Abnormalities of elastic and transporting properties of red blood cells under development of apoptosis

The functional properties of erythrocytes under development of apoptotic process in these cells were investigated by the low angle light scattering technique. Apoptosis induced by ionomycin was associated with an initial decrease of cell volume and caused formation of echinocytes. After that the cells restored their volume forming rounded erythrocytes with rugged membrane capable to agglomerate with each other. At the late stages of apoptosis, small fragmented cells can be revealed. Preapoptotic red blood cells (at all stages of apoptosis) manifested an enormous tolerance to hypotonic loading, whereas control cells hemolyzed just after reaching a critical volume (∼150 fl). Acidic hemolysis cannot differentiate between control and preapoptotic erythrocytes, the cells being hemolyzed not reaching the critical volume. Placing the control erythrocytes to a medium with ammonia ions instead of sodium ions caused an initial increase of cell volume above the critical point, and then it was also followed by hemolysis. Under ammonia loading, an initial rate of the cell volume growth and a ratio of the hemolyzed cells were significantly reduced in preapoptotic cells.     

Binding of 4'-aminomethyl 4,5',8-trimethyl psoralen to DNA, RNA and protein in HeLa cells and Drosophila cells.

In Drosophila cells and HeLa cells treated with 4'-aminomethyl trioxsalen and ultraviolet light, this compound binds covalently to DNA and RNA. The maximum number of molecules bound to 10(3) base pairs in DNA is 60 and in RNA it is 20. In nuclei treated likewise the number of molecules bound to 10(3) base pairs in DNA can be as high as 376. When cells are irradiated in the frozen state the number of 4'-aminomethyl trioxsalen molecules bound per 10(3) base pairs in DNA is about 40 and in RNA about 20. DNA molecules from cells or nuclei treated with 4'-aminomethyl trioxsalen and ultraviolet light are highly crosslinked and appear as loops interspersed by double stranded regions when analyzed in the electron microscope under denaturing conditions. The loop sizes are heterogeneous and the fraction of double stranded regions increases to almost complete double-strandedness at high degrees of reaction. No secondary structures could be found in ribosomal RNA from Drosophila cells or HeLa cells after treatment with 4'-aminomethyl trioxsalen and ultraviolet light. In cells treated with 4'-aminomethyl trioxsalen and ultraviolet light the RNAase activity is increased considerably suggesting a release of lysosomal enzymes. 4'-aminomethyl trioxsalen and its photodecomposition products bind strongly to cellular proteins.     

Ultrastructure of large granule-containing lymphocytes possessing natural killer activity

By means of light and electron microscopy, ultrastructural cytochemistry and immune cytochemistry methods, contents and ultrastructure of large granule-containing lymphocytes (LGL) have been studied in human blood--this is cell population possessing natural killer and, partly, antibody-depending cytotoxicity. The LGL concentrates are isolated from blood applying successive physical-chemical methods, differential centrifugation in the density gradient of pack-phycoll and percoll included. Separate LGL populations are marked by means of rosette-forming reaction with sheep erythrocytes and monoclonal antibodies OKT4 and OKT8. Relative and absolute amount of the LGL in 1 1 of blood is 5.4 +/- 0.5% and 0.319 +/- 0.28 X 10(9), respectively. The LGL ultrastructure is characterized with a low nuclear-cytoplasmic ratio, with presence of osmiophilic (azurophilic) granules in cytoplasm and specific parallel-tubular structures, with a well developed Golgi complex, an essential number of mitochondria, vesicles with smooth wall and vacuoles, as well as multivesicular bodies and Gallo bodies. The LGL subpopulations, expressing various membrane antigens (E+, E-, OKT8+, OKT8-) differ in their ultrastructure, that is evidently stipulated by the degree of their differentiation and their function.     

Vesicle-mediated transport of membrane and proteins in malaria-infected erythrocytes

