A fluorescence double-quenching study of native lipoproteins in an animal model of manganese deficiency

Iodide and acrylamide were applied simultaneously in a doublequenching experiment to compare acrylamide quenching constants for internal and external fluorophores of high-density lipoproteins (HDL₁ and HDL₂) from manganese-adequate (MnA) and deficient (MnD) rats, free of the electrostatic effects associated with iodide. In MnA HDL₁ compared to MnD HDL₁, the acrylamide quenching constant for external fluorophores was different (P < 0.1). In MnA HDL₂, there were two populations of fluorophores accessible to acrylamide, whereas in MnD HDL₂, all fluorophores were accessible to both quenchers. We concluded that there were structural (local environmental) differences, possibly charge-related, around the external fluorophores, and a slightly larger population of buried fluorophores in the MnD HDL₁ compared with MnA HDL₁. In MnA HDL₂, one-third of the fluorophores were accessible to iodide, and all external and internal fluorophores were accessible to acrylamide, whereas in MnD HDL₂, all fluorophores were accessible to both quenchers.     

Spectral fluctuation of a single fluorophore conjugated to a protein molecule

We have measured the fluorescence spectra of a single fluorophore attached to a single protein molecule in aqueous solution using a total internal reflection fluorescence microscope. The most reactive cysteine residue of myosin subfragment-1 (S1) was labeled with tetramethylrhodamine. The spectral shift induced by a change in solvent from aqueous buffer to methanol in both single-molecule and bulk measurements were similar, indicating that, even at the single molecule level, the fluorescence spectrum is sensitive to microenvironmental changes of fluorophores. The time dependence of the fluorescence spectra of fluorophores attached to S1 molecules solely showed a fluctuation with time over a time scale of seconds. Because the fluorescence spectra of the same fluorophores directly conjugated to a glass surface remained constant, the spectral fluctuation observed for the fluorophores attached to S1 is most likely due to slow spontaneous conformational changes in the S1 molecule. Thus, single-molecule fluorescence spectroscopy appears to be a powerful tool to study the dynamic behavior of single biomolecules.     

The abnormality of glucose transporter in the erythrocyte membrane of Chinese type 2 diabetic patients

Type 2 diabetes mellitus is characterized by impaired glucose uptake. With a photometric method of recording the erythrocyte suspension absorption during the course of glucose transport across the membranes, we observed that the initial rate of glucose zero-trans entry was decreased significantly in 30 Chinese type 2 diabetic patients as compared to 25 healthy controls. The rate of glucose infinite-cis efflux exhibited no difference between the patients and controls. The measurement of temperature dependence of glucose transport showed that the activation energy for glucose entry was increased in diabetic patients. The inhibitory constant of glucose entry by cytochalasin B (CB) in patients was similar to that of the controls. However, we found that the inhibitory constant was increased significantly in the patient erythrocytes after phloretin treatment. After the erythrocytes were made into stripped white ghosts, the fluorescence quenching experiment was performed. Glucose, CB and phloretin can quench the fluorescence of tryptophan residues in the glucose transporter 1, GLUT1. The abnormality of fluorescence quenching in the erythrocyte membranes of patients was observed. The transfer tendency of tryptophan residues from the hydrophilic environment to the hydrophobic environment was decreased in patient ghosts as binding with glucose, and the opposite tendency appeared as CB and phloretin instead of glucose. We conclude that the decreased in glucose entry in the erythrocyte membranes of diabetic patients was due to the GLUT1 change in structure - mostly the outer domain of the glucose transporter.     

