The cosmic reality check.
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Journal Article↗
Biomedical subjects
R Gilli
Publications and source records attributed to R Gilli.
Apocalmodulin binds to the myosin light chain kinase calmodulin target site.
The interaction of a 20-residue-long peptide derived from the calmodulin-binding domain of the smooth muscle myosin light chain kinase with calcium-free calmodulin (apocalmodulin) was studied using a combination of isothermal titration calorimetry and differential scanning calorimetry. We showed that: (i) a significant binding between apocalmodulin and the target peptide (RS20) exists in the absence of salt (Ka = 10(6) M-1), (ii) the peptide interacts with the C-terminal lobe of calmodulin and adopts a partly helical conformation, and (iii) the presence of salt weakens the affinity of the peptide for apocalmodulin, emphasizing the importance of electrostatic interactions in the complex. Based on these results and taking into account the work of Bayley et al. (Bayley, P. M., Findlay, W.A., and Martin, S. R. (1996) Protein Sci. 5, 1215-1228), we suggest a physiological role for apocalmodulin.
Calcium↗
Heat-shock protein 90 (hsp90) binds in vitro to tubulin dimer and inhibits microtubule formation.
Hsp90 interacts with steroid hormone receptors, protein kinases, and cytoskeletal proteins. The mode of action of hsp90 on microtubules and tubulin has not been investigated. Using isolated purified hsp90 and isolated tubulin, we demonstrated in vitro by difference absorption and fluorescence spectroscopy that hsp90 bound to tubulin with an apparent affinity constant of 5 x 10(5) M-1, assuming an apparent stoichiometry of 1 at 25 degrees C. Using microcalorimetry, we found a delta H of -9.8 +/- 0.8 kJ.mol-1. The binding of hsp90 to tubulin was confirmed by a sedimentation assay. Moreover, we showed that hsp90 inhibited tubulin polymerisation.
Animals↗
The two-state process of the heat shock protein 90 thermal denaturation: effect of calcium and magnesium.
Scanning microcalorimetry, native PAG electrophoresis, and circular dichroism were used to characterize thermal denaturation and oligomerization of heat shock protein 90 (hsp90) and the calcium and magnesium effect on these processes. The calorimetric curve of the hsp90 dimer consists of two transitions centered at 53.8 and 63.1 degrees C. Using specific ligand geldanamycin, we have found that N-terminal domains in the hsp90 dimer are melted independently in the lower-temperature peak, while the higher-temperature one comprises unfolding of two non-interacting parts of the middle domains and dimerization region. Unfolding of the N-terminal domain gives start to oligomerization of dimers; oligomers consist of dimers not dissociating upon denaturation. Calcium and magnesium strongly decrease the hsp90 thermostability and thereby cause oligomerization at lower temperature. We suggest that calcium affects the hsp90 oligomerization, known to be important for its chaperone activity, by shifting the unfolding temperature of the hsp90 N-terminal domain close to the heat shock temperature range.
Animals↗
Thermodynamic analysis of calcium and magnesium binding to calmodulin.
To elucidate some aspects still debated concerning the interaction of Ca2+ and Mg2+ with CaM, the thermodynamic binding parameters of Ca2+-CaM and Mg2+-CaM complexes were characterized by flow dialysis and isothermal microcalorimetry under different experimental conditions. In particular, the enthalpy and entropy changes associated with Ca2+ and Mg2+ binding to their sites were determined, allowing a better understanding of the mechanism underlying cation-CaM interactions. Ca2+-CaM interaction follows an enthalpy-entropy compensation relationship, suggesting that CaM explores a subspace of isoenergetical conformations which is modified by Ca2+ binding. This Ca2+-induced change in CaM dynamics is proposed to play a key role in CaM function, i.e. in its interaction with and/or activation of target proteins. Furthermore, data show that Mg2+ does not act as a direct competitor for Ca2+ binding on the four main Ca2+ binding sites, but rather as an allosteric effector. This implies that the four main Mg2+ binding sites are distinct from the EF-hand Ca2+ binding sites. Finally, Ca2+ is shown to interact with auxiliary binding sites on CaM. These weak affinity sites were thermodynamically characterized. The results presented here challenge the current accepted view of CaM ion binding.
Binding Sites↗
Conformation and thermal denaturation of apocalmodulin: role of electrostatic mutations.