Malaria parasites during intraerythrocytic development change the ultrastructure, biophysics, and the antigens of the host red blood cell membrane. Parasite-encoded proteins are associated with, inserted into, or secreted across the infected erythrocyte membrane. Since parasites of the genus Plasmodium are eukaryotic cells, it must be assumed that they possess essentially eukaryotic modes of vesicle-mediated transport and translocation of proteins and membranes. Numerous studies have demonstrated vesicular structures in the cytoplasm of malaria-infected red blood cells and an assortment of parasite proteins associated with the different vesicles, membranes, and membrane-defined compartments. Some parasite polypeptides remain trapped between the parasite and the parasitophorous vacuole membranes PVM, whereas others are associated with morphologically distinct membrane-limited vesicles and vacuoles. Some of these same parasite protein antigens also associate with the erythrocyte membrane or with parasite-induced ultrastructural modifications in the membrane of the parasitized red blood cells. This implies that intracellular transport occurs in malaria-infected erythrocytes, a capacity that uninfected red blood cells normally lose upon enucleation. The specific locations of parasite antigens within the infected cell also implys the existence of targeting signals in the translocated parasite polypeptides and perhaps transport-mediating proteins. The genes corresponding to some of these translocated proteins have been sequenced. Typical (and in some cases atypical) signal peptide sequences occur, as well as a number of sequences that may result in posttranslational modifications. How or if these features figure in to the translocation across, and targeting to a particular membrane compartment of the intraerythrocytic parasite remains unknown.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of photochemotherapy-induced apoptotic cell death in lymphoid cells.

Previous studies we performed showed that 8-methoxypsoralen in combination with ultraviolet A light (photochemotherapy) caused DNA damage and that this caused nucleotide depletion in peripheral blood leukocytes, secondary to an active form of programmed cell death, poly(ADP-ribosyl)ation. Further studies revealed that 24 h after exposure to 10 J/cm2 ultraviolet A light and 8-methoxypsoralen (300 ng/mL), apoptotic cells increased from 3 (control) to 31% (p less than 0.001). Ultraviolet A light alone also significantly increased the number of apoptotic cells. These morphological changes were confirmed by parallel findings on DNA electrophoresis. Treatment with 2 to 5 J/cm2 of ultraviolet A light and 8-methoxypsoralen caused an approximately 30% increase in cytosolic free calcium levels in peripheral blood leukocytes 1 h after exposure. Associated with this was a 51% increase in 45Ca2+ uptake over the first 60 min. Similar findings in a different lymphoid cell (CCRF-CEM) confirmed the results obtained with peripheral blood leukocytes. The use of calcium-free medium prevented a rise in cytosolic free calcium and decreased the number of cells undergoing apoptotic cell death. Cycloheximide inhibited ultraviolet A light - 8-methoxypsoralen induced apoptosis in CCRF-CEM cells; it also decreased calcium levels in control CCRF-CEM cells. This study shows that ultraviolet A light - 8-methoxypsoralen caused apoptotic cell death in lymphoid cells; this appeared to be associated with calcium influx, presumably because of the requirement of endogenous endonucleases for calcium.     

Freeze-fracture electron microscopic observations on the effects of sulphydryl group reagents on human erythrocyte membranes

Freeze-fracture electron microscopy of human red blood cells at pH 7.4 and 5.5 reveals the presence of membrane elevations (50-100 nm diameter). These are also observed after incubation of the erythrocytes with N-ethylmaleimide but not after incubation with p-chloromercuribenzene sulphonate. Neither of the sulphydryl-group reagents affects the distribution or size of intramembrane particles. The findings are discussed in the light of the effects of mercurials on erythrocyte membrane proteins.     

Changes of physiological and biochemical characteristics of rat erythrocytes after blood loss

In experiments of Wistar male rats, changes are studied of erythrocyte hematological, biochemical (activitities of transport ATPases), and rheological properties (capability for aggregation and deformability) 7 days after bloodletting of 12-15 % of the total blood mass. It has been shown that alongside with an elevation of erythrocyte volume and of the number of immature cells - reticulocytes, there was a statistically significant increase of Na,K-ATPase and Ca-ATPase activities in the whole erythrocytes and their membrane preparations - ghosts, the increment of activity in the case of Na,K-ATPase being essentially higher in the whole cells. This indicates the appearance of an enzyme activator inside the erythrocytes. There are also revealed a decrease of firmness of erythrocyte aggregates, a deceleration of spontaneous aggregation, and an increase of index of erythrocyte deformability. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are directed to optimization of blood circulation in large vessels and capillary network.     

