On the analysis of protein self-association by sedimentation velocity analytical ultracentrifugation

Analytical ultracentrifugation is one of the classical techniques for the study of protein interactions and protein self-association. Recent instrumental and computational developments have significantly enhanced this methodology. In this paper, new tools for the analysis of protein self-association by sedimentation velocity are developed, their statistical properties are examined, and considerations for optimal experimental design are discussed. A traditional strategy is the analysis of the isotherm of weight-average sedimentation coefficients s(w) as a function of protein concentration. From theoretical considerations, it is shown that integration of any differential sedimentation coefficient distribution c(s), ls-g(*)(s), or g(s(*)) can give a thermodynamically well-defined isotherm, as long as it provides a good model for the sedimentation profiles. To test this condition for the g(s(*)) distribution, a back-transform into the original data space is proposed. Deconvoluting diffusion in the sedimentation coefficient distribution c(s) can be advantageous to identify species that do not participate in the association. Because of the large number of scans that can be analyzed in the c(s) approach, its s(w) values are very precise and allow extension of the isotherm to very low concentrations. For all differential sedimentation coefficients, corrections are derived for the slowing of the sedimentation boundaries caused by radial dilution. As an alternative to the interpretation of the isotherm of the weight-average s value, direct global modeling of several sedimentation experiments with Lamm equation solutions was studied. For this purpose, a new software SEDPHAT is introduced, allowing the global analysis of several sedimentation velocity and equilibrium experiments. In this approach, information from the shape of the sedimentation profiles is exploited, which permits the identification of the association scheme and requires fewer experiments to precisely characterize the association. Further, under suitable conditions, fractions of incompetent material that are not part of the reversible equilibrium can be detected.     

Size-distribution analysis of proteins by analytical ultracentrifugation: strategies and application to model systems.

Strategies for the deconvolution of diffusion in the determination of size-distributions from sedimentation velocity experiments were examined and developed. On the basis of four different model systems, we studied the differential apparent sedimentation coefficient distributions by the time-derivative method, g(s*), and by least-squares direct boundary modeling, ls-g*(s), the integral sedimentation coefficient distribution by the van Holde-Weischet method, G(s), and the previously introduced differential distribution of Lamm equation solutions, c(s). It is shown that the least-squares approach ls-g*(s) can be extrapolated to infinite time by considering area divisions analogous to boundary divisions in the van Holde-Weischet method, thus allowing the transformation of interference optical data into an integral sedimentation coefficient distribution G(s). However, despite the model-free approach of G(s), for the systems considered, the direct boundary modeling with a distribution of Lamm equation solutions c(s) exhibited the highest resolution and sensitivity. The c(s) approach requires an estimate for the size-dependent diffusion coefficients D(s), which is usually incorporated in the form of a weight-average frictional ratio of all species, or in the form of prior knowledge of the molar mass of the main species. We studied the influence of the weight-average frictional ratio on the quality of the fit, and found that it is well-determined by the data. As a direct boundary model, the calculated c(s) distribution can be combined with a nonlinear regression to optimize distribution parameters, such as the exact meniscus position, and the weight-average frictional ratio. Although c(s) is computationally the most complex, it has the potential for the highest resolution and sensitivity of the methods described.     

Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.

Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.     

Mutational analysis of the energetics of the GrpE.DnaK binding interface: equilibrium association constants by sedimentation velocity analytical ultracentrifugation

DnaK, the prokaryotic Hsp70 molecular chaperone, requires the nucleotide exchange factor and heat shock protein GrpE to release ADP. GrpE and DnaK are tightly associated molecules with an extensive protein-protein interface, and in the absence of ADP, the dissociation constant for GrpE and DnaK is in the low nanomolar range. GrpE reduces the affinity of DnaK for ADP, and the reciprocal linkage is also true: ADP reduces the affinity of DnaK for GrpE. The energetic contributions of GrpE side-chains to GrpE-DnaK binding were probed by alanine-scanning mutagenesis. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) was used to measure the equilibrium constants (Keq) for GrpE binding to the ATPase domain of DnaK in the presence of ADP. ADP-bound DnaK is the natural target of GrpE, and the addition of ADP (final concentration of 5 microM) to the preformed GrpE-DnaK(ATPase) complexes allowed the equilibrium association constants to be brought into an experimentally accessible range. Under these experimental conditions, the substitution of one single GrpE amino acid residue, arginine 183 with alanine, resulted in a GrpE-DnaK(ATPase) complex that was weakly associated (Keq =9.4 x 10(4) M). This residue has been previously shown to be part of a thermodynamic linkage between two structural domains of GrpE: the thermosensing long helices and the C-terminal beta-domains. Several other GrpE side-chains were found to have a significant change in the free energy of binding (DeltaDeltaG approximately 1.5 to 1.7 kcal mol(-1)), compared to wild-type GrpE.DnaK(ATPase) in the same experimental conditions. Overall, the strong interactions between GrpE and DnaK appear to be dominated by electrostatics, not unlike barnase and barstar, another well-characterized protein-protein interaction. GrpE, an inherent thermosensor, exhibits non-Arrhenius behavior with respect to its nucleotide exchange function at bacterial heat shock temperatures, and mutation of several solvent-exposed side-chains located along the thermosensing indicated that these residues are indeed important for GrpE-DnaK interactions.     

