insertation of the LEU2 gene. These strains were generated from the CS1− cells by transformation with vectors encoding site‐specific mutants of CS1 possessing very low levels of enzymatic activity. One such strain in this study was subsequently found to have undergone reversion to produce a strain which had activity very similar to wild type. Positive correlation between citrate uptake and the rate of citrate oxidation was found, suggesting coupling of the two processes. Both mitochondrial citrate uptake and oxidation were decreased in the mutant lacking any form of CS1 protein. Reintroduction of mutagenized CS1 into yeast causes an enhancement in the rate of state 3 oxygen consumption and of citrate uptake. The observed respiratory increase produces a state 3 oxygen consumption rate which is 1.5‐ to 17‐fold greater than the rate of reintroduced mutagenized CS1 activity; conversely, wild‐type CS1 enzyme levels are about 30‐fold greater than the respiratory rate in wild‐type cells. Further evidence for uncoupling of the process of citrate utilization from CS1 activity is also seen in the revertant strain which has lower respiratory rate than the wild‐type strain. This difference is not due to decreased concentration of the citrate carrier of those cells with lower respiratory rates, a change in equilibrium for citrate across the mitochondrial membrane, or to decreased citrate saturability of such mitochondria. These results, supported by the interaction observed during column chromatography between matrix‐immobilized pig citrate synthase and the protein from yeast mitochondrial membrane preparations which exhibits citrate transport activity, strongly suggest physical interaction between CS1 and the mitochondrial citrate transport protein.
Binding mode of azide to ferric Aplysia limacina myoglobin. Crystallographic analysis at 1.9 Å resolution
Authors: Mattevi Andrea , Gatti Giuseppina , Coda Alessandro , Rizzi Menico , Ascenzi Paolo , Brunori Maurizio , Bolognesi Martino
Published: May 2005

The binding mode of azide to the ferric form of Aplysia limacina myoglobin has been studied by X‐ray crystallography. The three‐dimensional structure of the complex has been refined at 1.9 Å resolution to a crystallographic R‐factor of 13.9%, including 126 ordered solvent molecules. Azide binds to the heme iron, at the sixth co‐ordination position, and is oriented towards the outer part of the distal site crevice. This orientation is stabilized by an ionic interaction with the side‐chain if Arg66 (E10) which, form an outer orientation in the ‘aquo‐met’ ligand‐free myoglobin, folds back towards the distal site in the presence of the anionic ligand. In the absence of a hydrogen bond donor residue at the distal E7 position in Aplysia limacina myoglobin, a different polar residue, Arg66 at the E10 topological position, has been selected by molecular evolution in order to grant ligand stabilization.
Production and characterization of antibodies to mouse scrapie‐amyloid protein elicited by non‐carrier linked synthetic peptide immunogens
Authors: Di Martino Alessandro , Bigon Emilio , Corona Giuseppe , Callegaro Lanfranco
Published: May 2005

Two polyclonal antibodies were raised by immunizing rabbits with two non carrier‐linked synthetic peptides whose amino acid sequences corresponded to codons 89–107 (peptide P1) and 219–233 (peptide P2) of the translated cDNA sequence of murine PrP protein. These free peptides, whose structural characteristics in solution were studied by circular dichroism, elicited a reasonable immunologic response in animals. Both antibodies still recognized the corresponding immunogens after affinity chromatography purification. However, only antibodies raised to the former sequence reacted by immunobolt with a purified preparation of murine scrapie amyloid protein. These findings are discussed together with their correlation to peptide structure and the effectiveness of this simplified immunization procedure.
Lymphoblastoid cell adhesion mediated by a dimeric and polymeric endogenous β‐galactoside‐binding lectin (galaptin)
Authors: Ahmed Hafiz , Sharma Ahsu , DiCioccio Richard A. , Allen Howard J.
Published: May 2005

