Lipid binding to cytoglobin leads to a change in haem co-ordination: a role for cytoglobin in lipid signalling of oxidative stress

Cytoglobin is a recently discovered hexa-co-ordinate haemoglobin that does not appear to function as a classical oxygen-binding protein. Its function is unknown and studies on the effects of changes in its expression have not decisively determined its role within the cell. In the present paper, we report that the protein is transformed from hexa-co-ordinate to penta-co-ordinate on binding a lipid molecule. This transformation occurs with the ferric oxidation state of the protein, but not the ferrous state, indicating that this process only occurs under an oxidative environment and may thus be related to redox-linked cell signalling mechanisms. Oleate binds to the protein in a 1:1 stoichiometry and with high affinity (K(d)=0.7 μM); however, stopped-flow kinetic measurements yield a K(d) value of 110 μM. The discrepancy between these K(d) values may be rationalized by recognizing that cytoglobin is a disulfide-linked dimer and invoking co-operativity in oleate binding. The lipid-induced transformation of cytoglobin from hexa-co-ordinate to penta-co-ordinate does not occur with similar hexa-co-ordinate haemoglobins such as neuroglobin, and therefore appears to be a unique property of cytoglobin among the haemoglobin superfamily. The lipid-derived transformation may explain why cytoglobin has enhanced peroxidatic activity, converting lipids into various oxidized products, a property virtually absent from neuroglobin and much decreased in myoglobin. We propose that the binding of ferric cytoglobin to lipids and their subsequent transformation may be integral to the physiological function of cytoglobin, generating cell signalling lipid molecules under an oxidative environment.     

The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering

The aggregation behavior of zinc-free insulin has been studied by small-angle neutron scattering as a function of pH and ionic strength of the solution. The pair distance distribution functions for the 12 samples have been obtained by indirect Fourier transformation. The results show that the diameter of the aggregates is 40 Å at pH 11 and 10 mM NaCl, independent of the protein concentration. The largest diameter of about 120 Å is found for pH 8, 100 mM NaCl, and a protein concentration of 10 mg/ml. Estimates of the pair distance distribution functions, free of inter-particle correlation effects, were obtained by an indirect Fourier transformation, omitting the data at small scattering vectors, which are influenced by these effects. By this procedure the weight-averaged molecular mass and the average radius of gyration were determined. These parameters vary from 1.3 times the monomer mass and 14 Å, to 6.8 times the monomer mass and 31 Å, respectively. The mass distribution between the oligomers was determined by a model based on the crystal structure of zinc-free insulin. The results from this model and the Fourier transformations have been compared to an equilibrium model recently introduced by Kadima et al. (1993). The neutron scattering results agree well with the predictions of this model except that broader mass distributions are suggested by neutron scattering. Correspondence to: J. Skov Pedersen     

Short range order of hydrocarbon chains in fluid phospholipid bilayers studied by x-ray diffraction from highly oriented membranes

We present a study of the short range ordering of hydrocarbon chains in phospholipid bilayers. The x-ray peak associated with the hydrocarbon chains has been probed by means of reciprocal space mappings. Using 20 keV undulator radiation and samples of negligible mosaicity (orientational disorder), the intensity distribution is probed as a function of two coordinates, the momentum transfer parallel and perpendicular to the bilayer, over a wide range and at high resolution. Structural results are obtained concerning the distribution of tilted segments, the correlation length and the radial distribution function of the quasi two-dimensional liquid structure. A comparison is made with published molecular dynamics data (H. Heller, M. Schaefer, and K. Schulten. 1993. J. Phys. Chem. 97:8343-8360) by direct Fourier transformation of the atomic coordinates. The exact prefactor in the relationship between interchain distance and peak position is derived.     

Bilayer structure of dihexadecyl phosphate

The X-ray scattering data of dihexadecyl phosphate (DHP) were analyzed by the distance distribution function directly obtained from the scattered intensity by Fourier transformation with no prior assumption about size and shape. The detergent in aqueous solutions is a random distribution of lamellae with a thickness of 58 Å and consistent with a hollow vesicular structure of an outer diameter of 340 Å with some overall size heterogeneity. The electron density profile perpendicular to the lamellar plane indicates a bilayer as the underlying structural principle.     