Ferritin in erythrocytes and plasma of patients with iron overload

Erythrocyte and plasma ferritin was followed in 13 patients with iron overload undergoing phlebotomies for at least 6 months in comparison with untreated patients and normal males. Plasma ferritin was widely scattered with an average of only twice the normal, whereas erythrocyte ferritin was highly elevated to about twelve times the normal (p less than 0.0001). - The time course of plasma and erythrocyte ferritin during phlebotomy therapy was analyzed in 3 patients with idiopathic hemochromatosis. Three stages were established: 1. plasma ferritin dropped gradually into the normal range while erythrocyte ferritin remained high, 2. appropriate phlebotomies maintained normal plasma ferritin and high erythrocyte ferritin, and indicated a monthly uptake of dietary iron of 150-200 mg at a steady state, 3. at low plasma ferritin levels, erythrocyte ferritin was rapidly decreased by further intensive phlebotomy therapy. Based on the presumed net removal of iron, 1 microgram/l plasma ferritin was equivalent to 3-6 mg of body iron and 1 microgram/l erythrocyte ferritin to somewhat less than 1 mg of body iron. - An elevated erythrocyte ferritin during phlebotomy therapy in iron overload not only depends on body iron stores like plasma ferritin but may also be regulated by the activity of erythropoiesis.     

Impact of Zn,Cu, and Fe on the Activity of Carbonic Anhydrase of Erythrocytes in Ducks

The impact of zinc, copper, and iron on the duck erythrocyte carbonic anhydrase (CA) activity and the hemoglobin content in vitro culture were studied. The increase of zinc or iron addition at a low level induced the rise of CA activity, and the CA activity was inhibited by zinc or iron at a high addition level. The duck erythrocyte CA was strongly inhibited by cupric ion. The inhibition constant of duck erythrocyte CA to cupric ion is about 3.5 μM. Carbonic anhydrase compared to hemoglobin is more sensitive to zinc and copper in the environment. These findings suggest that some characteristics of duck erythrocyte CA are different from both CAI and CAII of mammals. The increase of Fe addition below 8 μM in the minimal essential medium brought about the rise of CA activity and resulted in the maximum of CA activity exceeding that induced by Zn. It provided a new evidence for the role of ferrous ion in CA.     

Age-related parallel decline in beta-adrenergic receptors, adenylate cyclase and phosphodiesterase activity in rat erythrocyte membranes.

The activities of three components of the cyclic AMP system were compared in erythrocyte ghost membranes prepared from the blood of rats at various ages from 1.5 to 15 months. The apparent number of β-adrenergic receptor sites, adenylate cyclase activity and cyclic AMP phosphodiesterase activity all declined about 50% in the membranes from the older animals (>5 months) as compared to the 1.5 month ones. The soluble erythrocyte phosphodiesterase also declined with age, but the decline did not parallel that of the membrane-associated activity. In contrast, there was no age-related change in the number of β-adrenergic receptors in membranes from the brains of the same animals. In erythrocyte ghosts, both the ratio of isoproterenol-stimulated adenylate cyclase activity to basal activity and the ratio of sodium fluoride-stimulated activity to basal were constant with age. Neither the dissociation constant for the β-adrenergic receptor nor the Michaelis constant for the phosphodiesterase changed as a function of age. Together with other data in the literature, these results suggest a close functional association of the components of the cyclic AMP system in the mature erythrocyte membrane, and support a physiological role for the cyclic AMP mediated β-adrenergic effects in the red blood cell.     

Effect of omega-3 fatty acids on the modification of erythrocyte membrane fatty acid content including oleic acid in peritoneal dialysis patients

Erythrocyte membrane fatty acids (FA), such as oleic acid, are related to acute coronary syndrome. There is no report about the effect of omega-3 FA on oleic acid in peritoneal dialysis (PD) patients. We hypothesized that omega-3 FA can modify erythrocyte membrane FA, including oleic acid, in PD patients. In a double-blind, randomized, placebo-controlled study, 18 patients who were treated with PD for at least 6 months were randomized to treatment for 12 weeks with omega-3 FA or placebo. Erythrocyte membrane FA content was measured by gas chromatography at baseline and after 12 weeks. The erythrocyte membrane content of eicosapentaenoic acid and docosahexaenoic acid was significantly increased and saturated FA and oleic acid were significantly decreased in the omega-3 FA supplementation group after 12 weeks compared to baseline. In conclusion, erythrocyte membrane FA content, including oleic acid, was significantly modified by omega-3 FA supplementation for 12 weeks in PD patients.     

Distance between Cys-201 in erythrocyte band 3 and the bilayer measured by single-photon radioluminescence.