Self-association of human and porcine relaxin as assessed by analytical ultracentrifugation and circular dichroism.

The self-association properties of recombinant DNA derived human relaxin, and porcine relaxin isolated from porcine ovaries, have been studied by sedimentation equilibrium analytical ultracentrifugation and circular dichroism (CD). The human relaxin ultracentrifuge data were adequately defined by a monomer-dimer self-association model with an association constant of approximately 6 x 10(5) M-1, whereas porcine relaxin was essentially monomeric in solution. An approximate 5-fold increase in weight fraction of human relaxin monomer elicited by dilution of the protein resulted in no change in the far-UV CD spectrum at 220 nm. In contrast, after the same increase in weight fraction of monomer, the near-UV circular dichroism spectra for human relaxin exhibited a significant decrease in the amplitude for the CD bands near 277 and 284 nm. These CD bands, which may be assigned to the lone tyrosine in human relaxin, are superimposed on a broad envelope that is probably due to the three disulfide chromophores. Although both the human and porcine proteins contain two tryptophan residues, the near-UV CD spectra exhibit only a broad shoulder near 295 nm rather than the strong CD bands often found for tryptophan. Moreover, there is little change in this broad band after dilution of human relaxin to concentrations that resulted in a 4-fold increase in monomer weight fraction. These data suggest that dissociation of the human relaxin dimer to monomer is not accompanied by large overall changes in secondary structure or alteration in the average tryptophan environment, whereas there is a significant change in the tyrosine environment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct analysis of the kinetic profiles of organophosphate-acetylcholinesterase adducts by MALDI-TOF mass spectrometry

A sensitive matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry procedure has been established for the detection and quantitation of acetylcholinesterase (AChE) inhibition by organophosphate (OP) compounds. Tryptic digests of purified recombinant mouse AChE (mAChE) were fractionally inhibited by paraoxon to form diethyl phosphoryl enzyme. The tryptic peptide of mAChE that contains the active center serine residue resolves to a molecular mass of 4331.0 Da. Phosphorylation of the enzyme by paraoxon results in covalent modification of the active center serine and a corresponding increase in molecular mass of the tryptic peptide by 136 Da. The relative abundance of AChE peptides containing a modified active center serine strongly correlates with the fractional inhibition of the enzyme, achieving a detection range of phosphorylated to nonphosphorylated enzyme of 5-95%. Modifications of AChE by OP compounds resulting in dimethyl, diethyl, and diisopropyl phosphoryl adducts have been monitored with subpicomole amounts of enzyme. The individual phosphorylated adducts of AChE that result from loss of one alkyl group from the inhibited enzyme (the aging reaction) and the reappearance of unmodified AChE (spontaneous reactivation) have been resolved by the kinetic profiles and relative abundance of species. Further, the tryptic peptide containing the active center serine of AChE, isolated from mouse brain by anion-exchange and affinity chromatography, has been monitored by mass spectrometry. Native brain AChE, purified from mice treated with sublethal doses of metrifonate, has demonstrated that enzyme modifications resulting from OP exposure can be detected in a single mouse brain. For dimethyl phosphorylated AChE, OP exposure has been monitored by the ratio of tryptic peptide peaks that correspond to unmodified (uninhibited and/or reactivated), inhibited, and aged enzyme.     