Some principles in the organization of sigma70-specific promoters on the E. coli genome on the basis of electrostatic patterns of promoter DNA

The distribution of electrostatic potential of the complete sequence of the E. coli genome was calculated. It was found that DNA is not a uniformly charged molecule. There are some local inhomogeneities in its electrostatic profile, which correlate with the position of promoters in the genome. Electrostatic patterns of promoter DNAs can be specified due to the presence of some distinctive motifs, which may be involved as promoter signal elements in RNA-polymerase-promoter recognition.     

Molecular size and shape of the native and reassociated forms of the extracellular haemoglobin of Tubifex tubifex

The extracellular haemoglobin of Tubifex tubifex and the product of its reassociation at neutral pH subsequent to dissociation at alkaline pH, were examined by small-angle X-ray scattering. The following molecular parameters were determined for the native and reassociated molecules, respectively: maximum diameter 30.0±1.0 and 32.0±1.0nm; radius of gyration 10.66±0.15 and 11.07±0.15 nm; molecular weight (3.09±0.15) × 10⁶ and (2.99±0.15) × 10⁶ dalton. Although the scattering curves of the native and reassociated haemoglobin possess similar shapes the distance distribution functions exhibit slight differences in their shape as well as in the position of their maximum. The best fit with the experimental distribution functions was obtained with models consisting of 12 spheres arranged in two hexagonal layers. In the case of the native haemoglobin each of the 12 spheres has a diameter of 9.3 nm while for the reassociated haemoglobin each of the 12 spheres has a diameter of 11.5 nm. The results suggest that although their molecular weights are the same, the reassociated molecule is slightly larger than the native molecule     

Preference of oxygenation between α and β subunits of haemoglobin: Results of multidimensional spectroscopic observation

The distribution of oxygen between the subunits of haemoglobin was studied spectrophotometrically. The difficulty in discriminating the spectral changes upon oxygen binding to the α or β subunit can be surmounted by means of multidimensional spectroscopic observations and a correlation analysis of the data. M-type abnormal haemoglobins are used as a control against normal haemoglobin because only one type of its subunits can bind oxygen.A multidimensional spectroscopic measuring system, which has been developed in our laboratory, makes it possible to carry out simultaneous and continuous acquisition of a set of spectroscopic data at several wavelengths on one sample solution during the course of increasing or decreasing the partial pressure of oxygen. The data-storing function of a magnetic disk memory provides enough precision for a rigorous investigation of the correlation of oxygen equilibrium curves measured at several wavelengths. No chemical modification to enhance the spectral difference between subunits is necessary.In conclusion, by detecting slight differences between the oxygenation-sensitive bands of α and β subunits, the β subunits are found to have a higher affinity for oxygen than the α subunits.     

A role for haemoglobin in all plant roots?

Abstract. We have found haemoglobin in plant roots whereas previously it has been recorded only in nitrogen fixing nodules of plants. Haemoglobin occurs not only in the roots of those plants that are capable of nodulation but also in the roots of species that are not known to nodulate. We suggest that a haemoglobin gene may be a component of the genome of all plants. The gene structure and sequence in two unrelated families of plants suggests that the plant haemoglobins have had a single origin and that this origin relates to the haemoglobin gene of the animal kingdom. At present we cannot completely rule out the possibility of a horizontal transfer of the gene from the animal kingdom to a progenitor of the dicotyledonous angiosperms but we favour a single origin of the gene from a progenitor organism to both the plant and animal kingdoms. We speculate about the possible functions of haemoglobin in plant roots and put the case that it is unlikely to have a function in facilitating oxygen diffusion. We suggest that haemoglobin may act as a signal molecule indicating oxygen deficit and the consequent need to shift plant metabolism from an oxidative to a fermentative pathway of energy generation.     

