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Biomedical subjects

R Cassoly

Publications and source records attributed to R Cassoly.

At least 37 records · Page 2Linked to original sources
PubMed1983

Spectral and oxygen-release kinetic properties of human hemoglobin bound to the cytoplasmic fragment of band 3 protein in solution.

Binding of the cytoplasmic fragment of band 3 protein to oxyhemoglobin in solution caused a spectral change in the absorbance of the hemoglobin beta chain at a ratio of one monomer of band 3 protein per alpha beta dimer of hemoglobin. This spectral change was reversed at higher ratios of cytoplasmic fragment to hemoglobin. The unusual dependence on protein concentration was interpreted as indicating the formation of higher aggregates of the complex between hemoglobin and the cytoplasmic fragment of band 3 protein. Oxygen-release kinetic measurements also showed marked changes as a function of the concentration of the cytoplasmic fragment of band 3 protein. The higher ratio mixture had significantly different kinetic properties as compared with the lower ratio one, which in turn was different from oxyhemoglobin in solution. The significance of the formation of aggregates of band 3 protein containing oxyhemoglobin dimers is discussed in context with evidence suggesting that band 3 protein may exist as an equilibrium mixture of tetramers and dimers in the membrane.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

Studies on the fragmentation of erythrocyte ghost membrane with p-chloromercuribenzoate in the micromolar range.

The effects of nonsaturating amounts (5-60 nmol/mg membrane protein) of p-chloromercuribenzoate on the stability of unsealed erythrocyte ghosts were studied by turbidimetric measurements and direct observation by phase contrast microscopy. The organic mercurial provokes drastic disorganization of the membrane involving vesicle formation by inter- and externalization of the bilayer. These effects are not associated with a release in solution of membrane proteins which was shown in previous studies to occur at higher p-chloromercuribenzoate concentration. Attempts have been made to identify the proteins involved in this phenomenon by the use of nonsaturating amounts of radioactively-labelled p-chloromercuribenzoate. Actin and band 3 protein which are the first to be labelled, represent plausible candidates as sensitive targets for the disrupting organic mercurial. Stroma obtained from spherocytes did not show significant differences with normocytes in their stability with regard to p-chloromercuribenzoate. Other reagents including N-ethylmaleimide, diamide and DNAase I were also studied. The results suggest strongly that the integrity of the sulfhydryl groups of actin, as well as those of band 3 protein, is essential for the stability of the erythrocyte membrane.

Chloromercuribenzoates↗
PubMed1983

Quantitative analysis of the association of human hemoglobin with the cytoplasmic fragment of band 3 protein.

The association of the isolated cytoplasmic fragment of band 3 protein with human hemoglobin was studied by rate zonal centrifugation in sucrose density gradients, by quenching of fragment fluorescence by hemoglobin, and by flash photolysis of carbon monoxidebound hemoglobin as a function of fragment concentration. The centrifugation results showed that both proteins interact and that the interaction is abolished upon addition of glyceraldehyde-3-phosphate dehydrogenase. The fractions eluted from the density gradient were analyzed further by spectrophotometric and gel electrophoretic methods. Two types of complexes could be identified, one containing the equivalent of 1 hemoglobin tetramer/dimer of cytoplasmic fragment and another containing 2 tetramers of hemoglobin/dimer of fragment. Flash photolysis and fluorescence-quenching experiments showed that liganded hemoglobin is stabilized as the alpha beta dimer when bound to the fragment, a result almost identical with that seen for membranebound hemoglobin in previous studies. The results further suggest that there are two binding sites for the alpha beta dimer of hemoglobin on one monomer of the cytoplasmic fragment but only one mutually exclusive hemoglobin tetramer binding site, suggesting the possibility of conformational isomerism when the fragment with two dimers bound isomerises to a fragment monomer with one hemoglobin tetramer bound. Finally, despite the stabilization of the dimeric state of liganded hemoglobin when bound to the fragment, estimates of the hemoglobin dimer and tetramer binding constants suggest that the hemoglobin tetramer binds more tightly by about 2 orders of magnitude.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1983

A dynamical study on the interactions between the cytoskeleton components in the human erythrocyte as detected by saturation transfer electron paramagnetic resonance of spin-labeled spectrin, ankyrin, and protein 4.1.

