Impermeant maleimides. Oriented probes of erythrocyte membrane proteins

Maleimides impermeant to human erythrocyte membranes have been synthesized and applied to studies of the sulfhydryl groups of the membrane. Reaction of radioactive dextran-maleimide and glutathione-maleimide with either intact erythrocytes or ghosts yields sulfhydryl titers for the outer (exofacial) and inner (endofacial) surfaces, respectively, of 1.5 to 1.7 and 27 to 28 amol/cell. Corresponding values for sulfhydryl groups within the membrane interior, as estimated with radioactive N-ethylmaleimide, are 16 to 22 amol/cell. After exofacial labeling of intact cells with [35S]glutathione-maleimide, autoradiography of sodium dodecyl sulfate-polyacrylamide gels demonstrates four bands (alpha, beta, gamma, and delta) containing, respectively, 13%, 63%, 11%, and 13% of the radioactivity. The major beta-band corresponds in position to polypeptides of molecular weight 40,000 to 70,000 and to Coomassie brilliant blue-stained Band 5. Selective extraction demonstrates that the major Band 5 protein is not identical with the labeled beta-band polypeptides. Following endofacial labeling of ghosts with [35S]glutathione-maleimide, autoradiography reveals radioactivity in all of the major Coomassie brilliant blue bands. The impermeant maleimides described are also applicable to studies of discrete functional proteins of the erythrocyte membrane, including the hexose transport mechanism and the major Rho antigenic site.     

Fractionation of human erythrocyte membranes. Presence of the nucleoside transport complex in an insoluble residue

(1) Human erythrocyte membranes, when dialysed against water at pH 9.5, were partly solubilized, losing 80% of the membrane proteins and 65% of the membrane lipids. Sodium dodecyl sulphate gel electrophoresis of the particulate material revealed selective removal of proteins from the membrane. (2) The lipid-rich particulate material remaining retained the ability to bind specifically the nucleoside transport inhibitor, nitrobenzylthioinosine, previously shown to bind selectively to the nucleoside transport mechanism of whole erythrocytes and erythrocyte ghosts.     

Manganese transport through human erythrocyte membranes. An EPR study

Manganese uptake by human erythrocytes was investigated in the concentration range 0.5-20 mM in the suspending solution, by using the EPR technique. S shaped dependencies of manganese influx on manganese doping solution concentration for both fresh and vanadate treated erythrocytes were found, with maximum influx values of 4.1 +/- 1.9 x 10(-10) mol/m2 x s and 2.1 +/- 0.3 x 10(-9) mol/m2 x s, respectively. At low manganese concentrations (< 2 mM) the manganese permeability coefficient increases with increasing the doping concentration, the ions cooperate for achieving a transport event. For high manganese concentration (> 5 mM) the permeability coefficient decreases with increasing the doping concentration, the ions competing for the limited amount of transport system. A similar increase in manganese uptake as in vanadate treated erythrocytes was measured for 'in vitro' aged erythrocytes. These results might suggest that human erythrocytes possess an active transport mechanism by which, they oppose to manganese influx. This hypothesis is also supported by the 10-15 min time lag between the moment of doping and the start of the manganese influx into the fresh erythrocytes. The manganese uptake inhibition by nifedipine, a calcium channel blocker, for the case of vanadate treated erythrocytes, suggests that, at least partially, manganese uptake by the cells occurs via the 'calcium channels'.     

Identification of Streptococcus mutans antigen D as the HPr component of the sugar-phosphotransferase transport system

Fosfomycin tromethamine is an orally administered fosfomycin that may be used for single-dose therapy of uncomplicated urinary tract infections. At breakpoint concentrations [< or = 128 micrograms/ml plus 25 micrograms/ml glucose-6-phosphate (G-6-P)], fosfomycin tromethamine inhibited > 90% of the 350 bacterial isolates tested. When testing Escherichia coli, Klebsiella spp., and Enterobacter spp., we note that the performance of fosfomycin disks improved when G-6-P was added to the disks. The interpretive error rates were minimized when 200-micrograms fosfomycin disks were supplemented with either 50 or 100 micrograms G-6-P. Using < or = 128 and > or = 256 micrograms/ml as the susceptible and resistant MIC breakpoints, respectively, the regression-analysis-derived disk diffusion zone diameter breakpoints for the 200-micrograms fosfomycin disk supplemented with 50 micrograms of G-6-P are as follows: susceptible, > or = 16 mm; intermediate, 13-15 mm; and resistant, < or = 12 mm.     

