Parameters of binding of fluorescent probe pyrrone red with human serum albumin

The constant of binding of a new fluorescent probe pyrrone red to human serum albumin (Kb = 4.7.10(5) M-1) and the number of binding sites (N = 2) were determined by the method of double fluorimetric titration at a Fex/Fem intensity ratio of 560/625 nm. The affinity of pyrrone red for albumin was by 20% lower than that of 8-anilinonaphthalenesulfonate and 2.4 times higher than that of another probe K35. Thus, pyrrone red is almost identical to amlinonaphthalenesulfonate in affinity for albumin and can be considered as a long-wavelength "red" analogue of anilinonaphthalenesulfonate.     

The ferritin content of human red blood cells during the replacement of embryonic cells by fetal cells

Mature human embryonic erythrocytes (hemoglobin is ≥ 90% of the cellular protein) contained at least 20 times as much ferritin as human adult erythrocytes, suggesting the possibility that the embryonic red cells participate in iron storage as they do in other embryonic or larval vertebrates. The ferritin content of mature red cells in the circulation declined when fetal red cells replaced embryonic red cells; the cell replacement was monitored by the disappearance of embryonic ε-chains and the appearance of the fetal globin chains, γ^A and γ^G. A constant ratio of 0.67 was obtained for $\text{γ}{}^{\mathrm{<mtext>G</mtext>}}\text{γ}{}^{\mathrm{<mtext>A</mtext>}}\text{+ γ}{}^{\mathrm{<mtext>G</mtext>}}$ from the first detectable appearance (4 weeks after conception) until 13 weeks, a value which is similar to the value previously obtained at 20 weeks gestation and birth but higher than that observable in adults; thus, both γ^G and γ^A chains are produced in similar amounts throughout gestation. The high levels of ferritin in normal human embryonic erythrocytes emphasize the similarity of erythropoiesis in human embryos and other vertebrates. In addition, the results show that red cell ferritin can be used as a marker for studying the mechanism of induction of embryonic erythropoiesis in cultured cell lines, such as K562 from human chronic myelocytic leukemia, and that ferritin content may also serve as a marker for cellular transformations involving reversions to embryonic erythropoiesis.     

Sedimentation of single human red blood cells. Differences between normal and glutaraldehyde fixed cells

The sedimentation behaviour of single human red blood cells fixed with glutaraldehyde at pH 7.4 and 6.4 was studied and compared to resiflts previously reported for normal fresh cells. The cells fixed at pH 7.4 were observed to have normal shapes while those fixed at pH 6.4 were more spherical and less disc-like. Fixation of glutaraldehyde removed “membrane flicker,” resulting in increased stability as indicated by a decrease in the number of orientation changes per minute from 2.93 ± 0.16 (SEM) to 1.74 ± 0.10 (SEM). An orientation change was defined as a change of 45° in any direction. Fixation also increased the edge-to-flat time preference ratio from 2.5 to 4.7, and increased the sedimentation velocities in all three orientations, despite a measured 2.5% decrease in mean cell density. Fixation of cells at pH 6.4 showed that the decrease in stability and preference for the on-edge orientation was associated with an increase in the sphericity of the cell.     

Preparation techniques for electron probe microanalysis of cells

Abstract

Electron probe X-ray microanalysis can be used to determine the elemental composition of cells and tissues in defined functional states. However, reliable quantitative data can be expected only after appropriate cryopreparation techniques. The most versatile preparation and analysis technique can be outlined by four succeeding steps: 1. Cryofixation, 2. Cryoultramicrotomy, 3. Cryotransfer including freeze-drying, 4. Energy dispersive X-ray microanalysis.     

Ion distribution in roots of barley seedlings measured by electron probe x-ray microanalysis

The distribution of ions, particularly K and Na, was studied in roots of barley seedlings grown on various ionic solutions. Analyses were made by means of electron probe x-ray microanalysis using frozen, fractured bulk specimens. By this technique, it was demonstrated that there can be variability in the ratio K/Na measured in the vacuoles of cortical cells, with this ratio often being lower in epidermal cells of the root than in the inner cortex. A sharp difference in the K/Na ratio was also found between cells of the endodermis and those of the adjacent cortex, and generally higher ratios of K/Na occurred in the stele than in the cortex. Estimation of the concentrations in the cytoplasm was at the limit of resolution of this technique, but it can be shown that the K/Na ratio in the cytoplasm was higher than that in the vacuole. In low salt roots, the K concentration in the cytoplasm was higher than that in the vacuoles. The results with the x-ray microprobe confirm other measurements based on flux analysis or analysis of small samples of the root.     