Isolated human erythrocyte spectrin, ankyrin, and protein 4.1 have been labeled with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl, and studied by saturation transfer electron paramagnetic resonance spectroscopy. The presence of the labels does not affect the reassociation of these proteins with erythrocyte membranes selectively depleted of either spectrin-actin or of all the extrinsic proteins. When maleimide spin-labeled spectrin is reassociated with the erythrocyte membrane in presence of all the cytoskeleton components, including endogeneous or purified muscle actin, spectrin still preserves its flexible character. The rotational mobilities of maleimide spin-labeled ankyrin and maleimide spin-labeled protein 4.1 are of the same order of magnitude (tau c (L"/L) approximately 5 X 10(-5) and 8 X 10(-5) s, respectively, at 2 degrees C), while protein 4.1 is almost three times smaller in size than ankyrin. This result indicates that the movements of membrane-bound maleimide spin-labeled protein 4.1 are more restricted than those of ankyrin. This suggests that their respective binding sites have different structural properties. The rotational movements of both proteins are slowed down on the addition of spectrin indicating that protein 4.1 as well as ankyrin also represents one of the links of the cytoskeleton to the membrane.

Actins↗
PubMed1982

Interaction of hemoglobin with the red blood cell membrane. A saturation transfer electron paramagnetic resonance study.

Human hemoglobin has been labeled on cysteine 93(beta) with the maleimide spin label, 3-maleimido-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl and reassociated with erythrocyte membrane previously stripped of hemoglobin and glyceraldehyde-3-phosphate dehydrogenase. The affinity of hemoglobin for the membrane is not affected by the presence of the label. Saturation transfer electron paramagnetic resonance measurements show that the diffusion rotational movements of hemoglobin are considerably slowed down when it is bound to the erythrocyte membrane. The correlation time of rotation, tau c, is found to be 8 . 10(-6) s as compared with 2 . 10(-8) s when the hemoglobin molecule is in solution. The same values are obtained whether the protein is associated with its high- or low-affinity binding sites. They depend on the viscosity of the solution. The high-affinity sites are presumably located on the segment of the band 3 protein which extends into the cytoplasm and which links through ankyrin, the spectrin-actin cytoskeleton to the membrane. When band 3 is cross-linked into a dimer after reaction with the copper-ortho-phenanthroline chelate, the correlation time of rotation of spin-labelled hemoglobin is unchanged. It is also independent of the presence of the spectrin-actin network and ankyrin. These results show tha the movements of hemoglobin bound by ionic linkage to different part (protein or phospholipid) of the cytoplasmic surface of the membrane are similarly highly restricted by some potential or energetic barrier. They give also evidence for independent movements and flexibility in the assembly of the macromolecules which link the spectrin-actin cytoskeleton to the erythrocyte membrane.

Cyclic N-Oxides↗
PubMed1982

[Interaction between oxyhemoglobin and cytoplasmic fragments of band 3 protein purified from human erythrocyte membranes].

The cytoplasmic fragment of the band 3 protein strongly interacts with oxyhemoglobin, as shown by rate zonal centrifugation and fluorescence quenching measurements. According to their relative concentrations two different types of complexes can be formed with a molar ratio of the band 3 fragment per oxyhemoglobin tetramer of 0.9 and 1.8 respectively. The equilibrium binding constant of oxyhemoglobin for the peptide is 1.4 x 10(-7) M-1 in 5 mM phosphate buffer at pH 6. Glyceraldehyde-3-phosphate dehydrogenase or an increase in pH in ionic strength prevent this association.

Anion Exchange Protein 1, Erythrocyte↗
PubMed1982

Differences in the electric birefringence of spectrin dimers and tetramers as shown by the fast reversing electric pulse method.