Changes of physiological and biochemical characteristics of rat erythrocytes after blood loss

In experiments on Wistar male rats, changes are studied of erythrocyte hematological, biochemical (activities of transport ATPases), and rheological properties (capability for aggregation and deformability) 7 days after bloodletting of 12–15% of the total blood mass. It has been shown that alongside with an elevation of erythrocyte volume and of the number of immature cells—reticulocytes, there was a statistically significant increase of Na,K-ATPase and Ca-ATPase activities in the whole erythrocytes and in their membrane preparations—ghosts, the increment of activity in the case of Na,K-ATPase being essentially higher in the whole cells. This indicates the appearance of an enzyme activator inside the erythrocytes. There are also revealed a decrease of firmness of erythrocyte aggregates, a deceleration of spontaneous aggregation, and an increase of index of erythrocyte deformability. The conclusion is made that changes of erythrocyte rheological properties are interconnected with changes of the Na,K-ATPase activity and are aimed at optimization of blood circulation in large vessels and capillary network.     

Aggregation of human red blood cells after moderate heat treatment

The aggregation behaviour of normal and heat treated (48.4 degrees C, 48.8 degrees C, 49.5 degrees C) red blood cells (RBCs) suspended in dextran-saline solutions (Dx 70, Dx 173) was investigated by a laser light reflectometric method over a wide range of bridging energies. The characteristic times of rouleau formation were found to be increased after RBC heat treatment. The disaggregation shear stress is not significantly different between normal RBCs and heat treated RBCs. The loss of cell deformability is nevertheless shown to improve slightly the dissociation efficiency of the flowing liquid in a shear flow resulting in a small reduction of the disaggregation shear rate after heat treatment. Heat treatment is also shown to alter the structure of RBC network at equilibrium. These results indicate that heat induced alterations of erythrocytes only affects the mechanical properties of the cell membrane without significant changes in the macromolecular bridging energy.     

Mitochondria are involved in apoptosis induced by ultraviolet radiation in lepidopteran Spodoptera litura cell line

Abstract Mitochondria are involved in apoptosis of mammalian cells and even single‐cell organisms, but mitochondria are not required in apoptosis in cultured Drosophila cells such as S2 and BG2 cell lines. It is not very clear whether mitochondria are involved in apoptosis in other insect cells such as lepidopteran cell lines. Thus, we determined to elucidate the role of mitochondria in apoptosis induced by ultraviolet radiation in Spodoptera litura (Lepidoptera: Noctuidae) cell line (SL‐ZSU‐1). The Western blot results suggested that cytochrome c in the ultraviolet‐treated SL‐1 cells was released from the mitochondria to cytosol as early as 4 h after the induction of ultraviolet radiation and increased in the cytosolic fractions in a time‐dependent manner. Flow cytometric analysis of mitochondrial membrane potential (ΔΨm) of SL‐ZSU‐1 cell treated with ultraviolet‐C (UV‐C) light indicated the decrease in mitochondrial membrane potential was dependent on the times of ultraviolet treatment. Both of them are different from apoptosis in cultured Drosophila melanogaster cell lines (S2 and BG2) and it appears evident mitochondria are involved in apoptosis of the studied lepidopteran cells.     

Effect of tributyltin on trout blood cells: changes in mitochondrial morphology and functionality

The aquatic environment is the largest sink for the highly toxic organotin compounds, particularly as one of the main sources is the direct release of organotins from marine antifouling paints. The aim of this study was to investigate the mitochondrial toxicity and proapoptotic activity of tributyltin chloride (TBTC) in teleost leukocytes and nucleated erythrocytes, by means of electron microscopy investigation and mitochondrial membrane potential evaluation, in order to provide an early indicator of aquatic environmental pollution. Erythrocytes and leukocytes were obtained from an inbred strain of rainbow trout (Oncorhynchus mykiss). Transmission electronic micrographs of trout red blood cells (RBC) incubated in the presence of TBTC at 1 and 5 microM for 60 min showed remarkable mitochondrial morphological changes. TBTC-mediated toxicity involved alteration of the cristae ultrastructure and mitochondrial swelling, in a dose-dependent manner. Both erythrocytes and leukocytes displayed a consistent drop in mitochondrial membrane potential following TBTC exposure at concentrations >1 microM. The proapoptotic effect of TBTC on fish blood cells, and involvement of mitochondrial pathways was also investigated by verifying the release of cytochrome c, activation of caspase-3 and the presence of "DNA laddering". Although mitochondrial activity was much more strongly affected in erythrocytes, leukocytes incubated in the presence of TBTC showed the characteristic features of apoptosis after only 1 h of incubation. Longer exposures, up to 12 h, were required to trigger an apoptotic response in erythrocytes.     