Single-photon radioluminescence (SPR), the excitation of fluorophores by short-range beta-decay electrons, was developed for the measurement of submicroscopic distances. The cytoplasmic domain of band 3 (cdb3) is the primary, multisite anchorage for the erythrocyte skeleton. To begin to define the membrane arrangement of the highly asymmetrical cdb3 structure, the distance from the bilayer of Cys-201 next to the "hinge" of cdb3 was measured by both SPR and resonance energy transfer (RET). cdb3 was labeled at Cys-201 with fluorescein maleimide. For SPR measurements, the bilayer was labeled with [3H]oleic acid. The corrected cdb3-specific SPR signal was 98 +/- 2 cps microCi-1 [mumol band 3]-1. From this and the signal from a parallel sample in which 3H2O was substituted for [3H]oleic acid to create uniform geometry between 3H and the fluorophores, a Cys-201-to-bilayer separation of 39 +/- 7 A was calculated. Confirmatory distances of 40 and 43 A were obtained by RET between fluorescein on Cys-201 and eosin and rhodamine B lipid probes, respectively. This distance indicates that Cys-201 lies near band 3's vertical axis of symmetry and that the subdomain of cdb3 between the hinge and the membrane is not significantly extended. In addition, these results validate SPR as a measure of molecular distances in biological systems.     

Soft X-ray microscopy analysis of cell volume and hemoglobin content in erythrocytes infected with asexual and sexual stages of Plasmodium falciparum

Plasmodium falciparum, the most virulent agent of human malaria, undergoes both asexual cycling and sexual differentiation inside erythrocytes. As the intraerythrocytic parasite develops it increases in size and alters the permeability of the host cell plasma membrane. An intriguing question is: how is the integrity of the host erythrocyte maintained during the intraerythrocytic cycle? We have used water window cryo X-ray tomography to determine cell morphology and hemoglobin content at different stages of asexual and sexual differentiation. The cryo stabilization preserves native structure permitting accurate analyses of parasite and host cell volumes. Absorption of soft X-rays by protein adheres to Beer–Lambert’s law permitting quantitation of the concentration of hemoglobin in the host cell compartment. During asexual development the volume of the parasite reaches about 50% of the uninfected erythrocyte volume but the infected erythrocyte volume remains relatively constant. The total hemoglobin content gradually decreases during the 48 h cycle but its concentration remains constant until early trophozoite stage, decreases by 25%, then remains constant again until just prior to rupture. During early sexual development the gametocyte has a similar morphology to a trophozoite but then undergoes a dramatic shape change. Our cryo X-ray tomography analysis reveals that about 70% of the host cell hemoglobin is taken up and digested during gametocyte development and the parasite eventually occupies about 50% of the uninfected erythrocyte volume. The total volume of the infected erythrocyte remains constant, apart from some reversible shrinkage at stage IV, while the concentration of hemoglobin decreases to about 70% of that in an uninfected erythrocyte.     

Steady-state kinetics of the catalase reaction in the presence of cyanide

Under carefully controlled experimental conditions, the Michaelis constant for H2O2 was measured to be 1.39 and 1.29 M in the reactions of beef erythrocyte and liver catalases, respectively. These values remained unchanged at temperatures between 1 and 26 degrees C. The turnover number of the Michaelis complex was about 2.25 X 10(7) s-1 for either enzyme at 26 degrees C. The cyanide inhibition in the catalase reaction has been reported to be noncompetitive in spite of the fact that cyanide and H2O2 compete for the same site on the catalase molecule. At high concentrations of H2O2, however, the inhibition became clearly competitive. The existence of the Michaelis complex and the anomalous features of cyanide inhibition were clearly accounted for on the basis of simple kinetic models. At H2O2 concentrations below 100 mM, the catalase reaction obeyed first order kinetics with respect to H2O2 and its apparent second order rate constant was measured to be 7.6 X 10(6) and 7.9 X 10(6) M-1 . S-1 for erythrocyte and liver catalases, respectively.     

