Biomedical subjects

C Poyart

Publications and source records attributed to C Poyart.

At least 55 records · Page 3Linked to original sources

PubMed1996

Hb Bruxelles, deletion of Phebeta42, shows a low oxygen affinity and low cooperativity of ligand binding.

Functional studies of partially purified hemoglobin (Hb) Bruxelles, Phebeta42 (CD1) --> 0 indicate a major shift in the allosteric equilibrium toward the deoxy (T state) conformation. While Hb A shows a roughly symmetrical oxygenation curve with maximum cooperativity near half-saturation, Hb Bruxelles shows mainly properties of the low affinity (T state) form. The oxygen equilibrium curves for purified (>80%) Hb Bruxelles show little cooperativity and a P50 (without 2,3-diphosphoglycerate) about twice that of Hb A. The low cooperativity for Hb Bruxelles is partially compensated by an increase in oxygen affinity of the deoxy conformation and a lower 2,3-diphosphoglycerate effect. The beta chains of normal Hb have consecutive phenylalanine residues at positions 41 and 42. DNA sequencing studies of Hb Bruxelles showed a deletion of the codon TTT, which corresponds to residue Phe42. The CO rebinding kinetics after flash photolysis show mainly the slow phase, characteristic of CO binding to the deoxy conformation. In phosphate buffer at pH 7, the slow phase dominates even at low photolysis levels, where the main reaction is ligand binding to the triply liganded form. This indicates a switchover point, from the deoxy to oxy conformation, occurring beyond three ligands for Hb Bruxelles. There are few natural mutants that show a change in the oxygen affinity and cooperativity as large as that observed for Hb Bruxelles.

Adult↗

PubMed1996

Heme-CO binding to tryptophan-containing calmodulin mutants.

The binding of heme-CO to genetically engineered calmodulin containing a single tryptophan residue has been studied. A tryptophan residue was integrated at one of five positions: 26 or 62 of the N-terminal, 81 in the central helix, or 99 or 135 of the C-terminal. As for the wild type, the mutant calmodulins bind four molecules of heme-CO with an average affinity of 1 microM. (i) Homotropic effect. The quenching of the tryptophan fluorescence by energy transfer to the hemes indicates that there is no preference between the N- or C-terminal pockets for heme binding. The quenching is less than expected for a binomial distribution of four sites. This could indicate a lower energy transfer rate due to a specific orientation factor. The weak quenching as a function of the number of hemes bound may also reveal a cooperativity in the heme binding; the data can be simulated assuming two pairs of sites, where each pocket shows a cooperative binding for two hemes. (ii) Heterotropic effect. As observed for the wild type, addition of melittin does not displace the hemes from the mutant calmodulins; the affinity of heme-CO for the calmodulin.melittin complex is higher than that for calmodulin alone. The affinity of heme-CO for native calmodulin is also higher in the presence of trifluoperazine.

Animals↗

PubMed1996

Functional studies and polymerization of recombinant hemoglobin Glu-alpha2beta26(A3) --> Val/Glu-7(A4) --> Ala.

In hemoglobin (Hb) S the hydrophobic mutated residue Val-beta6(A3) (donor site) closely interacts with the hydrophobic side groups of Phe-beta85(F1) and Leu-beta88(F4) (EF pocket, acceptor site) of a neighboring tetramer, resulting in decreased solubility and polymerization of the deoxy-Hb. The beta6(A3) residue is followed by two charged residues Glu-beta7(A4) and Lys-beta8(A5). This cluster has no attraction for the hydrophobic EF pocket. We have modified the beta7(A4) residue next to the donor site Val-beta6(A3), replacing the charged Glu by a hydrophobic Ala-(rHb betaE6V/E7A). The single mutant Glu-beta7 --> Ala-(rHb betaE7A) was also engineered. Both rHbs exhibit a heat instability and an increased oxygen affinity compared to Hb A and Hb S. There was a concentration dependence of the ligand binding properties (1-300 microM in heme) indicating an increased amount of dimers relative to Hb A. The deoxy form of rHb betaE6V/E7A polymerizes in vitro, with a decreased rate of polymer formation relative to Hb S, while the single mutant betaE7A does not polymerize in the same experimental conditions. The Glu-beta7(A4) --> Ala substitution does not increase the hydrophobic interaction between donor and acceptor site. We speculate that the loss of the normal saline bridge between Glu-beta7(A4) and Lys-beta132(H10) leads to an increased flexibility of the A helix and may account for the difference of the polymerization for this Hb S mutant.