A cereal haemoglobin gene is expressed in seed and root tissues under anaerobic conditions

Legumes, and a very few non-legume plant species, are known to possess functioning haemoglobin genes. We describe here the characterization of a haemoglobin cDNA isolated from barley. The deduced amino acid sequence shows 71% amino acid identity with a non-legume haemoglobin gene, a further 16% of the residues being conservative replacements. The barley cDNA also hybridizes to genomic sequences in rye, maize and wheat. The demonstration of a gene from a monocotyledon with close sequence homology to the known non-legume plant haemoglobins fills a major gap in the known distribution of haemoglobin genes in the plant kingdom. The expression of the gene is induced in isolated barley aleurone layers exposed to anaerobic conditions, and the roots of flooding-stressed barley plants. The expression of the RNA under anoxic conditions is similar to that of a known anaerobic response gene, alcohol dehydrogenase. Our results suggest that the increased expression of haemoglobin RNA is an integral part of the normal anaerobic response in barley. The findings are discussed in the light of current theories of haemoglobin function and evolution.     

A fast SEQUEST cross correlation algorithm

The SEQUEST program was the first and remains one of the most widely used tools for assigning a peptide sequence within a database to a tandem mass spectrum. The cross correlation score is the primary score function implemented within SEQUEST and it is this score that makes the tool particularly sensitive. Unfortunately, this score is computationally expensive to calculate, and thus, to make the score manageable, SEQUEST uses a less sensitive but fast preliminary score and restricts the cross correlation to just the top 500 peptides returned by the preliminary score. Classically, the cross correlation score has been calculated using Fast Fourier Transforms (FFT) to generate the full correlation function. We describe an alternate method of calculating the cross correlation score that does not require FFTs and can be computed efficiently in a fraction of the time. The fast calculation allows all candidate peptides to be scored by the cross correlation function, potentially mitigating the need for the preliminary score, and enables an E-value significance calculation based on the cross correlation score distribution calculated on all candidate peptide sequences obtained from a sequence database.     

A BASIC program for the removal of noise from reaction traces using Fourier filtering

Software for the removal of noise from reaction curves usingthe principle of Fourier filtering has been written in BASICto execute on a PC. The program inputs reaction traces whichare subjected to a rotation -inversion process, to produce functionssuitable for Fourier analysis. Fourier transformation into thefrequency domain is followed by multiplication of the transformby a rectangular filter function, to remove the noise frequencies.Inverse transformation then yields a noise-reduced reactiontrace suitable for further analysis. The program is interactiveat each stage and could easily be modified to remove noise froma range of input data types. Received on October 20, 1988; accepted on January 10, 1989     

Reducing noise in computed correlation functions using techniques from signal processing

Time correlation functions invariably suffer from random noise, especially at longer time intervals for which fewer data pairs are available. This noise is particularly of concern when calculating correlations that cannot be averaged over per-molecule contributions, such as stress in molecular simulations. In this work, a set of methods based in signal processing has been developed to reduce the inherent noise that is present in time- and frequency-domain representations of correlation functions. The stress time autocorrelation function, which leads to stress relaxation modulus and complex modulus, is used as an example. The difference between initial and final values of a time correlation function over a finite time domain is found to create so-called ‘leakage’ of noise from disallowed into harmonic frequencies during fast Fourier transformation. Decreasing this leakage effect through reflection to negative time and through applying a window function reduces noise levels significantly. Removing frequency components of insignificant magnitudes also provides significant noise reduction. Applying moving averages in the frequency and time domains also contributes to noise reduction. Specific results obtained by applying these methods to a model asphalt system enable more clear physical interpretations of the underlying relaxations after dramatic noise level reductions were attained.     

Haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) structure and function.

The characterization of haemoglobin Saki alpha 2 beta 2 14 Leu-Pro(a11) is described. This new mutation is unique since it only induces modification of the stability of the molecule. In vitro precipitation of haemoglobin Saki upon heat or in the presence of chemicals is compared to the stability of haemoglobin A and haemoglobin S.     