Differential effects of lectins mediating erythrocyte attachment and ingestion by macrophages

Lectin-mediated interaction of erythrocytes and macrophages was brought about in two steps. Step I involved macrophage treatment with lectin, and step II is the incubation of lectin-treated macrophages with mouse erythrocytes. The extent and nature of lectin-mediated macrophage erythrocyte interaction was studied using concanavalin A (ConA), wheat germ (WGA), soybean (SBA) and waxbean (WBA) agglutinins. The parameters affecting the interaction were studied in detail with the first two lectins.Under comparable conditions of lectin interaction with macrophages (step I), WGA mediates rosette formation involving interaction with several times the number of erythrocytes than those interacting with ConA-treated macrophages. The interaction mediated by WGA reaches, at 37 °C, a saturation value after 30 min of step II, whereas that mediated by ConA is still linear and exhibits half the amount of attached erythrocytes at 60 min. ConA-mediated attachment of erythrocytes is highly temperature-dependent being at 37 °C twice that observed at 24 °C. The temperature dependence of attachment is not affected by changes of either ConA concentration (5–40 μg/ml) or the temperature in step I. An optimum is observed, however, when the temperature of incubation in step I ranges between 14–18 °C. WGA-mediated attachment of erythrocytes is markedly less temperature-sensitive, exhibiting 70% of optimal attachment already at 8 °C. Only when the attachment phase follows incubation with a low concentration of WGA (2 μg/ml) high temperature sensitivity is exhibited. At 37 °C, however, the number of attached erythrocytes is the same for macrophages treated with WGA at concentrations of 2, 5, 10 and 40 μg/ml.ConA-mediated erythrocyte-macrophage interaction does not lead to erythrophagocytosis. When mediated by WGA, the attachment step is followed by a temperature-dependent ingestion step, i.e. 10% and 50% of the erythrocytes that attach to macrophages during the 60 min incubation at 24 °C and 37 °C, respectively, are ingested. There is a lag period of 10–20 min between attachment and ingestion implicating involvement of additional cellular processes preceding engulfment. Electron microscope images of areas of interaction of attached erythrocytes with macrophages indicate a significantly tighter binding (a thinner gap at membrane-membrane apposition areas) in the case of WGA-mediated rosette formation as compared with that established in ConA-mediated rosettes. Attachment via WGA is followed by a rapid change in the relative position of the attached erythrocytes on the macrophage, from a primary attachment at the distal peripheral regions of the cell, to a perinuclear position. In contrast, erythrocytes attached via ConA remain at the primary attachment point (at 37 °C) for extended periods. This differential behaviour does not stem from effects of ConA on macrophages, since when yeast cells were attached to ConA treated macrophages, the yeast cells showed the same movement as that exhibited by erythrocyte when attached via WGA.The different interaction patterns of erythrocytes with macrophages coated with ConA and WGA can be fitted into the following working hypothesis: the number of WGA-binding sites on the plasma membrane of macrophages is at least three times that of ConA-binding sites. Stable cell-cell interactions involve multibridge formation at the contact area of the two cells and this involves a delicate balance between number of lectin-receptor conjugates and their aggregation state within the membrane phase. A certain amount of clustering is a prerequisite for attachment, while a high degree of clustering reduces the chance of fruitful interactions. The engulfment step depends on the ability of membrane areas adjacent to primary contact area to establish additional stable bridges in the entire circumference of the attached cell. ConA-receptor conjugates appear to be less abundant and more aggregated within the membrane plane, preventing the completion of fruitful circumferential interaction of the adjacent membrane. WGA-receptor conjugates, being more abundant and apparently less aggregated are available at membrane areas needed for cell enclosure and provide the additional bridging without which engulfment does not take place. Change in relative position of attached erythrocytes seems to be a step in the manifold events occurring from attachment to ingestion.     

Principles of changes of structural organization of cell membranes and functional properties of erythrocytes in neurotic disorders

Investigation into structural, metabolic, and functional conditions of red blood cells was performed in 24 patients with a neurosis (neurasthenia, disturbance of asaptation) with the aid of electrophoretic division of proteins of the erythrocyte membrane, thin-layer chromatography, fluorescent probing of membranes, evaluation of peroxidative oxidation process, scanning and transmission electron microscopy, laser diphractometry, photometry. The patients with neurotic disorders at the early period after the influence of psychogenic factors (up to 3 months) revealed disorganization of lipid and protein composition of the red cell membrane, increase in microviscosity of its lipid phase, impairment of surface architectonics and ultrastructure of red cells, decrease of a deformation ability and increase of aggregate properties of erythrocytes. The authors treat stability of erythrocytes' homeostasis under the long-term influence of psychogenic factors from a viewpoint of adaptive changes in organism under the influence of neurogenic factors.     