Alanine↗

PubMed1996

Picosecond geminate recombination of CO to the complexes calmodulinheme-CO and calmodulinheme-CO*melittin.

Picosecond CO recombination kinetics have been measured after photodissociation of the artificial complexes calmodulin*heme-CO and calmodulin*heme-CO*melittin. These systems show an enhancement of the geminate fraction of kinetics relative to unbound heme-CO, due in part to fast geminate kinetics (tau=50ps for the initial phase), as well as a decrease in the rate of migration of CO away from the binding site. This indicates that calmodulin provides a complete pocket around the heme group. Rather than competing with the hemes for binding to calmodulin, the melittin seems to act as a cap to further enclose the hemes; melittin increases the affinity of calmodulin for heme-CO, but only weakly affects the CO recombination kinetics.

Amino Acid Sequence↗

PubMed1996

Two mutations in recombinant Hb beta F41(C7)Y, K82 (EF6)D show additive effects in decreasing oxygen affinity.

Based on the properties of two low oxygen affinity mutated hemoglobins (Hb), we have engineered a double mutant Hb (rHb beta YD) in which the beta F41Y substitution is associated with K82D. Functional studies have shown that the Hb alpha 2 beta 2(C7)F41Y exhibits a decreased oxygen affinity relative to Hb A, without a significantly increased autooxidation rate. The oxygen affinity of the natural mutant beta K82D (Hb Providence-Asp) is decreased due to the replacement of two positive charges by two negative ones at the main DPG-binding site. The functional properties of both single mutants are interesting in the view of obtaining an Hb-based blood substitute, which requires: (1) cooperative oxygen binding with an overall affinity near 30 mm Hg at half saturation, at 37 degrees C, and in the absence of 2,3 diphosphoglycerate (DPG), and (2) a slow rate of autooxidation in order to limit metHb formation. It was expected that the two mutations were at a sufficient distance (20 A) that their respective effects could combine to form low oxygen affinity tetramers. The double mutant does display additive effects resulting in a fourfold decrease in oxygen affinity; it can insure, in the absence of DPG, an oxygen delivery to the tissues similar to that of a red cell suspension in vivo at 37 degrees C. Nevertheless, the rate of autooxidation, 3.5-fold larger than that of Hb A, remains a problem.

Hemoglobins↗

PubMed1996

Hemolytic anemias due to hemoglobinopathies.

Hemoglobinopathies responsible for hemolytic anemias may be divided into two groups. The first one corresponds to thalassemias and the second to the presence of a structurally abnormal hemoglobin (Hb). In thalassemia, the primary biochemical abnormality is a quantitative defect in the biosynthesis of one type of Hb chain. This defect leads to an overall deficit of Hb accumulation in the erythrocyte (hypochromia) together with the presence of an excess of the normally synthesized chains. The unpaired subunits which are less soluble than HbA precipitate, bind to the membrane and ultimately lead to hemolysis. In the second group, the hemolytic anemia is a direct consequence of the physicochemical properties of the structurally abnormal Hb. This molecule may polymerize, precipitate or crystallize within the red blood cell (RBC) leading to membrane alterations and to the destruction of the cell. This chapter will emphasize several examples of structurally abnormal Hbs, such as sickle cell disease and congenital Heinz body hemolytic anemia (CHBHA).