Scanning microcalorimetry and circular dichroism were used to study conformational state and heat denaturation of Ca2+-free synthetic calmodulin (SynCaM) and three charge reversal mutants. We produced evidence for the major role of the electrostatic potential in the stability and flexibility of SynCaM. The substitution of 118DEE120 by 118KKK120 (SynCaM12A) does not influence the flexibility of the protein; the replacement of 82EEE84 by 82KKK84 (SynCaM8) decreases its level, while the combination of these two mutations in SynCaM18A significantly increases the flexibility. The heat denaturation of apoSynCaM and its mutants is well approximated by two two-state transitions with the lower-temperature transition corresponding to C-terminal lobe melting and the higher-temperature one to N-terminal lobe melting. The difference in transition temperatures for the two lobes decreases in SynCaM8 and increases in SynCaM18A, suggesting a modification in the influence of one lobe to the other. The electrostatic mutations change the parameters of thermal denaturation of SynCaM lobes in a similar way as pH conditions affect thermal transition parameters of multidomain proteins, leading to a linear temperature dependence of transition enthalpy. One domain of the N-terminal lobe in apoSynCaM18A is unfolded in the native state. Near-UV CD spectra point out the invariability of the local structure of aromatic residues upon mutations, although the secondary structure undergoes striking transformations. Cacodylate ions strongly and specifically alter the helical content of SynCaM. Our data unambiguously demonstrate that the two lobes are not independent, and interactions between the lobes are mediated by the electrostatic potential of the molecule.
Calcium↗
Feasibility of peripheral blood stem cell (PBSC) and peripheral blood mononuclear cell (PBMNC) separation in children with a body weight below 20 KG.
Nine children from 10 to 76 months (median 28.0), weighing 8.5 to 19.7 kg (median 13.0 kg) underwent peripheral blood stem cell separation (PBSCS) or peripheral blood mononuclear cell separation (PBMNCS), after insertion of a double-lumen central venous catheter (8-10 French). Separations were performed with a continuous flow blood separator (Fen-wall CS 3000 plus), running a specially adopted separation-program. In 7 children (5 with neuroblastoma IV, 1 with multifocal Ewing's sarcoma, and 1 with rhabdomyosarcoma IV), stem cells were mobilized by application of G-CSF at a dosage of 15-27.7 micrograms/kg body weight (median 16.25) subcutaneously following high-dose chemotherapy, according to the disease-related protocols, whereas 2 children had PBMNCS to induce graft vs. leukemia (GvL)-reaction in the HLA-identical sibling suffering from relapsed chronic myelogenous leukemia (CML: n = 1), or chronic myelomonocytic leukemia (CMML: n = 1) after allogeneic BMT. In all cases, the collecting procedure was performed after filling the cell separator with priming solution consisting of 2 U of irradiated and washed packed red cells, 250 ml human albumin, and 0.9% NaCl. In the 7 patients with solid tumors between 0.45 and 62.7 x 10(6) CD-34 positive cells/kg body weight were separated; the patient who had the lowest yield was separated twice after another mobilizing course. Three patients (2 with neuroblastoma IV and 1 with multifocal Ewing's-sarcoma) underwent a double transplantation with 1-3 portions of the collected stem cells within a 5- to 6-week interval. Two children had a rapid engraftment on both peripheral blood stem cell transplantations (PBSCTs). The third child, who had the lowest yield and was separated twice had prompt engraftment at the first PBSCT but delayed and incomplete engraftment at the second PBSCT. One patient after adoptive immunotransfer with PBMNCs for relapsed CML is now 40 months in complete cytogenetic and molecular biological remission, whereas the other patient treated for relapsed CMML did not respond to the PBMNC-transfusion. The results indicate that PBSCS and PBMNCS can be performed in children with a body weight below 20 kg.
Antigens, CD34↗
Effect of monomer composition on proton dissociation of weak polyacids.
The study of the proton dissociation process of weak polyacids (eg a carboxylic poly(monoprotic)acid) is based on the knowledge of the change in electrostatic free energy, G(el), as a function of the variation of the number of charges on the polymer chain. The original treatment proposed by Manning can be used to describe the proton dissociation process of weak poly(monoprotic)acids, in the absence of pH-induced conformational transitions. In order to describe the alpha dependence of pKa of weak co-poly(monoprotic)acids containing two different acidic groups in different amounts along the polymer chain, a simple modification of the model is proposed.
Carboxylic Acids↗
Structural characterization and solution properties of an acidic branched (1-->3)-beta-D-glucan from Aureobasidium pullulans.