Direct photoaffinity labeling by dolastatin 10 of the amino-terminal peptide of beta-tubulin containing cysteine 12

Tubulin with bound [5-3H]dolastatin 10 was exposed to ultraviolet light, and 8-10% of the bound drug cross-linked to the protein, most of it specifically. The primary cross-link was to the peptide spanning amino acid residues 2-31 of beta-tubulin, but the specific amino acid could not be identified. Indirect studies indicated that cross-link formation occurred between cysteine 12 and the thiazole moiety of dolastatin 10. An equipotent analog of dolastatin 10, lacking the thiazole ring, did not form an ultraviolet light-induced cross-link to beta-tubulin. Preillumination of tubulin with ultraviolet light, known to induce cross-link formation between cysteine 12 and exchangeable site nucleotide, inhibited the binding of [5-3H]dolastatin 10 and cross-link formation more potently than it inhibited the binding of colchicine or vinblastine to tubulin. Conversely, binding of dolastatin 10 to tubulin inhibited formation of the cross-link between cysteine 12 and the exchangeable site nucleotide. Dithiothreitol inhibited formation of the beta-tubulin/dolastatin 10 cross-link but not the beta-tubulin/exchangeable site nucleotide cross-link. Modeling studies revealed a highly favored binding site for dolastatin 10 at the + end of beta-tubulin in proximity to the exchangeable site GDP. Computational docking of an energy-minimized dolastatin 10 conformation at this site placed the thiazole ring of dolastatin 10 8-9 A from the sulfur atom of cysteine 12. Dolastatin 15 and cryptophycin 1 could also be docked into positions that overlapped more extensively with the docked dolastatin 10 than with each other. This result was consistent with the observed binding properties of these peptides.     

Molecular architecture of E. coli purine nucleoside phosphorylase studied by analytical ultracentrifugation and CD spectroscopy

Purine nucleoside phosphorylase (PNP) is a key enzyme of the nucleoside salvage pathway and is characterized by complex kinetics. It was suggested that this is due to coexistence of various oligomeric forms that differ in specific activity. In this work, the molecular architecture of Escherichia coli PNP in solution was studied by analytical ultracentrifugation and CD spectroscopy. Sedimentation equilibrium analysis revealed a homohexameric molecule with molecular mass 150+/-10 kDa, regardless of the conditions investigated-protein concentration, 0.18-1.7 mg/mL; presence of up to 10 mM phosphate and up to 100 mM KCl; temperature, 4-20 degrees C. The parameters obtained from the self-associating model also describe the hexameric form. Sedimentation velocity experiments conducted for broad protein concentration range (1 microg/mL-1.3 mg/mL) with boundary (classical) and band (active enzyme) approaches gave s(0)20,w=7.7+/-0.3 and 8.3+/-0.4 S, respectively. The molecular mass of the sedimenting particle (146+/-30 kDa), calculated using the Svedberg equation, corresponds to the mass of the hexamer. Relative values of the CD signal at 220 nm and the catalytic activity of PNP as a function of GdnHCl concentration were found to be correlated. The transition from the native state to the random coil is a single-step process. The sedimentation coefficient determined at 1 M GdnHCl (at which the enzyme is still fully active) is 7.7 S, showing that also under these conditions the hexamer is the only catalytically active form. Hence, in solution similar to the crystal, E. coli PNP is a hexameric molecule and previous suggestions for coexistence of two oligomeric forms are incorrect.     

Comparison of the toxicities of patulin and patulin adducts formed with cysteine.

The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg.     

Studies on the structure and mechanism of a bacterial protein toxin by analytical ultracentrifugation and small-angle neutron scattering.