Anemia, Hemolytic↗

PubMed1996

Serodiagnosis of listeriosis based upon detection of antibodies against recombinant truncated forms of listeriolysin O.

Amino-terminal fragments of listeriolysin O (LLO) of 240 and 411 residues (fragments LLO240 and LLO411, respectively) were expressed in Escherichia coli as fusion polypeptides with maltose-binding protein (MBP) with the aim of producing specific antigens for use in serological tests. In Western blots (immunoblots) with crude bacterial extracts of the fusion polypeptides, the reactivities of MBP-LLO240 and MBP-LLO411 with anti-LLO antibody (ALLO)- and anti-streptolysin O antibody (ASLO)-positive human sera were first compared with that of the entire LLO (LLO530) also fused to MBP (MBP-LLO530). Sixteen of 17 (94.1%) ALLO-positive samples reacting with MBP-LLO530 also reacted with MBP-LLO411, whereas this proportion dropped to 11 of 17 (64.7%) with MBP-LLO240. Alternatively, 18 of 19 (94.7%) ASLO-positive samples giving an interpretable result reacted with MBP-LLO530, whereas 1 of 19 (5.3%) of these samples reacted with MBP-LLO240 or MBP-LLO411. The fusion polypeptide MBP-LLO411 was purified by maltose affinity chromatography and was further evaluated as a diagnostic antigen in a Western blot assay. Twenty-one of 21 (100%) serum samples obtained from patients with listeriosis and found to be positive for ALLO by a reference dot blot test reacted with MBP-LLO411, whereas 1 of 20 (5%) ASLO-positive serum samples and 1 of 100 (1%) serum samples from healthy adults were reactive. Thus, a polypeptide limited to the 411 amino-terminal residues of LLO is a specific and sensitive antigen for the detection of ALLO.

Adolescent↗

PubMed1996

The role of proline beta 5(A2) in the functional properties of human adult hemoglobin.

The replacement of beta 5(A2)Pro by Arg in Hb Warwickshire appears to be without an effect on the functional properties of human Hb A, despite adding two external positive charges close to the central cavity of the hemoglobin tetramer, along the dyad axis. To clarify the role of this portion of the molecule involved in oxygen-linked anion binding, we have engineered the recombinant hemoglobin alpha 2 beta (2)5(A2)Pro-->Ala[rHb beta 5(A2)Pro-->Ala]. The rHb beta 5(A2)Pro-->Ala exhibits an increased oxygen affinity compared to Hb A, with normal heterotropic effects in standard conditions. The increased oxygen affinity may be attributed to the absence of proline, which would render the A helix more flexible, thus destabilizing the T structure. The normal functional properties of Hb Warwickshire may be due to the regulation of oxygen affinity by electrostatic effects involving diffusible anions not bound to any specific site.

2,3-Diphosphoglycerate↗

PubMed1995

How allosteric effectors can bind to the same protein residue and produce opposite shifts in the allosteric equilibrium.

Monoaldehyde allosteric effectors of hemoglobin were designed, using molecular modeling software (GRID), to form a Schiff base adduct with the Val 1 alpha N-terminal nitrogens and interact via a salt bridge with Arg 141 alpha of the opposite subunit. The designed molecules were synthesized if not available. It was envisioned that the molecules, which are aldehyde acids, would produce a high-affinity hemoglobin with potential interest as antisickling agents similar to other aldehyde acids reported earlier. X-ray crystallographic analysis indicated that the aldehyde acids did bind as modeled de novo in symmetry-related pairs to the alpha subunit N-terminal nitrogens. However, oxygen equilibrium curves run on solutions obtained from T- (tense) state hemoglobin crystals of reacted effector molecules produced low-affinity hemoglobins. The shift in the allosteric equilibrium was opposite to that expected. We conclude that the observed shift in allosteric equilibrium was due to the acid group on the monoaldehyde aromatic ring that forms a salt bridge with the guanidinium ion of Arg 141 alpha on the opposite subunit. This added constraint to the T-state structure that ties two subunits across the molecular symmetry axis shifts the equilibrium further toward the T-state. We tested this idea by comparing aldehydes that form Schiff base interactions with the same Val 1 alpha residues but do not interact across the dimer subunit symmetry axis (a new one in this study with no acid group and others that have had determined crystal structures). The latter aldehydes shift the allosteric equilibrium toward the R-state. A hypothesis to predict the direction in shift of the allosteric equilibrium is made and indicates that it is not exclusively where the molecule binds but how it interacts with the protein to stabilize or destabilize the T- (tense) allosteric state.