Glutaraldehyde‐polymerized human splenic galaptin, a β‐galactoside‐binding lectin, was demonstrated to have enhanced hemagglutinating and asialofetuin binding activity relative of native dimeric galaptin when these lectins were present in solution. The polymerized lectin consisted primarily of 2‐, 4‐ and 12‐membered species after reductive alkylation. Both forms of galaptin bound, at 4 °C, to saturable B lymphoblastoid cell surface receptors. Estimates obtained by Scatchard analyses, with the binding data expressed in terms of 14.5 kDa subunit molarity, were 5 × 107 binding sites/cell with affinity constant Ka = 2.2 × 105 M for dimeric galaptin and 17 × 107 binding sites/cell with Ka = 3.4 × 105 M−1 for polymeric galaptin. Both forms of galaptin adsorbed to polystyrene with high efficiency; however, only plastic‐adsorbed polymeric galaptin mediated adhesion of lymphoblastoid cells. Cell adhesion was inhibited by lactose. Plastic‐adsorbed polymeric galaptin bound asialofetuin more efficiently than dimeric galaptin. Asialofetuin binding was inhibited 65% and 30–50% by lactose for plastic‐adsorbed polymeric and dimeric galaptin, respectively, Native fetuin bound to the adsorbed dimeric galaptin in a lactose‐insensitive manner. These data indicate that cell surface receptor‐galaptin interaction is carbohydrate specific whereas polystyrene‐adsorbed galaptin may demonstrate protein‐protein interactions with soluble ligands.
MHC Class I/peptide interactions: Binding specificity and kinetics
Authors: Margulies David H. , Corr Maripat , Boyd Lisa F. , Khilko Sergei N.
Published: May 2005

Recent developments in the preparation of soluble analogues of the major histocompatibility complex (MHC) class l molecules as well as in the applications of real time biosensor technology have permitted the direct analysis of the binding of MHC class l molecules to antigenic peptides. Using synthetic peptide analogues with cysteine substitutions at appropriate positions, peptides can be immobilized on a dextran‐modified gold biosensor surface with a specific spatial orientation. A full set of such substituted peptides (known as ‘pepsicles’, as they are peptides on a stick) representing antigenic or self peptides can be used in the functional mapping of the MHC class l peptide binding site. Scans of sets of peptide analogues reveal that some amino acid side chains of the peptide are critical to stable binding to the MHC molecule, while others are not. This is consistent with functional experiments using substituted peptides and three‐dimensional molecular models of MHC/peptide complexes. Details analysis of the kinetic dissociation rates (kd) of the MHC molecules from the specifically coupled solid phase peptides revels that the stability of the complex is a function of the particular peptide, its coupling position, and the MHC molecule. Measured kd values for antigenic peptide/class I interactions at 25°C are in the range of ca 10−4–10−6/s. Biosensor methodology for the analysis of the binding of MHC class I molecules to solid‐phase peptides using real time surface plasmon resonance offers a rational approach to the general analysis of protein/peptide interactions.
A novel computational tool for automated structure‐based drug design
Authors: Böhm Hans‐Joachim
Published: May 2005

The computer program LUDI for automated structure‐based drug design is described. The program constructs possible new ligands for a given protein of known three‐dimensional structure. This novel approach is based upon rules about energetically favourable non‐bonded contact geometries between functional groups of the protein and the ligand which are derived from a statistical analysis of crystal packings of organic molecules. In a first step small fragments are docked into the protein binding site in such a way that hydrogen bonds and ionic interactions can be formed with the protein and hydrophobic pockets are filled with lipophilic groups of the ligands. The program can then append further fragments onto a previously positioned fragments or onto an already existing ligand (e.g., a lead structure that one seeks to improve). It is also possible to link several fragments together by bridge fragments to form a complete molecule. All putative ligands retrieved or constructed by LUDI are scored. We use a simple scoring function that was fitted to experimentally determined binding constants of protein–ligand complexes. LUCI is a very fast program with typical execution times of 1–5 min on a work station and is therefore suitable for interactive usage.
From crystals to enzymes: Simple models of molecular recognition
Authors: Czugler Mátyás
Published: May 2005