Potassium influx in human neonatal red blood cells. Partition into its major components.

The potassium influx in human neonatal red blood cells (nRBC) shows an approximately 25% lower value compared to the total potassium influx in adult red blood cells (aRBC). The ouabain-sensitive potassium influx component represents approximately 70-75% of the total potassium influx for both types of cells but with an absolute value significantly lower in nRBC. In nRBC, the half maximum inhibitory effect for ouabain was obtained at a 10(-9) M concentration. The ouabain-insensitive nRBC potassium influx fractions showed two components: (i) a bumetanide-sensitive component, significantly lower than that of aRBC, (ii) a ouabain-bumetanide-insensitive (leak) component with a similar value in both cell types. The sum of the ouabain-sensitive and furosemide-sensitive components amounted in nRBC to a greater value than the total potassium influx. This behaviour could be interpreted as a superposition of the action of the inhibitors on the components affected.     

A note on osmotic pressure measured with ionophore treated red blood cells

A method is described by which the osmotic pressure of macromolecules or many low molecular weight substances can be measured relative to the known osmotic pressure of a reference substance. Measurements can also be made in the presence of univalent electrolytes. The method involves the use of ionophore treated mammalian red blood cells as osmometers. Details are given for the establishment of the isosmotic identity line for dextran M_w = 9 400, M_n = 5 500 and sucrose using nystatin treated human red blood cells. The sucrose concentrations used were from 20 to 33 mOsm (50–80 kPa).     

IgE receptor-mediated depolarization of rat basophilic leukemia cells measured with the fluorescent probe bis-oxonol

Receptor-mediated changes in plasma membrane potential were recorded in rat basophilic leukemia (RBL) cells with the potential-sensitive fluorescent indicator bis-oxonol. Depolarization of the mitochondria with metabolic inhibitors was not detected by bis-oxonol, suggesting that only potential changes across the plasma membrane were being measured. The resting membrane potential of RBL cells was largely generated by the equilibrium distribution of K+ and not through electrogenic activity of the sodium pump. Depolarization was maintained as long as IgE receptors remained aggregated. We believe that at physiologic calcium concentrations a large portion of the measured potential change may be due to calcium influx across the plasma membrane. Prevention of calcium influx by lanthanum, disruption of aggregated receptors, or prior depolarization in a high K+ saline solution completely inhibited the antigen-induced depolarization. The time course of the antigen-stimulated increase in bis-oxonol fluorescence was similar, but not identical, to the antigen-stimulated rise in cytoplasmic free ionized calcium measured with fura-2. Antigen-stimulated depolarization was inhibited by removing both calcium and sodium and could be restored by the addition of either ion. Reduction of total cellular adenosine triphosphate inhibited depolarization in response to antigen stimulation.     

Total sialic acid content of glycophorins during senescence of human red blood cells.

Glycophorins extracted from membranes of young and old human red blood cells have within an error of +/- 1.5% the same sialic acid content when referred to a relative measure of the number of glycophorins. The degree of surface iodination in glycophorins, which was shown to be the same in young and old cells, served as this relative measure. This finding implies that senescent human red blood cells hardly reveal desialylated surface proteins (less than or equal to 3%). However, the sialic acid content per cell was repeatedly reported to be 10 to 15% lower in old than in young cells. Therefore, we conclude 1) that human red blood cells lose intact glycophorin together with membrane during red blood cell senescence, and 2) that removal of desialylated and senescent red blood cells from the circulation proceeds by different routes.     

Amine and carboxylate spin probe permeability in red cells

Summary Permeabilities for a homologous series of amine and carboxylate nitroxide spin probes were measured in human red blood cells by an electron paramagnetic resonance (EPR) method. Permeabilities determined in this study are much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. This suggests that the rate-limiting region for permeation of these nonelectrolytes is somewhere in the membrane periphery rather than in the center of the membrane. The red cell membrane does not discriminate between these probes on the basis of molecular volume, as might be predicted by a simple free-volume theory of membrane permeation.     

Cation and ATP content of ferret red cells

Ferret red cells were shown to have the following properties: 1. They have a high sodium (96 mmol/l cell) and low potassium (3.9 mmol/l cell) content. 2. The majority do not appear to have an active sodium pump in their membranes. 3. Their membranes are highly permeable to rubidium indicating that they are probably also highly permeable to potassium. 4. Their magnesium (3.01 mmol/l cell) and calcium (0.01 mmol/l cell) contents are similar to those of red cells from other species. 5. Their ATP content (0.6 mmol/l cell) is similar to that of cat and dog red cells and is sufficiently high to activate known ion transport systems.