Isolation of human erythrocyte membranes in glucose solution

A method is described for the preparation or removal of erythrocyte membranes from hemolysates by a glucose solution. The procedure is simple and rapid, requiring centrifugation at 8000g for 2 min. The preparation has microscopic shape and two-dimensional peptide patterns similar to those of the membrane isolated by conventional procedures (10,000g for 20 min). The present procedure is suitable for dealing with a bulky preparation or for removal of erythrocyte membranes from large volumes of hemolysates to purify enzymes and proteins of soluble or membrane fractions.     

Topography and functions of sulfhydryl groups of the human erythrocyte glucose transport mechanism

Summary Membrane-impermeant and -permeant maleimides were applied to characterize the location and function of the sulfhydryl (SH) groups essential for the facilitated diffusion mediated by the human erythrocyte glucose transport protein. Three such classes have been identified. Type I SH is accessible to membrane-impermeant reagents at the outer (exofacial) surface of the intact erythrocyte. Alkylation of this class inhibits glucose transport; D-glucose and cytochalasin B protect against the alkylation. Type II SH is located at the inner (endofacial) surface of the membrane and is accessible to the membrane-impermeant reagent glutathione maleimide only after lysis of the erythrocyte. D-glucose enhances, while cytochalasin B reduces, the alkylation of Type II SH by maleimides. Reaction of Types I and II SH with an impermeant maleimide increases the half-saturation concentration for binding of D-glucose to erythrocyte membranes. By contrast, inactivation of Type III SH markedly decreases the half-saturation concentration for the binding of D-glucose and other transported sugars. Type III SH is inactivated by the relatively lipid-soluble reagents N-ethylmaleimide (NEM) and dipyridyl disulfide, but not by the impermeant glutathione maleimide. Type III SH is thus located in a hydrophobic membrane domain. A kinetic model constructed to explain these observations indicates that Type III SH is required for the translocation event in a hydrophobic membrane domain which leads to the dissociation of glucose bound to transport sites at the membrane surfaces.     

Characterization of the glucose transporter from human erythrocytes

The D-glucose transporter from human erythrocytes has been purified and reconstituted by Kasahara and Hinkle (J Biol Chem 252:7394–7390). Using a similar purification scheme, we have isolated the protein with 65% of the extracted phospholipid at a lipid-protein ratio of 14:1 by weight. The K_D (0.14 μM) and extent (11 nmoles/mg protein) for binding of ³H-cytochalasin B was determined by equilibrium dialysis. Glucose was a linear competitive inhibitor of binding of cytochalasin B, with an inhibition constant of 30 mM. To further characterize the protein, samples were filtered in the presence of sodium dodecyl sulfate (SDS) through Sepharose 6B to remove 95% of the lipid followed by filtration of Sephadex G150 to remove the remaining lipid and a contaminating amount of a minor, lower-molecular-weight protein. This preparation contains only 24% acidic and basic amino acids. The protein also contains 5% neutral sugars (of which 3% is galactose), 7% glucosamine, and 5% sialic acid.     

Identification of the facilitative glucose transporter 12 gene Glut12 in mouse preimplantation embryos

In this study we report the cloning and characterisation of the mouse Glut12 gene and examine for the first time its expression pattern in the earliest stages of development. Mouse Glut12 (mGlut12) was cloned from preimplantation embryos by 5'RACE RT-PCR using primers designed from an EST clone corresponding to a human GLUT12 antigenic sequence after positive immunoreactivity was observed in mouse two-cell embryos by western immunoblotting. The mGlut12 gene contains an open reading frame of 1869 base pairs, potentially encoding a polypeptide of 622 amino acids. The predicted mGLUT12 protein bears all the hallmarks of the SLC2A family of hexose transporters and shares an 83% sequence homology to human GLUT12. Consistent with its human homolog mGlut12 mRNA is found highly expressed in skeletal and cardiac muscle and fat. Additionally, it was also found in the uterus and during early embryogenesis. During early development in the mouse, Glut12 expression is clearly apparent in ovulated oocytes and two-cell embryos but declines in day 3 morulae. With the exception of some Glut12 expression apparent in blastocysts, Glut12 mRNA remains at low to undetectable levels until E11.     