An acidic exopolysaccharide was isolated from a selected strain of Aureobasidium pullulans. On the basis of spectroscopic and chromatographic techniques, the polymer was identified as a beta-D-glucan containing a main chain of (1-->3)-linked beta-D-glucopy-ranosyl units substituted at the O-6 position by single beta-D-glucopyranosyl side chains. The ratio of units in the main chain to units in the side chain was found to be 1.4:1. The ionic character of this exopolysaccharide is due to the presence of malate residues which are linked to the polymer through ester bonds. The degree of substitution was estimated to be very low (0.05). In aqueous solution no signals are present in the NMR spectra strongly suggesting that the polymer adopts a rigid ordered conformation as further confirmed by rheological data. A solvent-induced conformational transition was observed in DMSO in which NMR spectra with good signal-to-noise ratio were obtained. The solution behaviour of the polymer is similar to that of other branched (1-->3)-beta-D-glucans in spite of both the degree of branching and the substitution with malate groups.
Carbohydrate Conformation↗
Thermal denaturation of bacterial and bovine dihydrofolate reductases and their complexes with NADPH, trimethoprim and methotrexate.
Scanning microcalorimetry was used for the study of thermal denaturation of E.coli and bovine liver dihydrofolate reductases (cDHFR and bDHFR, respectively) and their complexes with NADPH, trimethoprim (TMP) and methotrexate (MTX) at pH 6.8. It was shown that the denaturation temperature of bDHFR is 7.2 degrees C less than that of cDHFR and that ionic strength is equally important for the thermostability and cooperativity of the denaturation process of the two proteins. Binding of antifolate compounds significantly stabilizes DHFR against heat denaturation. The stabilizing effect and the transition cooperativity depend on the nature of the inhibitor, the presence of NADPH and the origin of the enzyme. The dependence of calorimetric denaturation enthalpy (calculated per gram of protein) on denaturation temperature for DHFRs, their complexes with NADPH and binary/ternary complexes with TMP/MTX fits to the same straight line with the slope of 0.66 J/Kg. This relatively high value indicates an essential role of hydrophobic contacts in the stabilization of DHFR structure. The change of denaturation temperatures in binary complexes with MTX/TMP (in comparison with the free enzymes) is as much as 14.2 degrees C/8.5 degrees C and 13.3 degrees C/3.2 degrees C for cDHFR and bDHFR, respectively. The same change in ternary complexes with MTX/TMP is much more pronounced and equals to 21.9 degrees C/16.8 degrees C and 29.0 degrees C/16.4 degrees C. The vast difference of binary and ternary complexes thermostability demonstrates the important role of cofactor in the stabilization of enzyme. Moving from binary to ternary systems causes a significant increase in denaturation temperatures, even when corresponding association constants do not change (cDHFR binary/ternary complexes with MTX) or increases very slightly (bDHFR binary/ternary complexes with TMP). In all other cases the increase of denaturation temperature for each protein in complex with ligands correlates with the association constant for the corresponding complex.
Animals↗
NMR analysis of succinoglycans from different microbial sources: partial assignment of their 1H and 13C NMR spectra and location of the succinate and the acetate groups.
In order to obtain information on the location of succinate and acetate groups, comparative NMR analyses were carried out on succinoglycans from different microbial sources by using conventional and advanced NMR techniques. In particular, one-dimensional, 1H and 13C NMR spectra were recorded for qualitative and quantitative analysis on native high-molecular-weight succinoglycans (both in the Na+ salt and free-acid forms) from Pseudomonas sp. NCIB 11592, Agrobacterium radiobacter A201-25, Rhizobium meliloti YE-2, and Rhizobium sp. isolated from Vicia faba and compared with those of the deacylated and deacylated-depyruvated, partially depolymerised exopolysaccharides from Rhizobium meliloti YE-2. Moreover, a series of two-dimensional experiments was performed on all the exopolysaccharides aiming at the partial assignment of the NMR spectra. The NMR data showed that succinate is located on O-6 of either one or both of the two side chain 3-linked beta-D-Glc residues, whereas the acetate (when it is present) is located on one of the O-6 of backbone 4-linked beta-D-Glc units, but the specific site could not be determined. In addition, the spectral features of the succinate substituent were found to be sensitive to pH changes.
Acetates↗
FTIR studies of sodium hyaluronate and its oligomers in the amorphous solid phase and in aqueous solution.
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Carbohydrate Conformation↗
[Quality control of blood components by means of flow cytometers].