Pneumolysin, an important virulence factor of the human pathogen Streptococcus pneumoniae, is a pore-forming toxin which also possesses the ability to activate the complement system directly. Pneumolysin binds to cholesterol in cell membrane surfaces as a prelude to pore formation, which involves the oligomerization of the protein. Two important aspects of the pore-forming activity of pneumolysin are therefore the effect of the toxin on bilayer membrane structure and the nature of the self-association into oligomers undergone by it. We have used analytical ultracentrifugation (AUC) to investigate oligomerization and small-angle neutron scattering (SANS) to investigate the changes in membrane structure accompanying pore formation.Pneumolysin self-associates in solution to form oligomeric structures apparently similar to those which appear on the membrane coincident with pore formation. It has previously been demonstrated by us using site-specific chemical derivatization of the protein that the self-interaction preceding oligomerization involves its C-terminal domain. The AUC experiments described here involved pneumolysin toxoids harbouring mutations in different domains, and support our previous conclusions that self-interaction via the C-terminal domain leads to oligomerization and that this may be related to the mechanism by which pneumolysin activates the complement system.SANS data at a variety of neutron contrasts were obtained from liposomes used as model cell membranes in the absence of pneumolysin, and following the addition of toxin at a number of concentrations. These experiments were designed to allow visualization of the effect that pneumolysin has on bilayer membrane structure resulting from oligomerization into a pore-forming complex. The structure of the liposomal membrane alone and following addition of pneumolysin was calculated by the fitting of scattering equations directly to the scattering curves. The fitting equations describe scattering from simple three-dimensional scattering volume models for the structures present in the sample, whose dimensions were varied iteratively within the fitting program. The overall trend was a thinning of the liposome surface on toxin attack, which was countered by the formation of localized structures thicker than the liposome bilayer itself, in a manner dependent on pneumolysin concentration. At the neutron contrast match point of the liposomes, pneumolysin oligomers were observed. Inactive toxin appeared to bind to the liposome but not to cause membrane alteration; subsequent activation of pneumolysin in situ brought about changes in liposome structure similar to those seen in the presence of active toxin. We propose that the changes in membrane structure on toxin attack which we have observed are related to the mechanism by which pneumolysin forms pores and provide an important perspective on protein/membrane interactions in general. We discuss these results in the light of published data concerning the interaction of gramicidin with bilayers and the hydrophobic mismatch effect.     

Comparison of the toxicities of patulin and patulin adducts formed with cysteine.

The toxicities of patulin and of the patulin adducts formed with cysteine were compared using the mutation-sensitive strain Escherichia coli W3110 thy polA1 and its polA1+ revertant. The acute toxicities of patulin and of the adduct mixture were also compared using NMRI mice. The adduct mixture was shown by thin-layer chromatography to consist of one ninhydrin-positive, one ninhydrin- and MBTH (3-methyl-2-benzothiazolinone hydrazone)-positive, three MBTH-positive, and two ninhydrin- and MBTH-negative components. The results showed that patulin was over 100 times more toxic to E. coli than the adduct complex. Neither patulin nor the adduct mixture was found to induce the repair effect in E. coli. In the mouse feeding tests, the oral 50% lethal dose for patulin was 29 mg/kg, while that of the adduct mixture was greater than 2,370 mg/kg.     

Reversible dimer formation and stability of the anti-tumour single-chain Fv antibody MFE-23 by neutron scattering,analytical ultracentrifugation,and NMR and FT-IR spectroscopy

Cre recombinase uses two pairs of sequential cleavage and religation reactions to exchange homologous DNA strands between 34 base-pair (bp) LoxP recognition sequences. In the oligomeric recombination complex, a switch between "cleaving" and "non-cleaving" subunit conformations regulates the number, order, and regio-specificity of the strand exchanges. However, the particular sequence of events has been in question. From analysis of strand composition of the Holliday junction (HJ) intermediate, we determined that Cre initiates recombination of LoxP by cleaving the upper strand on the left arm. Cre preferred to react with the left arm of a LoxP suicide substrate, but at a similar rate to the right arm, indicating that the first strand to be exchanged is selected prior to cleavage. We propose that during complex assembly the cleaving subunit preferentially associates with the LoxP left arm, directing the first strand exchange to that side. In addition, this biased assembly would enforce productive orientation of LoxP sites in the recombination synapses. A novel Cre-HJ complex structure in which LoxP was oriented with the left arm bound by the cleaving Cre subunit suggested a physical basis for the strand exchange order. Lys86 and Lys201 interact with the left arm scissile adenine base differently than in structures that have a scissile guanine. These interactions are associated with positioning the 198-208 loop, a structural component of the conformational switch, in a configuration that is specific to the cleaving conformation. Our results suggest that strand exchange order and site alignment are regulated by an "induced fit" mechanism in which the cleaving conformation is selectively stabilized through protein-DNA interactions with the scissile base on the strand that is cleaved first.     