Oligomeric fluorescent labels for DNA

In an effort to find fluorescent labels that have large Stokes shifts and increased emission intensity, a strategy for fluorescence labeling of DNA was explored in which multiple individual fluorophores are incorporated at adjacent positions at the end of a DNA probe. To encourage close interactions, hydrocarbon and heterocycle fluorophores were substituted at C-1 of deoxyribose, replacing the DNA base. The C-glycosides studied contained the well-known fluorophores terphenyl, pyrene, and terthiophene. For comparison, a commercial fluorescein-dU nucleotide was examined. Oligomeric labels containing up to five fluorophores were tested. Interestingly, all four dyes behaved differently on multiple substitution. Fluorescein displayed strong self-quenching properties, with the quantum yield dropping severalfold with each additional substitution and with a constant, small Stokes shift. In contrast, pyrene showed increases in quantum yield on addition of more than one fluorophore and yielded efficient long-wavelength emission on multiple substitution, with Stokes shifts of >130 nm. Oligomeric terphenyl labels gave a small progressive red shift in absorption and a marked red shift in emission wavelength and showed a strong increase in brightness with more monomers. Finally, terthiophene oligomers showed self-quenching combined with increasing Stokes shifts. Overall, the results suggest that some oligomeric fluorescent labels exhibit properties not available in common fluorescein class (or other commercial) labels, such as large Stokes shifts and increasing brightness with increasing substitution.     

An ENDOR study of human and bovine erythrocyte superoxide dismutase: 1H and 14N interactions

Hyperfine interactions (1H and 14N) with the paramagnetic Cu(II)-site obtained from frozen solutions of human and bovine erythrocyte superoxide dismutase (superoxide:superoxide oxidoreductase, EC 1.15.1.1) as well as from their derivatives produced by anion binding (N3-, CN-) and by depletion of the Zn(II) site were studied using electron nuclear double resonance (ENDOR) spectroscopy at about 15 K. Both interactions were found to be identical in human and bovine erythrocyte superoxide dismutase. In all compounds, an anisotropic, exchangeable 1H interaction with a nearly constant coupling value (approximately 3 MHz along g perpendicular ) was observed which is due to either histidine NH- or water protons. Other proton interactions were tentatively assigned to H beta 1 of His-44, H delta 2 of His-46 and to H beta 2 of His-44. Depletion of the Zn(II) site did not alter appreciably the pattern of the proton interactions. The 14N couplings of the native specimen indicated equivalent coordination, whereas Zn(II) depletion and CN- addition were found to produce either some or drastic inequivalences, respectively. For N3- addition to either the native or the Zn(II)-depleted sample only minor effects on the respective 14N coupling pattern were observed.     

Erythrocyte Membrane Gold Levels After Treatment with Auranofin and Sodium Aurothiomalate

Triethylphosphine gold-2,3,4,6-tetra-o-acetyl-L-thio-D-glucopyranoside (auranofin and sodium aurothiomalate; Myocrisin are two chemically different gold compounds used to treat rheumatoid arthritis. This study highlights the interaction, in vivo, of these drugs with erythrocyte membrane in patients with rheumatoid arthritis. Fifty-eight patients with definite or classical rheumatoid arthritis were included in this study and randomly allocated to three groups as 18 patients in the Myocrisin group, 20 patients in the auranofin group, and 20 patients in the placebo group. The drugs appeared to react, in vivo, in different ways. With Myocrisin, the level of gold in erythrocyte membrane was, initially, very high and decayed exponentially afterwards, whereas auranofin produced a constant high level up to 36 weeks. The erythrocyte membrane gold level in nonsmokers was higher than that in smokers in the auranofin group, and it decreased with an increase in the number of cigarettes smoked (r = 0.836 P < 0.01); no such correlation was observed in the Myocrisin group. In a changeover study, auranofin appeared to change the nature of erythrocyte membrane after reacting with it and rendering it incapable of picking up any gold from Myocrisin. In the case of auranofin, the hemolysate membrane gold level was found to correlate with clinical improvement.     