Studies on the osmotic resistance and the viability of amidinated erythrocytes]

The present studies are concerned with properties of amidinated erythrocytes. The reactions of dimethyladipimidate with proteins in solution and red blood cells, respectively, result in an intermolecular cross-linking. Following an amidination of human serum albumin or human gamma-globulin cross-linked products of increased molecular weight have been demonstrated by polyacrylamide gel and immune electrophoresis. Human erythrocytes previously amidinated intensely, exhibit a restricted motility of membrane particles and cross-linked hemoglobin. Intensely amidinated erythrocytes are resistant against distilled water, and they do no longer agglutinate. The findings presumably indicate an increased permeability of the amidinated red cell membrane. The glycolytic activity was found to be normal in moderately amidinated erythrocytes. In comparison with normal red blood cells, previously moderately amidinated erythrocytes of the rat become sequestered more quickly after re-injection into the vascular system.     

Rosette formation of concanavalin A-treated erythrocytes around polymorphonuclear leucocytes and lymphocytes.

Concanavalin A (Con A) induces rosette formation of erythrocytes around polymorphonuclear leucocytes and lymphocytes in cell suspensions of autologous human blood cells. The effect which is most characteristic in a concentration between 25 and 50 microgram/ml is due to Con A bound on the erythrocyte membrane. A similar effect, although less pronounced, was observed with phytohaemagglutinin at concentrations of 10 and 25 microgram/ml. The treated erythrocytes showed a higher affinity to polymorphonuclears when compared with lymphocytes. At the contact area, the membrane of the erythrocyte became highly folded while its free surface was smooth and spherical. The effect of the local concentration and immunobilization of the lectin on the erythrocyte membrane and the similarity of the contact pattern to that of erythrophagocytosis are discussed.     

Reactive effect of low intensity He-Ne laser upon damaged ultrastructure of human erythrocyte membrane in Fenton system by atomic force microscopy

To find out the mechanism of modulating the deformability of erythrocytes with low intensity He-Ne laser action, we studied the effect of low intensity He-Ne laser on the ultrastructure of human erythrocyte membrane. Erythrocytes were treated with free radicals from a Fenton reaction system before exposing them to low intensity He-Ne laser. The ultrastructure of damaged erythrocyte membrane was examined by atomic force microscopy. The results showed that the erythrocyte membrane became very rough and the molecules on the surface of the membrane congregated into particles of different magnitudes sizes after treating with free radicals. Comparing the degree of congregation of the molecular particles in the non-irradiated group and the He-Ne laser irradiated （9 mW and 18 mW） group, we found the average size of molecular particles in the laser irradiated group was smaller than that in the non-irradiated group, indicating that the low intensity laser had repairing function to the damage of erythrocyte membrane produced by the free radicals.     

The fate of female donor blastodermal cells in male chimeric chickens.

In previous experiments in our laboratories, chickens that are chimeric in their gamete, melanocyte, and blood cell populations have been produced by injection of dispersed stage X blastodermal donor cells into the subgerminal cavity of stage X recipient embryos. In some experiments, donor cells were transfected with reporter gene constructs prior to injection as a preliminary step in the production of transgenic birds. Chimerism was assessed by test mating, observation of plumage, and DNA fingerprinting. Methods were sought that would provide a relatively rapid analysis of the spatial distribution of descendants of donor cells in chimeras to assess the efficacy of various methods of chimera construction. To date, the sex of donor and recipient embryos was not known and, therefore, numerous mixed sex chimeras must have been constructed by chance, since donor cells were usually collected from several embryos rather than from individual embryos. The presence of female-derived cells was determined by in situ hybridization using a W-chromosome-specific DNA probe, using smears of washed erythrocytes from 16 phenotypically male chimeric chickens ranging in age from 4 days to 42 months posthatching. The proportion of female cells detected in the erythrocyte samples was zero (eight samples) or very low (0.020-0.083%), although 1% of the erythrocytes from a phenotypically male chick that was killed 4 days after hatch were female-derived. The low proportions of female-derived cells were surprising, considering that most of these chimeras had been produced by the injection of cells pooled from several donor embryos and most recipients had been exposed to gamma irradiation prior to injection, thus dramatically enhancing the level of incorporation of donor cells into the resulting chimeras. By contrast, 0-100% of the erythrocytes were female-derived in blood samples taken at 10 days of incubation from the chorioallantois of seven phenotypically normal male embryos that resulted from the injection of blastodermal cells pooled from five embryos into irradiated recipient embryos. Approximately 70% of the erythrocytes in a blood sample from a phenotypically normal female chimeric embryo were female-derived, and 100% of the erythrocytes examined from an intersex embryo bearing a right testis and a left ovary were female-derived. These results indicate that female-derived cells can contribute to the formation of erythropoietic tissue during the early development of what will become a phenotypically male chimeric embryo. It would appear, therefore, that female-derived cells are blocked in development or destroyed, or certain male-female combinations of cells may be lethal prior to hatching.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transformation of erythrocytes and their ultrastructure with respect to aging