Small numbers of leukocytes cannot be counted by automated methods. So we performed fluorescent staining of leukocyte DNA and analyzed 50 samples of single-donor PC by FACS. In 72% of these PC we found white blood cells (WBC) < 5/microliter = WBC < 10(6)/U. Nevertheless we do filter our PC. Should fresh frozen plasma (FFP) be irradiated for immunocompromised patients to prevent TA-GvHD? To evaluate the number and distribution of WBC in FFP we stained WBC with moAb and performed FACS analyses. We found WBC 7.164 +/- 5.66 x 10(6)/U, lymphocytes, 3.032 +/- 3.66 x 10(6)/U and T lymphocytes 2.135 +/- 2.02 x 10(6)/U. According to these data it should be considered to irradiate FFP for immunocompromised patients.
Blood Donors↗
Thermodynamic study of folate analogue binding to dihydrofolate reductases from different species.
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Animals↗
Thermodynamic study of the interaction of methotrexate, its metabolites, and new antifolates with thymidylate synthase: influence of FdUMP.
A microcalorimetric method was used for the direct study of the interaction of methotrexate, its metabolites, and new antifolates N10-propargyl-5,8-dideazafolate (CB 3717) and 2-methyl,2-desamino N10-propargyl-5,8-dideazafolate (CB 3819), with thymidylate synthase. We show that 7-hydroxymethotrexate and dideazafolates require the prior binding of dUMP or its fluorinated derivative FdUMP to bind to thymidylate synthase, as does methotrexate. Conversely, we show that methotrexate-G2 can interact directly with the enzyme alone. On the other hand, both dUMP and FdUMP exhibited a large cooperative effect on the affinity for thymidylate synthase of the inhibitors, and surprisingly, no significant difference was shown at this level between the natural substrate dUMP and its fluorinated derivative. It was demonstrated that this cooperative effect had an enthalpic origin. In the presence of FdUMP or dUMP, all the studied compounds except 7-hydroxymethotrexate exhibited a large negative enthalpy variation when binding to thymidylate synthase (from -44 to -91 kJ/mol). CB 3717 and methotrexate-G2 are competitors for the same protein binding site. Polyglutamation of methotrexate lead to compounds with higher affinity (association constants were 6.6 x 10(3) M-1 and 2.3 x 10(6) M-1 for methotrexate and methotrexate-G2 respectively) while hydroxylation has an unfavourable effect (association constant of 7-hydroxymethotrexate inferior to 500 M-1). Evidence for the influence of polyglutamation was also provided by the relatively low affinity of dideazofolates for thymidylate synthase (association constant equal to 1.4 and 1.7 x 10(7) M-1 for CB 3717 and CB 3819, respectively), whereas these compounds are known to be strong inhibitors of the enzyme in cells in their polyglutamated forms.
Calorimetry↗
A flow microcalorimetric method for enzyme activity measurements: application to dihydrofolate reductase.
A flow microcalorimetric method was developed for the analysis of enzymatic activities in crude tissue homogenates. It can be applied whenever a heat exchange is involved in an enzymatic reaction. The consequent sensitivity obviously depends on the enthalpy variation observed. Dihydrofolate reductase was chosen as an example; this enzyme is the molecular target of methotrexate, a widely used anticancer agent. This calorimetric method, whose sensitivity limit is 1.48 X 10(-4) units of dihydrofolate reductase per milliliter of reactant medium, allows enzyme activity measurements in tissues with low dihydrofolate reductase levels. A few examples of measurements in animal tissues are given. These measurements are of some interest; indeed, increased activity and increased levels of this enzyme are two of the mechanisms which may explain resistance to methotrexate.
Animals↗
Binding of 7-hydroxy-methotrexate to human serum albumin.
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Binding, Competitive↗
pH-Stat titration method for dihydrofolate reductase activity measurements: application to determination of substrate Michaelis constant and antifolate inhibition constant.
A pH-Stat titration method was developed for measuring dihydrofolate reductase (DHFR) activity; this method permits detection of very low DHFR activities corresponding to 100 pmol of substrate reduced per minute. This value is about ten times lower than those observed using the classical spectrophotometric method. This sensitivity makes it possible to measure the DHFR in crude tissue extracts. With beef liver DHFR, Michaelis constants for the cofactor NADPH and the natural substrate determined by this method were 1.9 +/- 0.3 X 10(-5) and 8.5 +/- 0.5 X 10(-7) M, respectively. The inhibition constant of methotrexate, a competitive inhibitor of dihydrofolate, was 3.4 +/- 1.3 X 10(-11) M.
Animals↗