Interactions of glycolytic enzymes with erythrocyte membranes

Partition equilibrium experiments have been used to characterize the interactions of erythrocyte ghosts with four glycolytic enzymes, namely aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphofructokinase and lactate dehydrogenase, in 5 mM sodium phosphate buffer (pH 7.4). For each of these tetrameric enzymes a single intrinsic association constant sufficed to describe its interaction with erythrocyte matrix sites, the membrane capacity for the first three enzymes coinciding with the band 3 protein content. For lactate dehydrogenase the erythrocyte membrane capacity was twice as great. The membrane interactions of aldolase and glyceraldehyde-3-phosphate dehydrogenase were mutually inhibitory, as were those involving either of these enzymes and lactate dehydrogenase. Although the binding of phosphofructokinase to erythrocyte membranes was inhibited by aldolase, there was a transient concentration range of aldolase for which its interaction with matrix sites was enhanced by the presence of phosphofructokinase. In the presence of a moderate concentration of bovine serum albumin (15 mg/ml) the binding of aldolase to erythrocyte ghosts was enhanced in accordance with the prediction of thermodynamic nonideality based on excluded volume. At higher concentrations of albumin, however, the measured association constant decreased due to very weak binding of the space-filling protein to either the enzyme or the erythrocyte membrane. The implications of these findings are discussed in relation to the likely subcellular distribution of glycolytic enzymes in the red blood cell.     

Fluorescent peptide hormones: development of high affinity vasopressin analogues.

Highly potent and specific peptide hormone analogues with fluorescent reporter groups are current research goals. Until now, however, only moderately potent analogues have been described. We report here several types of vasopressin (VP) analogues with different fluorophores attached to the peptide. In a first series, fluorophores were attached to the free epsilon amino function of [des-amino1-lysine8]VP (dLVP), producing agonistic analogues. In a second series, reporter groups were added to the N-terminal of open-chain antagonist structures. The biological activities of these analogues were assessed by two different sets of experiments: 1) The measurement of their binding affinities towards the V1a-vasopressin receptor subtype from WRK1 cells or rat liver membrane preparations; 2) Their ability to stimulate the phospholipase C activity in WRK1 cells. As expected, a simple acylation of fluorophores to dLVP resulted in a considerable loss of affinity. If however, the Lys8 side chain was extended through double Schiff-base formation with glutaraldehyde-ethylenediamine followed by reduction to an aminoalkyl aminoalkylamine, single fluorophores could be added without loss of affinity compared to VP. The open-chain analogues, on the other hand, while displaying weak affinity, nevertheless exhibited pure antagonistic behavior.     

Carbonic anhydrase C in white-skeletal-muscle tissue.

We investigated the activity of carbonic anhydrase in blood-free perfused white skeletal muscles of the rabbit. Carbonic anhydrase activities were measured in supernatants and in Triton extracts of the particulate fractions of white-skeletal-muscle homogenate by using a rapid-reaction stopped-flow apparatus equipped with a pH electrode. An average carbonic anhydrase concentration of about 0.5 microM was determined for white skeletal muscle. This concentration is about 1% of that inside the erythrocyte. Some 85% of the muscle enzyme was found in the homogenate supernatant, and only 15% appeared to be associated with membranes and organelles. White-skeletal-muscle carbonic anhydrase was characterized in terms of its Michaelis constant and catalytic-centre activity (turnover number) for CO2 and its inhibition constant towards ethoxzolamide. These properties were identical with those of the rabbit erythrocyte carbonic anhydrase C, suggesting that a type-C enzyme is present in white skeletal muscle. Affinity chromatography of muscle supernatant and of lysed erythrocytes showed that, whereas rabbit erythrocytes contain about equal amounts of carbonic anhydrase isoenzymes B and C, the B isoenzyme is practically absent from white skeletal muscle. Similarly, ethoxzolamide-inhibition curves suggested that white skeletal muscle contains no carbonic anhydrase A. It is concluded that white skeletal muscle contains essentially one carbonic anhydrase isoenzyme, the C form, most of which is probably of cytosolic origin.     

Effects of inner solution viscosity and membrane stiffness on erythrocyte deformability on passing through a microchannel: prediction by two-dimensional simulation

We studied erythrocyte deformability in an effort to develop diagnostic methods based on its measurement and thus aid in the development of therapies for circulatory diseases. In the reported work, we performed two-dimensional numerical simulations of blood flow through a microchannel (MC) to evaluate erythrocyte deformability, applying the immersed boundary method to simulate erythrocyte movement and deformation. To evaluate deformability, MC transit capacity and shape recoverability were considered, defined as the time required to pass through the MC and the time constant during the shape-recovery process after exiting the MC, respectively. The simulation results showed that the erythrocyte MC transit time increased when the viscosity of the inner solution or the stiffness of the membrane increased. The time constant for erythrocyte shape recovery increased as the inner solution viscosity increased. In contrast, the time constant decreased as the erythrocyte membrane stiffness increased. These time-constant trends were in agreement with a theoretical equation derived using the Kelvin model and with previous experimental results. This diagnostic method of measuring erythrocyte shape recoverability and MC transit capacity is anticipated to have clinical application.     