Identification of a sialoglycopeptide released by self-digestion from human erythrocyte membranes

Membranes from human O Rhesus-positive erythrocyte 'ghosts' were tested in vitro for their ability to digest their own glycoproteins. 'Ghost' membranes incubated in Tris/HCl buffer, pH 7.4, release a sialoglycopeptide, which contains glucosamine, galactosamine, galactose and mainly polar amino acids. Chemical composition, molecular size and aggregation properties suggest that this glycopeptide may be a fragment of glycophorin.     

Structural and physiologic determinants of human erythrocyte sugar transport regulation by adenosine triphosphate

Human erythrocyte sugar transport is mediated by the integral membrane protein GLUT1 and is regulated by cytosolic ATP [Carruthers, A., and Helgerson, A. L. (1989) Biochemistry 28, 8337-8346]. This study asks the following questions. (1) Where is the GLUT1 ATP binding site? (2) Is ATP-GLUT1 interaction sufficient for sugar transport regulation? (3) Is ATP modulation of transport subject to metabolic control? GLUT1 residues 301-364 were identified as one element of the GLUT1 ATP binding domain by peptide mapping and N-terminal sequence analysis of proteolytic fragments of azidoATP-photolabeled GLUT1. Nucleotide binding and sugar transport experiments undertaken with dimeric and tetrameric forms of GLUT1 indicate that only tetrameric GLUT1 binds and is subject to modulation by ATP. Reconstitution experiments indicate that nucleotide and tetrameric GLUT1 are sufficient for ATP modulation of sugar transport. Feedback control of GLUT1 regulation by ATP was investigated by measuring sugar uptake into erythrocyte ghosts containing or lacking ATP and glycolytic intermediates. Only AMP and ADP modulate ATP regulation of transport. Reduced cytosolic pH inhibits ATP modulation of GLUT1-mediated 3OMG uptake and increases Kd(app) for ATP interaction with GLUT1. We conclude that tetrameric but not dimeric GLUT1 is subject to direct regulation by cytosolic ATP and that this regulation is antagonized by intracellular AMP and acidification.     

The effect of gamma radiation on Li+ transport through human erythrocyte membranes

Human erythrocytes were exposed at room temperatures to 662 keV gamma radiation at doses up to 60 Gy. The total delta Li+ influx as well as the delta Li+ influx by passive diffusion and Na(+)-K+ pump mechanisms increased linearly with the dose. The rate constant K of Li+ efflux by the Li(+)-Na+ countertransport mechanism through irradiated erythrocyte membranes was reduced by about 35% at the highest dose.     

Complementation studies with Co-expressed fragments of the human red cell anion transporter (Band 3; AE1). The role of some exofacial loops in anion transport

We constructed cDNA clones encoding fragments of band 3 in which the membrane domain was truncated from either the N or the C terminus within each of the first four exofacial loops. The truncations containing the C terminus of the protein were fused with the cleavable N-terminal signal sequence of glycophorin A to facilitate the correct orientation of the most N-terminal band 3 membrane span. Cleavage of the glycophorin A signal sequence was observed, except when the truncation was in the first exofacial loop where the signal peptidase cleavage site was probably too close to the membrane. The anion transport activity of co-expressed complementary pairs of truncations which together contained the entire band 3 membrane domain was examined. The pairs of fragments divided in the third and fourth exofacial loops yielded transport activity, but the pair separated within the second exofacial loop was not active. We conclude that the integrity of the second exofacial loop, but not the third and fourth exofacial loops, is necessary for transport activity. The unusually stable association between the fragments divided in the second exofacial loop suggests that interactions may occur between polar surfaces on amphiphilic portions of the third and fifth transmembrane spans.     

Identification and characterization of PtlC, an essential component of the pertussis toxin secretion system

PtlC is a member of a set of proteins necessary for the secretion of pertussis toxin (PT) from Bordetella pertussis. Using polyclonal antibodies specific for PtlC, we identified PtlC as a protein with an apparent molecular weight of 85,000 that localizes to the membrane fraction of bacterial cell extracts. We found that a mutant strain of B. pertussis that contains an in-frame deletion in ptlC is unable to secrete PT and that PT secretion is fully restored by expressing ptlC in trans, indicating that PtlC is essential for transport of PT across the bacterial membrane(s). PT secretion was inhibited in wild-type B. pertussis after introduction of a plasmid expressing a mutant ptlC altered in the putative nucleotide-binding region, suggesting that this region of PtlC is essential for proper function. Moreover, the observed dominant negative phenotype suggests that PtlC either functions as a multimer or interacts with some other component(s) necessary for secretion of PT.     