Human haemoglobin: a new paradigm for oxygen binding involving two types of alphabeta contacts.

This review summarizes the most recent state of haemoglobin (Hb) research based on the literature and our own results. In particular, an attempt is made to form a unified picture for haemoglobin function by reconciling the cooperative oxygen binding with the stabilization of the bound dioxygen in aqueous solvent. The HbA molecule contains two types of alphabeta contacts. One type is the alpha1beta2 or alpha2beta1 contacts, called sliding contacts, and these are strongly associated with the cooperative binding of O2 to the alpha2beta2 tetramer. The other type is the alpha1beta1 or alpha2beta2 contacts, called packing contacts, but whose role in Hb function was not clear until quite recently. However, detailed pH-dependence studies of the autoxidation rate of HbO2 have revealed that the alpha1beta1 and alpha2beta2 interfaces are used for controlling the stability of the bound O2. When the alpha1beta1 or alpha2beta2 contact is formed, the beta chain is subjected to a conformational constraint which causes the distal (E7) histidine to be tilted slightly away from the bound dioxygen, preventing the proton-catalysed nucleophilic displacement of O2- from the FeO2 by an entering water molecule. This is one of the most characteristic features of HbO2 stability. Finally we discuss the role of the alpha1beta1 or alpha2beta2 contacts by providing some examples of unstable haemoglobin mutants. These pathological mutations are found mostly on the beta chain, especially in the alpha1beta1 contact regions. In this way, HbA seems to differentiate two types of alphabeta contacts for its functional properties.     

Osteopontin's colocalization with the adhesion molecule CEACAM5 in cytoplasm of carcinoma of tongue and its correlation with the invasion of that diease

ABSTRACT: The purpose of this study was to investigate the expression of carcinoembryonic antigenrelated cell adhesion molecule 5 (CEACAM5) and correlate it with OPN expression and function in squamous carcinoma of tongue. Paraffin were sections of 80 samples with squamous carcinoma of tongue and 40 samples with normal tissue of tongue for benign lesion having undergone surgery. Immunohistochemistry (IHC) was used to study the distribution of CEACAM5 and OPN, and double-labeling immunohistochemistry was used to observe the relationship between CEACAM5 and OPN expression. CEACAM5 and OPN are found in normal tissue of tongue, but with different expression pattern. CEACAM5 expression mainly with membranous staining is restricted on the superficial epithelium. However, OPN expression with mainly cytoplasmic staining is restricted on the deep epithelium. No colocalization of CEACAM5 and OPN have been observed in normal tissue of tongue. In squamous carcinoma of tongue, CEACAM5 expression with cytoplasmic staining is different from normal tongue tissue with membranous staining, and the transformation of CEACAM5 distribution from membrane to cytoplasm is an important incident for the invasion and differentiation of tumor. CEACAM5 and OPN are colocalized in cytoplasm, and a significant correlation was observed between the positive colocalization and the negative colocalization in the depth of invasion and the differentiation of the tumor.     

States of stability/lysis in human fetal and adults red blood cells

In human red blood cells from umbilical cord (NRBC) and from adult (ARBC) blood we measured: (i) the equilibrium distribution (medium/cells) for haemoglobin as a function of medium osmolality (osmotic fragility curve) and (ii) the rate of haemoglobin loss to a hypotonic medium of fixed osmolality. From the analysis of the osmotic (cumulative) fragility curve, a subpopulation of high resistance (young?) cells was individualized only in samples from cord blood. The differences presented in the rate of haemoglobin loss between samples of umbilical cord and adult's blood point to distinct cell surface restrictions (cytoskeleton and/or plasma membrane phospholipid) to haemoglobin leak in both cells. Uniformly distributed (among all cells in both samples) differences could explain the distinct rates of haemoglobin loss. However, marked differences restricted to only a subpopulation of cord blood cells could also explain these results.     

Structures of deoxy and carbonmonoxy haemoglobin Kansas in the deoxy quaternary conformation.