Location and dynamics of anthracyclines bound to unilamellar phosphatidylcholine vesicles

We have exploited the intrinsic fluorescence properties of the anthracycline antitumor antibiotics to study the dependence on drug structure of relative drug location and dynamics when the anthracyclines were bound to sonicated dimyristoylphosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) vesicles at 27.5 degrees C. Iodide quenching experiments at constant ionic strength were used to evaluate the relative accessibilities of the bound fluorophores to membrane-impermeable iodide. Iodide was found to quench the fluorescence of anthracyclines in free solution by both static and dynamic mechanisms, whereas quenching of membrane-bound fluorophores was predominantly due to the dynamic mechanism. Modified Stern-Volmer plots of anthracyclines bound to fluid-phase DMPC bilayers were linear, and the biomolecular rate constant (kq) values ranged from 0.6 X 10(9) to 1.3 X 10(9) M-1 s-1. Modified Stern-Volmer plots of anthracyclines bound to solid-phase DPPC bilayers were curved, indicative of a heterogeneous-bound drug population. A strong correlation between drug hydrophobicity and penetration of the fluorophore into the bilayer was observed for the daunosamine-containing anthracyclines. Steady-state fluorescence anisotropy measurements under iodide quenching conditions were used to investigate the diffusive motions of anthracyclines in isotropic solvent and in fluid-phase DMPC bilayers. Anthracycline derivatives free in solution exhibited limiting anisotropy (alpha infinity) values which decayed to zero at times long compared to the excited-state lifetime, in contrast to anthracyclines bound to fluid-phase DMPC bilayers, which showed nonzero alpha infinity values. Steady-state anisotropies of membrane-bound anthracyclines were found to be governed principally by alpha infinity and not by the mean rotational rate (R).(ABSTRACT TRUNCATED AT 250 WORDS)     

Periodic auto-immune hemolytic anemia: An induced dynamical disease

Experimentally induced auto-immune hemolytic anemia (AIHA) in rabbits is characterized either by constant depressed erythrocyte numbers, or by oscillatory erythrocyte numbers about a depressed level (periodic auto-immune hemolytic anemia). Here the experimetallys observed characteristics of AIHA are satisfactorily accounted for by a simple model for erythropoiesis, assuming only the peripheral erythrocyte destruction rate is elevated with all other parameters normal. The onset of periodic AIHA is identified with the occurrence of a Hopf bifurcation in the model dynamics for certain values of the erythrocyte destruction rate.     

Polymorphism of exon 4 in the CANP-3 gene in patients with primary myopathies

The structures of the gene for calpain (CANP-3) and of the DMD gene were analyzed in patients with primary myopathies [limb-girdle muscular distrophy (LGMD) and Duchenne-Becker myodystrophy (DBM)] from various regions of Russia. Via amplification of DNA isolated from the peripheral blood lymphocytes of 74 patients, extended deletions were found in 18 out of 55 patients with DBM. In none of the 19 patients with LGMD, were extended deletions in the CANP-3 gene found. In most patients with LGMD, the amplification of the promoter region and exons 1, 2, 3, 4, 5, and 6 of the CANP-3 gene yielded a single product of corresponding length, but in six patients (three sib pairs), amplification of exon 4 of the CANP-3 gene yielded two products of different size. The following single-strand conformation polymorphism (SSCP) analysis revealed a pronounced polymorphism of exon 4 of the CANP-3 gene in 14 out of 19 patients with LGMD. This structure of exon 4 of the CANP-3 gene was found neither in 16 patients with DBM who had deletions in the DMD gene nor in 16 patients with DBM who had no deletions in the DMD gene.