Structural models resulting from the x‐ray crystal structure determination of small molecules are shown to be closely mimicking some of the protein structure and function features. Crystals of inclusion compounds, a special type of solid molecular associates, lend special support to such a view. They are inherently suited for such modelling purpose due to easily visible association and molecular recognition effects. It is envisaged that combination of the analysis of high‐resolution small molecule crystallographic models and those of protein structures will yield to further fruitful analogies.
Analysis of molecular recognition: Steric electrostatic and hydrophobic complementarity
Authors: Náray‐Szabó Gábor
Published: May 2005

We discuss three important aspects of molecular recognition: steric, electrostatic and hydrophobic. Steric fit means that interacting atoms may not approach each other beyond their van der Waals radii and, simultaneously, crevices should be filled as densely as possible. Electrostatic fit requires the maximum ionic and polar (hydrogen bond or other) interaction between host and guest atoms while the hydrophobic fit corresponds to the association trend between apolar groups in an aqueous medium. Space‐filling models, obtained by molecular graphics, illustrate steric complementarity while we use molecular electrostatic potentials (MEPs) and fields (MEFs) to investigate electrostatic and hydrophobic matching. Molecular regions with negative and positive MEPs attract and repel a positive probe charge, respectively, so we consider them as attracting each other. Furthermore we postulate that regions with MEFs of similar magnitude tend to associate more strongly than those with very different fields (similis simili gaudet principle). We apply the above rules to the study of complementarity in the tryspin‐BPTI complex and in a crystalline association between styrene epoxide as guest and a comphor‐based anthracene derivative as host. We discuss molecular similarity on the same footing as complementarity and give some examples on the application of the concept of the rationalization of relative strengths of trypsin inhibitors.
Macromolecular crowding and molecular recognition
Authors: Minton Allen P.
Published: May 2005

The effects of macromolecular crowding upon macromolecular associations in solution, and upon binding of a macromolecular ligand to a surface site, ar ereviewed. It is demonstrated, by means of two examples, that crowding may lead to significant alterations of biochemical or biological recognition processes at the molecular level.
The making of the minibody: An engineered β‐protein for the display of conformationally constrained peptides
Authors: Tramontano Anna , Bianchi Elisabetta , Venturini Sara , Martin Franck , Pessi Antonelo , Sallozzo Maurizio
Published: May 2005

Conformationally constraining selectable peptides onto a suitable scaffold that enables their conformation to be predicted or readily determined by experimental techniques would considerably boost drug discovery process by reducing the gap between the discovery of a peptide lead and the design of a peptidomimetic with a more desirable pharmacological profile. With this in mind, we designed the minibody, a 61‐residue β‐protein aimed at retaining some desirable features of immunogloblin variable domains, such as tolerance to sequence variability in selected regions of the protein and predictability of main chain conformation of the same regions, based on the ‘canonical structures’ model. To test the ability of the minibody scaffold to support functional sites we also designed a metal binding version of the protein by suitably choosing the sequences of its loops. The minibody was produced both by chemical syntyhesis and expression in E. coli and charactgerized by size exclusion chromatography, UV CD (circular dichroism) spectroscopy and metal binding activity. All our data supported the model, but a more detailed structural characterization of the molecule was impaired by its low soubility. We were able to overcome this problem both by further; mutagenesis of the framework and by addition of a solublizing motif. The minibody is being used to select constrained human IL‐6 peptidic ligands from a library displayed on the surface of the f1 bacteriophage.
Analysis of the interaction between human interleukin‐5 and the soluble domain of its receptor using a surface plasmon resonance biosensor
Authors: Morton Thomas A. , Bennett Donald B. , Appelbaum Edward R. , Cusimano Donna M. , Johanson Kyung O. , Matico Rosalie E. , Young Peter R. , Doyle Michael , Chaiken Irwin M.
Published: May 2005