Haemoglobin Kansas (Asn102(G4)β → Thr) is the only widely studied mutant or modified haemoglobin having four functional haems and displaying lower than normal oxygen affinity. Two forms of this mutant have been investigated by X-ray diffraction. The deoxy form, whose crystals are isomorphous with the native form, has been examined directly by the difference Fourier technique (3.4 Å). In addition to the replaced residue itself, the difference electron density map shows signs of slight movements throughout the region between α and β haem pockets. However, neither type of chain shows stereochemical evidence of a very abnormal oxygen affinity in the tetramer. The nature of the perturbation is different from that proposed in the earlier, low-resolution study of Greer (1971a). Exposure of deoxy crystals to carbon monoxide produces two new crystal forms similar but not identical to the native deoxy form. One of these structures has been solved by rotation and translation function methods and a difference map between carbonmonoxy haemoglobin Kansas in the deoxy quaternary structure and native deoxy haemoglobin has been calculated at 3.5 Å resolution. Carbon monoxide molecules are observed at three of the four haems, and two unidentified large peaks³ appear next to the sulphydryl groups of Cys93β. None of the interchain salt bridges which stabilize the deoxy quaternary state appears to be broken, but large tertiary structural changes are seen in the liganded chains. It seems that when the molecule is subjected to the lattice constraints of the deoxy crystal, the salt bridges do not break upon ligand binding, even though the pH dependence of the first Adair constant and the linearity of proton release with ligand uptake both imply that this does happen to stripped HbA in solution.     

Separation of glycosylated haemoglobins using immobilized phenylboronic acid. Effect of ligand concentration, column operating conditions, and comparison with ion-exchange and isoelectric-focusing

Haemoglobins from diabetic and non-diabetic individuals have been separated by affinity chromatography using immobilized phenylboronate, which interacts specifically with diol-containing compounds such as glycosylated haemoglobin. The effects of ligand concentration, flow rate, column geometry, preincubation of sample, buffer composition and temperature have been investigated. Significant correlation was found between results from affinity-chromatography and ion-exchange and isoelectric-focusing methods. Isoelectric-focusing of the haemoglobin fractions obtained from affinity chromatography indicate that, in addition to haemoglobin A1c, some haemoglobin A is also bound to immobilized phenylboronic acid. Assays of haemolysates obtained from red blood cells incubated in glucose solutions suggest that unstable pre-(haemoglobin A1c) does not interfere. The assay is not affected by the presence of haemoglobin F.     

Motility evaluation of bull spermatozoa by photon correlation spectroscopy.

Quasi-elastic light scattering techniques are employed to evaluate motility of bovine spermatozoa. The electric field correlation function CE(k, tau) has two components CE(k, tau) = alphafm+(1-alpha)fd, where fm is the correlation function due to motile cells, fd is due to dead cells and alpha is the fraction of motile cells. A function which is a linear combination of the experimentally measured fd and an empirically determined function fm is fit to the CE(k, tau) data. In this way, alpha and the approximate analytic form of fm are determined. The distribution of swimming speeds P(v) is derived from fm using an inverse Fourier sine transform procedure. Results for the time dependence of motility of bull sperm in Hank's balanced salt solution are presented.     

Resistance of mammalian red blood cells of different size to hypertonic milieu

1. The resistance of different mammalian red blood cells (RBCs) to hyperosmotic environments was studied. RBCs of six mammalian species were exposed to 10 increasingly hyperosmotic NaCl solutions for 24 hr at 5 degrees C. 2. The osmolality at which the amount of liberated haemoglobin reached a preset level (e.g. 3-4% of the total haemoglobin) showed a linear correlation with negative slope with RBC volume. This indicates that small RBCs are more resistant to hyperosmotic milieu than large ones. 3. A similar relation can be found from literature data when maximal urinary tonicities are plotted as a function of RBC volume, i.e. animals with the ability to produce highly concentrated urine have small RBCs. 4. RBC volume and maximal urinary tonicity in mammals are therefore tightly linked. Future research will have to show whether this correlation is fortuitous or not and whether, as can be speculated, RBC size is directly or indirectly regulated by the kidney.