The electric birefringence of purified spectrin has been examined in medium of low ionic strength at 20 degrees C and for electric fields smaller than 4 X 10(4) V m(-1), using the reversing electric pulse method. This technique allows study of the permanent and induced dipole electric moment of macromolecules more easily than in measurements using only rectangular pulses. We show that spectrin heterodimers and heterotetramers have different electro-optical properties. The relaxation time of the tetramer (7 microseconds) is significantly longer than that of the dimer (4.5 microseconds). Tetramers and dimers have also different polarizability parameters.

Birefringence↗
PubMed1980

Spin labeling of human spectrin. Effects of temperature, divalent cations and reassociation with erythrocyte membrane.

Spectrin extracted from human red blood cells has been spin labeled in its dimeric and tetrameric forms with five different nitroxide derivatives of increasing chain length between their maleimide binding group and their nitroxide reporter group. Three molecules of spin label are bound per spectrin dimer. Electron spin resonance spectra show the simultaneous presence of strongly and weakly immobilized spin labels. Their relative proportion depends on the label length and is suddenly modified when it reaches 12 A This indicates the presence of cavities of approximately this size in the tertiary structure of spectrin in solution at 0 degrees C. The conformation of spectrin varies greatly with temperature. Reversible changes occur between 0 and 35 degrees C. At higher temperatures, partial denaturation is observed. Divalent cations (Mg2+ and Ca2+) stabilize spectrin in a more constrained conformation and protect it against thermal denaturation. The same behavior is observed when spin-labeled spectrin is reassociated with spectrin-depleted inside-out erythrocyte vesicles. When fatty acid spin labels are incorporated in the phospholipidic structure of these vesicles, the reassociation of spectrin does not change their electron spin resonance spectra. This result confirms the fact that spectrin interacts predominantly with proteins on erythrocyte membranes.

Calcium↗
PubMed1979

Binding of alkylisocyanides with soybean leghemoglobin. Comparisons with sperm whale myoglobin.

The binding of various linear and branched chain alkylisocyanides to soybean leghemoglobin has been studied with respect to association and dissociation kinetics and the results compared with those obtained in parallel on sperm whale and horse heart myoglobins; the linear ligands used (methyl to n-heptyl) cover a greater distribution of chain lengths than hitherto used. The association rate constants are much higher for leghemoglobin than for myoglobin, while the dissociation rates are slower. For a given protein, the dissociation rate constants are not much different when different isocyanides are used (except for methyl), whereas the association rates show complex behavior in relation with the alkyl chain length; singular differences are observed between leghemoglobin and sperm whale myoglobin in this regard. For myoglobin, the binding rate constants decrease from methyl to n-propyl, but remain approximately the same when the ligand carries a still longer alkyl chain. In contrast, for leghemoglobin, although the rate constants decrease from methyl to n-propyl, they show a progressive and important rise with longer alkyl substituents: n-butyl and n-pentyl.

Animals↗
PubMed1978

Use of nitric oxide as a probe for assessing the formation of asymmetrical hemoglobin hybrids. An attempted comparison between alphaNObetaNOalphadeoxybetadeoxy, alpha2NObeta2deoxy, and alpha2deoxybeta2NO hybrids.

It has been recently demonstrated that some nitrosyl hemoglobin derivatives have different optical spectrum according to the nature of their quaternary structure (Cassoly, R. (1974) C. R. Seances Acad. Sci., Paris 278, 1417-1420; Salhany, J. M., Ogawa, S., and Shulman, R. G. (1974) Proc. Natl, Acad. Sci. U.S.A. 71, 3359-3362; Cassoly, R. (1975) J. Mol. Biol. 98, 581-595). This property has been used in order to detect the presence of asymmetrical hybrids alphaNObetaNOalpha'O2beta'O2 in a mixture of the two hemoglobins alpha2NObeta2NO and alpha2'O2beta2'O2. When one changes, by deoxygenation, the conformation of the hybrid, there is a characteristic modification in the optical spectrum of the nitrosyl subunits. Quantitative analysis of this phenomenon shows that asymmetrical alphaNObetaNOalphadeoxybetadeoxy and symmetrical alpha2NObeta2deoxy hybrids have distinct properties. The structure-linked optical transition is different in rate and amplitude; it is faster and larger for the asymmetrical molecule. Carbon monoxide binding kinetics performed in absence of phosphate have also indicated that the allosteric equilibrium is more displaced in favor of the T state for alphaNObetaNOalphadeoxybetadeoxy by comparison with the symmetrical deoxygenated intermediates.