Aldehydes↗

PubMed1995

Bisaldehyde allosteric effectors as molecular ratchets and probes.

Four new series of monoaldehyde bisacids and bisaldehyde bisacids with varying chain lengths have been synthesized and evaluated as allosteric effectors of hemoglobin. Molecular modeling, oxygen equilibrium, and crystallographic studies were combined for structure/function studies. Crystallographic analyses of the bisaldehydes reveal that Schiff base interaction occurred exclusively between Val 1 alpha and Lys 99 alpha of the opposite alpha chain even though the two terminal Val 1 alpha nitrogens are ideally spaced to also form cross-links. The reason for the observed mode of binding appears to be the influence of chain direction set by key substitutions on the bisaldehyde molecule. Even longer chain derivatives that could overcome the direction set by the key functional groups bind in the same manner. These studies support the general conclusion that long flexible molecules prefer to bind along cavity walls, like double-sided molecular sticky tape, rather than span large open spaces with few chances for interaction. The cross-linked bisaldehydes bind at the same site when incubated under both allosteric states and exhibit reduced cooperativity with a significant decrease in oxygen affinity. The chain length acts as a molecular ratchet and dictates the degree of allosteric effect observed. The tighter the cross-link, the greater the constraint on the tense- (T-) state and the stronger the allosteric effect that is produced. The monoaldehyde bisacids bind in the same fashion with Schiff base formation at Val 1 alpha while the acid that replaces the second aldehyde moiety forms a salt bridge with Lys 99 alpha of the opposite subunit. This class of molecules has weaker allosteric effector activity as would be expected with replacement of one covalent bond by a salt bridge. The importance of Lys 99 alpha on the allosteric equilibrium is confirmed.

Aldehydes↗

PubMed1995

Loss of allosteric behaviour in recombinant hemoglobin alpha 2 beta 2(92)(F8) His-->Ala: restoration upon addition of strong effectors.

In the stereochemical model proposed by Perutz [1], the Fe-His(F8) bond plays a significant role in the allosteric transition in hemoglobin and the resulting cooperativity in ligand binding. When this bond is ruptured, there is a loss in the transmission of the information concerning ligand binding; examples are Hb(NO)4 in the presence of inositol hexakisphosphate (IHP), or nickel substituted Hb hybrids which, despite being liganded, exhibit deoxy-like properties. To study the effects of the loss of the iron proximal histidine bond, we have engineered the alpha 2 beta 2(F8)H92A recombinant Hb. The replacement of the highly conserved proximal histidine F8 residue by an alanine results in a low affinity for the heme group and a loss of the allosteric properties; kinetics of CO recombination after photodissociation show only the rapid bimolecular phase, characteristic of the high affinity R-state. However, a significant amount of deoxy (T-state) kinetics are observed after addition of external effectors such as IHP. The iron-histidine bond is apparently crucial for the heme-heme interaction, but the allosteric equilibrium may still be influenced by external constraints.

Alanine↗

PubMed1995

Characterization of superoxide dismutase genes from gram-positive bacteria by polymerase chain reaction using degenerate primers.

An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae, and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes, and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.