Calcium and temperature regulation of the stability of the human platelet integrin GPIIb/IIIa in solution: an analytical ultracentrifugation study

The human platelet integrin GPIIb/IIIa (228 kDa), a Ca-dependent heterodimer formed by the _IIb subunit (GPIIb, 136 kDa) and the ₃ subunit (GPIIIa, 92 kDa), serves as the fibrinogen receptor at the surface of activated platelets. The degree of dissociation of the GPIIb/IIIa heterodimer (s°₂₀ ^*, 8.9 S) into its constituent glycoproteins (GPIIb, 5.8 S; and GPIIIa, 3.9 S) has been assessed by analytical ultracentrifugation in Triton X100 buffers, and its Ca²⁺- and temperature-dependence correlated with Ca²⁺-binding to GPIIb/IIIa and its temperature dependence. At 21°C half-maximal dissociation of GPIIb/IIIa occurs at 5.5 ± 2.5 × 10^–8 M Ca²⁺, very close to the dissociation constant of the high affinity Ca-binding site of GPIIb/IIIa (Kd₁ 8 ± 3 × 10^–8 M) (Rivas and González-Rodríguez, 1991) and much lower than the Kd of the 3.4 medium affinity Ca-binding sites (Kd₂ 4 ± 1.5 × 10^–5 M), which seems to demonstrate that the stability of the heterodimer in solution at room temperature is regulated by the degree of saturation of the high-affinity Ca-binding site. At 4°C, the stability of the heterodimer is apparently Ca²⁺-independent, while at room and physiological temperatures (15–37°C) the degree of dissociation of the heterodimer is regulated by the degree of dissociation of the high- and medium-affinity Ca-binding sites, respectively. On increasing the Ca²⁺ concentration up to 1 × 10^–4 M after dissociation in Triton X100 solutions, the reconstitution of the GPIIb/IIIa heterodimer depends on the time and temperature at which the dissociated heterodimer was maintained, being almost complete within the first 5–10 min at 37°C and within the first 1–2 h at 21°C. After this time, a time- and temperature-dependent irreversible autoassociation of GPIIb (covalent) and GPIIIa (non-covalent) occurs, which hinders both the isolation of permanently stable monoamers of GPIIb and GPIIIa and the reconstitution of the GPIIb/IIIa heterodimer in Triton X100 solutions. Abbreviations: GPIIb, GPIIIa, and GPIIb/IIIa, glycoprotein IIb, IIIa, and the heterodimer formed by them, respectively; s°₂₀ ^*, the sedimentation coefficient of the glycoprotein-detergent complexes determined at 20°C, after extrapolation to zero-glycoprotein concentration Offprint requests to: J. González-Rodríguez     

The dimeric and trimeric solution structures of the multidomain complement protein properdin by X-ray scattering, analytical ultracentrifugation and constrained modelling

Properdin regulates the alternative pathway of the complement system of immune defence by stabilising the C3 convertase complex. It contains six thrombospondin repeat type I (TSR-1 to TSR-6) domains and an N-terminal domain. Properdin exists as either a dimer, trimer or tetramer. In order to determine the solution structure of multiple TSR domains, the molecular structures of dimeric and trimeric properdin were studied by X-ray scattering and analytical ultracentrifugation. Guinier analyses showed that the dimer and trimer have radii of gyration R(G) values of 7.5 nm and 10.3 nm, respectively, and cross-sectional radii of gyration R(XS) values of 1.3 nm and 1.5 nm, respectively. Distance distribution functions showed that the maximum lengths of the dimer and trimer were 25 nm and 30 nm, respectively. Analytical ultracentrifugation gave sedimentation coefficients of 5.1S and 5.2S for the dimer and trimer forms, respectively. Homology models for the TSR domains were constructed using the crystal structure of the TSP-2 and TSP-3 domains in human thrombospondin as templates. Properdin could be represented by seven TSR domains, not six as believed, since the crystal structure determined for TSP-2 and TSP-3 showed that the N-terminal domain (TSR-0) could be represented by a truncated TSR domain with the same six conserved Cys residues found in TSR-1 to TSR-6. Automated constrained molecular modelling revealed the solution conformations of multiple TSR domains in properdin at medium resolution. The comparison of 3125 systematically generated conformational models for the trimer with the X-ray data showed that good curve fits could be obtained by assuming that the linker between adjacent TSR domains possessed limited flexibility. Good trimer models correspond to partially collapsed triangular structures, and extended triangular shapes do not fit the data. The corresponding 3125 models for the dimer revealed a similar outcome in which a partially collapsed TSR structure gave good fits. The models account for the effect of mutations that cause properdin deficiencies, and suggest that the biologically active TSR-4, TSR-5 and TSR-6 domains are exposed for protein-protein interactions. The role of the other TSR domains in properdin may be to act as spacers to make TSR-4, TSR-5 and TSR-6 accessible for function.     

Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis

Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-alpha (alpha-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular alpha-SNAP failed to detect (32)P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser(10). PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser(10) phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.     

The lack of mutagenic properties of patulin and patulin adducts formed with cysteine in Salmonella test systems.

The mutagenic properties of patulin and the patulin adducts formed with cysteine were tested with histidine auxotroph Salmonella typhimurium strains as indicator organisms. The tests were performed by microsomal activation and host-mediated assay. Neither patulin nor patulin--cysteine reaction mixture was mutagenic in these test systems.     

The modulation of topoisomerase I-mediated DNA cleavage and the induction of DNA-topoisomerase I crosslinks by crotonaldehyde-derived DNA adducts

Crotonaldehyde is a representative alpha,beta-unsaturated aldehyde endowed of mutagenic and carcinogenic properties related to its propensity to react with DNA. Cyclic crotonaldehyde-derived deoxyguanosine (CrA-PdG) adducts can undergo ring opening in duplex DNA to yield a highly reactive aldehydic moiety. Here, we demonstrate that site-specifically modified DNA oligonucleotides containing a single CrA-PdG adduct can form crosslinks with topoisomerase I (Top1), both directly and indirectly. Direct covalent complex formation between the CrA-PdG adduct and Top1 is detectable after reduction with sodium cyanoborohydride, which is consistent with the formation of a Schiff base between Top1 and the ring open aldehyde form of the adduct. In addition, we show that the CrA-PdG adduct alters the cleavage and religation activities of Top1. It suppresses Top1 cleavage complexes at the adduct site and induces both reversible and irreversible cleavage complexes adjacent to the CrA-PdG adduct. The formation of stable DNA-Top1 crosslinks and the induction of Top1 cleavage complexes by CrA-PdG are mutually exclusive. Lastly, we found that crotonaldehyde induces the formation of DNA-Top1 complexes in mammalian cells, which suggests a potential relationship between formation of DNA-Top1 crosslinks and the mutagenic and carcinogenic properties of crotonaldehyde.     

Assessment of the composition and structure of covalent complexes of superoxide dismutase with aldehyde dextran by analytical ultracentrifugation]

Superoxide dismutase was covalently coupled wih aldehyde dextran, a polymeric carrier of molecular mass of 70 kDa. Modification produced an increase in the enzyme thermostability. Modified preparations retained a high specific activity. The composition of the thus obtained conjugates was analyzed by the ultracentrifugation and diffusion methods. The protein induced the destruction of aldehyde dextran, the enzyme being modified by its fragments. The presence of aldehyde dextran excess in the incubation medium promoted superoxide dismutase dissociation into individual subunits. At the enzyme/carrier ratio of 1:02 modification occurred as covalent coupling of the biocatalyst subunits and its one-point modification.     

Characterisation of the low affinity interaction between rat cell adhesion molecules CD2 and CD48 by analytical ultracentrifugation

CD2 is a cell adhesion molecule found on the plasma membrane of T-lymphocytes. Its counter-receptor in rat is the structurally related CD48. This interaction is believed to contribute to the adhesion of T-cells to other cells such as cytotoxic targets and antigen presenting cells. Cell-cell adhesion involves the formation of multiple cell adhesion molecule complexes at the cell surface and if cell-cell de-adhesion is to occur, these complexes need to be disrupted. The affinities of cell adhesion molecule interactions are suggested to be relatively weak to allow this de-adhesion of cell-cell interactions. The CD2/CD48 interaction has been studied using recombinant extracellular proteins and the affinity of the interaction of soluble recombinant rat CD2–CD48 has been determined (at 37°C) using surface plasmon resonance (and shown to be weak), with the dissociation constant K_d=60–90 μm. The values determined by surface plasmon resonance results could be affected by the immobilisation of the ligand on the chip and any self-association on the chip. We used three different analytical ultracentrifuge procedures which each allowed the interaction to be studied in free solution without the need for an immobilisation medium. Both sedimentation equilibrium (using direct analysis of the concentration distribution and also modelling of molecular weight versus concentration data) and sedimentation velocity at 5°C yielded dissociation constants in the range of 20– 110 μm, supporting the surface plasmon resonance findings showing that binding between these cell adhesion molecules is relatively weak. These studies also ruled out the presence of any significant self-association of the reactants which could lead to systematic error in the surface plasmon resonance results. Accepted: 19 November 1996