Identification of two leucine-sensitive lysine transport activities in human placental basal membrane

The recent demonstration of multiple high-affinity leucine-sensitive cationic transport systems prompted this investigation of their role in lysine uptake in basal cell membrane. Transport of lysine by basal membrane was saturable at both 22 and 37 degrees C and linear in time to 1 min and 30 sec, respectively. At 22 degrees C, at least two systems were active. The portion of uptake inhibited by the sulphydryl binding reagent N-ethylmaleimide (NEM) but not by leucine in the absence of sodium had a high K(m) and high Vmax and was attributed to system y+. NEM-insensitive uptake was fitted by a one-system model with K(m) (+/- s.e.) of 4 +/- 1 microM and a Vmax of 0.9 +/- 0.1 pmol/mg protein/min. This component was completely inhibited by leucine in the absence of sodium but not by glutamine in the presence of sodium. Therefore, it was attributed to system bo,+. At 37 degrees C, at least three systems were active. For essentially the same reasons as above the NEM inhibitable uptake was attributed to system y+. NEM-insensitive uptake was fitted by a one-system model with K(m) of 26 +/- 7 microM and Vmax of 11.1 +/- 2.8 pmol/mg protein/30 sec. Inhibition studies, however, indicated its heterogeneity. NEM-insensitive saturable uptake was only partially inhibited by either leucine in the absence of sodium (system bo,+) or by glutamine in the presence of sodium (system y+L). It is concluded that the NEM-insensitive portion of lysine uptake at 37 degrees C represents activity of both system bo,+ and the temperature-sensitive system y+L. As a previous investigation indicates, only one of these (system y+L) is present in the more specialized microvillous membrane. The demonstration of functional differences in the high affinity leucine transporters of basal and microvillous membrane in this and our previous investigations suggest that the two membranes possess different transport or modifier proteins.     

Functional significance of the early component of the human blink reflex.

The relationship between the size of the first electromyographic (EMG) component of the cutaneous blink reflex (Rl) and onset of eyelid closure in human adults was determined in 4 experiments in which R1 size was varied by different means: change in stimulus intensity, paired stimulation, and warning. Two-phase lid movements were frequently seen, with an early small movement followed by a large rapid movement. All experiments showed that larger R1s were associated with shorter latencies of both movements. This covariation was general across participants and was independent of shifts in the excitability of the blink reflex pathways indexed by R1 latency, R2 latency, and R2 area (R2 is the more prolonged, later EMG component). The results indicate that R1 acts first to evoke an early lid movement and second to facilitate eyelid closure by the later R2 burst. Identification of this second behavioral function for R1 aids the interpretation of other findings and encourages its use as a model system. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Autopsy. A crucial component of human clinical investigation

OBJECTIVE: To determine the proper role of human clinical investigation, including autopsy, in public health research policy. DATA SOURCES: Medical reports and reviews, and literature concerning the philosophy of medicine. CONCLUSIONS: Autopsy has always been a cornerstone of medical research. Although many pathologists appreciate the research value of autopsies, many other physicians do not. Declining federal support for human clinical research in general, and autopsy in particular, party reflects the popular view that laboratory experimentation-especially that performed on nonhuman animals-can reliably model human conditions. Human clinical research is often difficult, expensive, and time-consuming, but there seems to be no adequate substitute for studies of naturally occurring human conditions. Such studies address many critical medical questions. Autopsy remains an invaluable complement to evolving molecular biology techniques.     