Base Sequence↗

PubMed1995

Protection by lazaroids of the erythrocyte (Ca2+, Mg2+)-ATPase against iron-induced inhibition.

The calmodulin-stimulated (Ca2+, Mg2+)-ATPase (calmodulin-ATPase) of the erythrocyte membrane is susceptible to oxidative stress induced by heme and non-heme iron. There is a time-and concentration-dependent inhibition of the calmodulin-ATPase activity when the erythrocyte membranes are treated with either iron or hemin. In the present study, the calmodulin-ATPase has been used as a model system to evaluate the protective effects of a vitamin E analog (U83836E) and two 21-aminosteroids (U74500A and U74389G) against calmodulin-ATPase inhibition induced by iron and hemin. The drugs, lazaroids from Upjohn, can significantly protect the enzyme against iron-induced inhibition and also causes a decrease in the formation of thiobarbituric acid reactive species, with an IC50 of 0.4 microM for the drug U83836E and 4 microM for the drug U74500A. The 21-aminosteroid U74389G does not restore iron-inhibited calmodulin-ATPase activity under similar conditions. At higher concentrations (> 100 microM) all three drugs inhibit the calmodulin-ATPase activity. None of the drugs tested can restore hemin-inhibited calmodulin-ATPase activity.

Antioxidants↗

PubMed1995

Heme as an optical probe for studying the interactions between calmodulin and the Ca(2+)-ATPase of the human erythrocyte membrane.

The heme group was used as an optical probe to study the interactions between calmodulin and its targets: the peptide melittin and the enzyme Ca(2+)-ATPase. As already reported, melittin when present in Tris buffer binds hemin-CN which quenches the tryptophan fluorescence. Addition of calmodulin restores the fluorescence significantly accompanied by a blue shift. We show here that the recovery of fluorescence is very slow and takes about 120 min to become constant. In a hydrophobic buffer, the fluorescence spectrum of melittin is already shifted with a peak at 335 nm and intensity almost 2-fold relative to a similar concentration of melittin in Tris buffer. The quenching of tryptophan fluorescence is lesser in this buffer and further addition of calmodulin fails to restore the fluorescence. This indicates the absence of binding of calmodulin to melittin in hydrophobic conditions. Under similar conditions of hydrophobicity, hemin-CN quenches about 35% of the tryptophan fluorescence of the Ca(2+)-ATPase. The subsequent addition of calmodulin restores about half of the quenched fluorescence. The interaction of calmodulin with the Ca(2+)-ATPase even under hydrophobic conditions suggests its high specificity for the enzyme which may be expected for a physiological target.

Calcium-Transporting ATPases↗

PubMed1995

Intrinsic activity at the molecular level: E. J. Ariëns' concept visualized.

The concept of using affinity and intrinsic activity to analyze drug interactions with receptors has had a long history in pharmacological studies. In the simplest case, the biological response will be proportional to the amount of drug bound, i.e. its affinity. However, the biological response is also mediated by the ability of a drug when bound to exert its maximum effectiveness. This effectiveness is termed the intrinsic activity. Physicochemical processes have been thought to be at the basis of intrinsic activity. Detailed oxygen and solution binding experiments combined with X-ray crystallographic studies on allosteric effectors to hemoglobin demonstrate that these potential drug agents bind at the same site in hemoglobin with similar binding constants yet shift the allosteric equilibrium and the oxygen affinity of the T-structure by different degrees. Therefore some of the effectors with similar binding affinities for the same site exhibit varying degrees of affectiveness, i.e. they possess different intrinsic activities. The intrinsic activity of the effector is defined as the ratio of the oxygen affinity constant to the T-state with drug/oxygen affinity constant to the T-state without drug (KT+drug)/(KT control). The source of the intrinsic activity appears to be the ability of the effectors to interact with key residues such as Lys99 alpha at the binding site. These results suggest a general molecular mechanism for allosteric effector modulation of hemoglobin function that might be of use in other allosteric enzyme systems.