Haptoglobins↗
PubMed1978

[Studies of spin-labeled spectrin].

Spectrin isolated from human erythrocytes has been spin-labeled with five maleimide nitroxides. The mobility of the labels is strongly dependent on their size, and on the temperature. A thermal transition of spectrin is shown to occur above 30 degrees C. Calcium and magnesium provoke a strong immobilization of the labels. This effect is yet more pronounced when spectrin is allowed to reassociate with the cytoplasmic surface of the membrane.

Electron Spin Resonance Spectroscopy↗
PubMed1976

Unequivalence between hemoglobin subunits. The effects of inositol hexakisphosphate on the absorption spectrum of liganded valency hybrids.

The effects of inositol hexakisphosphate on the visible absorption spectrum of liganded valency hybrids alphaCO2 beta+H20, alpha+H2ObetaCO2 and alphaCO2beta+CN2, alpha+CN2beta CO2 (where alphaCO, alpha+H2O, alpha+CN and betaCO, beta+H2O, beta+CN represent the alpha and beta chain of hemoglobin, respectively, in their carbon monoxide, aquomet and cyanomet forms) have been examined in an attempt to determine if their alpha and beta chains are equally sensitive to the action of the anion. The difference spectra induced by inositol hexakisphosphate, are different for the two chains. The beta+H2O subunit contributes chiefly to the absorbance changes, rather than alpha+H2O. In contrast, it is the alphaCO chains which is more sensitive to inositol hexakisphosphate than betaCO. The question of whether such a selective response would also occur in aquomethemoglobin and carboxyhemoglobin is discussed. Criteria of changes in the quaternary structure of hybrids has been studied by measuring the effects of inositol hexakisphosphate on their ultraviolet absorption spectrum.

Carboxyhemoglobin↗
PubMed1976

[Study by fast kinetics of certain ligand combination and dissociation reactions with bis(maleimidomethyl)ether-hemoglobin (HbBME)].

Bis(N-maleimidomethyl)ether-hemoglobin (HbBME) is a well known derivative of hemoglobin obtained by reaction with bis(N-maleimidomethyl)ether, which forms an intra-chain bond in each of the beta subunit of the protein. Previous cristallographic determinations suggest that this chemical modification exerts an effect on the structure of the COOH-terminal region in the beta chain. As a consequence, the quaternary structure of deoxygenated HbBME is strongly destabilized in favor of the oxy conformation. Kinetic studies including oxygen dissociation and carbon monoxide binding reactions have been performed on HbBME in order to correlate these changes in structure with the reactivity of hemes in the protein. The disappearance of heme-heme interaction in HbBME is expressed in the character of the oxygen dissociation kinetics. With respect of this reaction, we show that the reactivity of alpha chains remains unchanged inside the chemically modified tetramer, whilst that of beta chains is increased. Carbon monoxide binding kinetics show heterogeneity with two well separated phases. The fast phase contributes about 75 per cent of the total absorbance change and has a rate of binding similar to that of fastly reacting hemoglobins. The slow phase show the same rate as in native deoxyhemoglobin. The effects of inositolhexaphospate on the these kinetics have been studied. They consist mainly in the decrease of fast phase which can disappear completely according to the experimental conditions. These experiments suggest that deoxy-HbBME exists in solution as a mixture of two isomers in slow equilibrium. Taking account of previous structural studies, these results are discussed in comparison with an hemoglobin mutant with very similar properties, hemoglobin Bethesda (Tyr 145beta leads to His). The importance of the COOH-terminal region on the functional properties of hemoglobin is once again emphasized.

Binding Sites↗