Changes in the signalling status of the small GTP-binding proteins Rac and Rho do not influence insulin-stimulated hexose transport

Post-receptor signalling molecules that convey the signal from the activated insulin receptor to the actual process of Glut4 translocation and hexose uptake are poorly understood. Various studies have suggested a requirement of the lipid kinase phosphatidylinositol-3 kinase (PI3-kinase) in this process. PI3kinase regulates the activation status of the small GTP-binding protein Rac which, in turn, is able to activate another G-protein Rho. Rac and Rho are known to regulate the structure of the membrane- and cytoplasmic actin-cytoskeleton. We have examined whether Rac and Rho transfer the signals generated by PI3kinase towards insulin-stimulated hexose uptake. For that purpose, we expressed in 3T3-L1 adipocytes the dominant-negative mutant of RacN17 using vaccinia virus-mediated gene transfer. The expression levels of the RacN17 protein were monitored by Western blotting. The abrogation of endogenous Rac signalling by expression of RacN17 was inferred from the observed loss of arachidonic acid release in response to insulin. Basal and insulin-stimulated hexose transport were not affected by expression of the RacN17 mutant. A possible contribution of Rho.GTP to stimulation of hexose uptake was examined by pre-incubation of adipocytes with lysophosphatidic acid (LPA). We observed a profound effect of LPA on the structure of the cytoskeleton and on the phosphorylation of Focal Adhesion Kinase (p125FAK), indicating that 3T3-L1 adipocytes respond to LPA and that Rho was activated by LPA. However, no effect was detected on the basal or on the insulin-stimulated hexose transport. We conclude that Rac and Rho are unlikely to be involved in insulin-stimulated hexose transport, suggesting a possible contribution of other signalling pathways, downstream of PI3kinase to this process.     

Lysosomal component in the mechanism of the toxic effect of sporofusarin

Mutant spores of Dictyostelium discoideum, strain SG-10, differ from wild type spores in their ability to spontaneously germinate, to be activated with 5% Dimethyl Sulfoxide (DMSO), and to be deactivated with 0.2 M sucrose. Both heat-activated wild type and mutant spores began to swell after a lag of 60-75 min at ambient temperature. Suspension of heat activated spores in 5% DMSO resulted in blockage of spore swelling and a concomitant severe inhibition of respiration; removal of 5% DMSO allowed resumption of respiration and the spores began to swell after a lag of only 15 min. It was concluded that 5% DMSO allowed the early reactions (M) to proceed but blocked the later reactions (R) of post-activation lag. Treatment of one day old spores with 20% DMSO solution for 30-120 min quantitatively activated the population. The post-activation lag time was directly dependent on the time of 20% DMSO treatment. Spores activated with 20% DMSO treatment could be deactivated by incubation at 0 degrees C; the spores most quickly deactivated at 0 degrees C were those within 10 min of swelling. Mitochondrial transport inhibitors such as azide and cyanide caused deactivation in an analogous manner. It is hypothesized that spores proceed to the second portion of the lag phase called (R) before the environment determines if dormancy is reimposed or if germination will proceed. The sensitive strain (SG-10) showed a greater degree of "damage" than the wild type after supraoptimal treatment with 40% DMSO. The spores became more resistant with age to the "damaging" action of 40% DMSO. All the observed effects of DMSO treatment were compatible with our multistate model of activation which suggests that the early portion of the lag phase (M) may involve a relative uncoupling of oxidative phosphorylation while the later portion (R) may require tight coupling.     

Self-digestion of human erythrocyte membranes. Role of adenosine triphosphate and glutathione

Intact human erythrocytes incubated at 37 degrees C, pH7.4, release a sialoglycopeptide similar in its chemical composition, immunological and aggregation properties to the glycopeptide released by isolated 'ghost' membranes. The presence of ATP or reduced glutathione at physiological concentrations in the incubation medium of 'ghost' membranes inhibits this self-digestion process.     

Receptor distribution and the mechanism of enhanced erythrocyte agglutination by soybean agglutinin

We have examined the role of receptor clustering in intact erythrocyte membranes exhibiting enhanced lectin-mediated cell agglutination by analyzing freeze-fracture and freeze-etch images of human erythrocytes labeled with ferritin-conjugated soybean agglutinin. We find that trypsinization and fixation of intact erythrocytes, in either order, causes no alteration of the random distribution of ferritin-conjugated soybean agglutinin on the surfaces of these cells as compared to their distribution on the surfaces of fixed erythrocytes and untreated erythrocyte ghosts. Furthermore, clustering of the intramembranous particles in the membrane of intact erythrocytes was not found with any of the cells described above. We conclude that clustering of the soybean agglutinin receptors is not a major factor involved in the enhanced agglutination of intact trypsinized erythrocytes. Caution is necessary in transferring information obtained with erythrocyte ghosts, where clustering can be induced, to intact erythrocytes.