Allosteric Regulation↗

PubMed1995

Correlation of carbon monoxide association rates and the position of absorption band III in hemoproteins.

We have examined the absorbance of a charge-transfer transition near 760 nm, known as band III, in several hemoproteins and heme complexes. The band III position correlates with the rate of carbon monoxide binding to the heme. A band III present at 760 nm indicates an unfavorable geometry of the heme for carbon monoxide binding; a red-shift of the band III to 765 nm indicates a less-constrained geometry of the heme as evidenced by higher carbon monoxide association rates. The band III position correlates well with the Raman frequency of the Fe-His(F8) bond as suggested previously for normal hemoglobin A [Sassaroli, M. & Rousseau, D. L. (1987) Biochemistry 26, 3092-3098]. Aplysia myoglobin and the chimeric heme protein kinase FixL from Bradyrhizobium japonicum, hemoproteins with an apolar residue in place of the highly conserved polar histidine E7, do not fit the relationship between the band III position and the rate of binding of carbon monoxide to the heme. With these few exceptions, the measurement of band III appears to be a practical means to probe the stretch frequency of the Fe-His(F8) bond.

Carbon Monoxide↗

PubMed1995

Evidence for sub-picosecond heme doming in hemoglobin and myoglobin: a time-resolved resonance Raman comparison of carbonmonoxy and deoxy species.

Separation of the photophysical aspects of the sub-picosecond (sub-ps) time-resolved resonance Raman signal from contributions due to conformation has been achieved by comparing deoxyhemoglobin (Hb) in the T state with (carbonmonoxy)hemoglobin (HbCO), deoxy-beta 4 (beta 4 CO) (All R state), and monomers deoxymyoglobin and (carbonmonoxy)myoglobin (MbCO) [beta 4 consists of a tetramer of four beta-subunits and shows no cooperativity]. In all photolyzed species, Hb*(CO), Mb*(CO), and beta 4*(CO), the iron-histidine out-of-plane mode (vFe-His), indicative of heme doming, achieves 90% of its full intensity in 1 ps. The frequency of this mode (223-228 cm-1) is shifted significantly relative to equilibrium deoxy-Hb (210-216 cm-1) in the T state, but not with respect to either equilibrium deoxy-Mb or deoxy-beta 4. A correlation between the +12 cm-1 bandshift of vFe-His and the -2 cm-1 shift of the electron density marker band (v4 at 1370 cm-1) relative to T-state deoxy-Hb is shown to hold on all time scales, including the sub-picosecond time scale. Photolyzed Hb*(CO) consists of R-state or weakly interacting tetramers on the picosecond time scale and is shown to have properties similar to those of photolyzed Mb*(CO) and beta 4*(CO) on the picosecond time scale. These results establish that heme doming occurs as an ultrafast reaction to ligand dissociation and that heme doming is the primary event in the sequence of conformational changes leading to the cooperative R-->T transition.

Carboxyhemoglobin↗

PubMed1995

Functional aspects of ultra-rapid heme doming in hemoglobin, myoglobin, and the myoglobin mutant H93G.

Heme iron out-of-plane displacement following ligand dissociation in hemoglobin, myoglobin, and the proximal cavity mutant H93G is shown to be as rapid as the heme iron out-of-plane vibrational period by sub-picosecond time-resolved resonance Raman spectroscopy. The results demonstrate that the effect of steric repulsion initiated by the spin change of the iron gives rise to heme doming independent of covalent attachment of the proximal ligand to the protein. It is concluded that the protein plays a passive role in the initial ultrafast heme iron motion toward the out-of-plane position observed in the deoxy structure of hemoglobin and myoglobin. The results suggest that the spin change of the heme iron is the primary cause of rapid heme doming and that steric repulsion of the proximal ligand with the heme plays a secondary role in forcing the iron out of the heme plane.

Animals↗