Transformation and ultrastructure of erythrocytes have been investigated in 58 white non-inbred rats on the 1st, 2nd, 3d days after birth and at the age of 1.5, 3, 8, 20 months. The transformation index of the erythrocytes drops by the 8th month of life and further it rises in old animals. In newborn rats echino-poikiloid forms predominate; they are mainly presented as reticulocytes with remnants of nuclei, mitochondria and other organells. During first three days of life cells of the erythroid line actively free themselves from the nucleus by means of its pyknosis and chromatinolysis. In newborn rats erythrocytes have folds of plasmolemma of linear and spotted form on their surface; their number sharply decreases in mature animals. The least changes in form and ultrastructure of erythrocytes are noticed in 3-8-month-old animals.     

Interaction of photosynthetic pigments with various organic solvents. Magnetic circular dichroism approach and application to chlorosomes

Reduction of extracellular ferricyanide by intact cells reflects the activity of an as yet unidentified trans-plasma membrane oxidoreductase. In human erythrocytes, this activity was found to be limited by the ability of the cells to recycle intracellular ascorbic acid, its primary trans-membrane electron donor. Ascorbate-dependent ferricyanide reduction by erythrocytes was partially inhibited by reaction of one or more cell-surface sulfhydryls with p-chloromercuribenzene sulfonic acid, an effect that persisted in resealed ghosts prepared from such treated cells. However, treatment of intact cells with the sulfhydryl reagent had no effect on NADH-dependent ferricyanide or ferricytochrome c reductase activities of open ghosts prepared from treated cells. When cytosol-free ghosts were resealed to contain trypsin or pronase, ascorbate-dependent reduction of extravesicular ferricyanide was doubled, whereas NADH-dependent ferricyanide and ferricytochrome c reduction were decreased by proteolytic digestion. The trans-membrane ascorbate-dependent activity was also found to be inhibited by reaction of sulfhydryls on its cytoplasmic face. These results show that the trans-membrane ferricyanide oxidoreductase is limited by the ability of erythrocytes to recycle intracellular ascorbate, that it does not involve the endofacial NADH-dependent cytochrome b(5) reductase system, and that it is a trans-membrane protein that contains sensitive sulfhydryl groups on both membrane faces.     

Phloretinyl-3′-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes: II. Irreversible transport inhibition is induced by photolysis

In the dark, phloretinyl-3′-benzylazide (PBAz), at a nominal concentration of 10 μM, will inhibit the transport of d-glucose in human erythrocytes by more than 90%. This inhibition can be completely reversed by percolating the cell suspension through a small column of Sephadex G-10; cells recovered after this treatment, and then loaded with 100 mM d-glucose, possess a transport capacity (glucose efflux) equal to untreated cells. The Sephadex matrix completely removes non-covalently bound inhibitor even though, under these conditions (subdued light, 0.2% hematocrit, 0°C, pH 6.2 or 7.8), from 70 to 80% of the PBAz added is bound to the cells (mostly non-specifically to hemoglobin). However, when erythrocytes exposed to 10 μM inhibitor are irradiated with long wavelength ultraviolet light, the glucose transporter is irreversibly inhibited; after 1 min irradiation, about 50% of transporter activity cannot be restored by Sephadex treatment. Under identical conditions, control cells (no PBAz,but irradiated and treated with Sephadex) retain over 90% of carrier activity. The photolytic conversion of the inhibition to an irreversible form is directly dependent on PBAz concentration. The results reaffirm our earlier conclusions that PBAz is a potentially useful photoaffinity labeling agent for the glucose transporter in erythrocyte membranes.