A surface plasmon resonance (SPR) biosensor was used to study the interaction of human interleukin‐5 (hIL5) with its receptor. IL5 is a major growth factor in the production and activation of eosinophilis. The receptor for IL5 is composed of two subunits, α and β. The α subunit provides the specificity for IL5 and consist of an extracellular soluble domain, a single transmembrane region and a cytoplasmic tail. We expressed the soluble domain of the human IL5 receptor α subunit (shIL5Rα) and human IL5 (hIL5) in Drosophila. Both hIL5 and shIL5Rα were immobilized separately through amine groups onto the carboxylated dextran layer of sensor chips of the BIAcore™ (Pharmacia) SPR biosensor after N‐hydroxysuccinimide/carbodiimide activation of the chip surface. Interactions were measured for the complementary macromolecule, either shIL5Rα or hIL5, in solution. Kinetics of binding of soluble analyst to immobilized ligand were measured and from this the association rate constant, dissociation rate constant and equilibrium dissociation constant (Kd) were derived. With immobilized shIL5Rα and soluble hIL5, the measured Kd was 2 nM. A similar value was obtained by titration calorimetry. The Kd for Drosophila expressed receptor and IL5 is higher than the values reported for proteins expressed in different systems, likely due to differences in the methods of interaction analysis used for differences in protein glycosylation. Receptor‐IL5 binding was relatively pH independent between pH 6.5 and 9.5. Outside this range the dissociation rate increased with compressibility little increased in association rate. The values obtained for the interaction of hIL5 and shIL5Rα were found to depend on which component was immobilized; the Kd was 5.5 nM with immobilized hIL5 and soluble shIL5Rα. The SPR biosensor provides a unified methodology to measure the interaction properties of shIL5Rα and hIL5 derivatives, mutants and mimetic as well as to evaluate potential antagonists of the receptor‐cytokine interaction.
Structural and physicochemical analysis of the reaction between the anti‐lysozyme antibody D1.3 and the anti‐idiotopic antibodies E225 and E5.2
Authors: Tello Diana , Eisenstein Edward , Schwarz Frederick P. , Goldbaum Fernando A. , Fields Barry A. , Mariuzza Roy A. , Poljak Roberto J.
Published: May 2005

The reaction between the mouse (BALB/c) anti‐idiotiopic monoclonal antibodies E225 and E5.2 and idiotopes on the (BALB/c) anti‐lysozyme monoclonal antibody D1.3 has been characterized by titration calorimetry, by equilibrium sedimentation and by the determination of binding association and dissociation rates. The reaction between E5.2 and D1.3 is driven by a large negative enthalpy and its rate and equilibrium association constants are comparable to those observed in other antigen–antibody reactions. In contrast, the reaction between E225 and D1.3 is entropically driven and characterized by slow association kinetic (1 × 103 M−1 sec−1) and a resulting low equilibrium constant (Ka = 2 × 105M−1). A correlation of these properties with the three‐dimensional structure of the Fab225‐FabD1.3 complex, previously determined by X‐ray diffraction methods to 2.5 Å resolution, indicates that conformational changes of several D1.3 contacting residues, located in its complementarity determining regions, may explain these features of the reaction.
DNA recognition by DNase I
Authors: Suck Dietrich
Published: May 2005

Bovine pancreatic DNase I shows a strong preference for double‐stranded substrates and cleaves DNA with strongly varying cutting rates suggesting that the enzyme recognises sequence‐dependent structural variations of the DNA double helix. The complicated cleavage pattern indicates that several local as well as global helix parameters influences the cutting frequency of DNase I at a given bond. The high resolution crystal structures of two DNase I‐DNA complexes showed that the enzyme binds tightly in the minor groove, and to the sugar‐phosphate backbones of both strands, and thereby induces a widening of the minor groove and a bending towards the major grooves. In agreement with biochemical data this suggests that flexibility and minor groove geometry are major parameters determining the cutting rate of DNase I. Experimental observations showing that the sequence environmental of a dinucleotide step strongly affects its cleavage efficiency can be rationalized by that fact that six base pair are in contact with the enzyme. Mutational analysis based on the structural results has identified critical residues for DNA binding and cleavage and has lead to a proposal for the catalytic mechanism.