Expression of the myc/His-tagged human peptide transporter hPEPT1 in yeast for protein purification and functional analysis

The human intestinal peptide transporter hPEPT1 has been expressed in the yeast Pichia pastoris using the promoter of the glyceraldehyde-3-phosphate-dehydrogenase gene. A myc-epitope fused to a polyhistidine-tag was introduced at the C-terminus of hPEPT1 for ease of detection and purification. Yeast cells transformed with tagged hPEPT1 exhibited 30-fold increased dipeptide uptake compared to control cells with a substrate specificity and pH dependence similar to the native transporter. The tagged hPEPT1 protein was detected in crude membrane fractions of Pichia cells with an apparent molecular mass of 66 kDa and an expression level of approximately 64 pmol/mg membrane protein. These studies demonstrate that tagged hPEPT1 can be expressed functionally in P. pastoris with unaltered phenotypical characteristics allowing the yeast cells to be used for functional analysis such as screening for compounds utilizing the peptide transporter for absorption in the human intestine. Moreover, recombinant hPEPT1 can now easily be detected for further purification purposes using immobilized metal-affinity chromatography.     

Physiological significance and bioenergetic aspects of glucose dehydrogenase

The regulation of the PQQ-linked glucose dehydrogenase in different organisms is reviewed. It is concluded that this enzyme functions as an auxiliary energy-generating mechanism, because it is maximally synthesized under conditions of energy stress. It is now definitively established that the oxidation of glucose to gluconate generates metabolically useful energy. The magnitude of the contribution of the oxidation of glucose to gluconate via this enzyme to the growth yield of organisms such asAcinetobacter calcoaceticus is not yet clear.     

Primary structure of the majorO-glycosidically linked carbohydrate unit of human von Willebrand factor

A reduced tetrasaccharide chain was obtained from human von Willebrand factor (vWF) by mild alkaline borohydride treatment. The purification of thisO-glycosidically-linked oligosaccharide was achieved by serial affinity chromatography on immobilized concanavalin A andLens culinaris agglutinin and finally gel filtration. Its structure was determined by a combination of methylation studies and 500 MHz¹H-NMR spectroscopy to be: NeuAc(2-3)Gal(1-3)[NeuAc(2-6)]GalNAc-ol.Abbreviations ConA concanavalin A - LCA Lens culinaris agglutinin - vWF von Willebrand factor - NeuAc N-acetylneuraminic acid - Gal d-galactose - GalNAc-ol N-acetyl-d-galactosaminitol - HMW high molecular weight - LMW low molecular weight     

Fluorescence studies on erythrocyte membrane isolated fromPlasmodium berghei infected mice

The erythrocyte host cell plays a key role in the well defined developmental stages of the malarial parasite growth and propogation in the erythrocyte cycle of malaria. The host cell serves the parasites by supplying metabolites and removing the catabolites produced by the obligatory parasites. It has been observed that the plasma membrane of the infected cells show a substantially higher fluidity probably due to the depletion of cholesterol content from the host cell. The protein component of the membrane is also modulated due to the insertion of new polypeptides of the parasitic origin, which confers upon it new antigenic properties. We have studied the membrane fraction isolated from mice erythrocytes infected withPlasmodium berghei using fluorescent probes like DPH, ANS and series of fluorenyl fatty acids, which permit depth dependent analysis of membrane. We have observed that there is a marked difference in the fluorescence emission wavelength maximum, the dissociation constant K_d of ANS when bound to normal and infected erythrocytes, though relatively small differences are observed in the fluorescence polarisation values of the two cell types. The fluorenyl fatty acids also show the differences when bound to normal and infected erythrocytes, indicating that either they are in a different environment or they have differing binding properties to the two cell types.Abbreviations DPH 1,6-Diphenyl-1,3,5-Hexatriene - ANS 8-Anilino-napthalene Sulfonic Acid - C2A-FL 2-Fluorenyl-acetic Acid - C4A-FL 2-Fluorenyl-butyric Acid - C6A-FL 2-Fluorenyl-hexanoic Acid - C8A-FL 2-Fluorenyl-octanoic Acid     

Dynamics of glucose uptake by single Escherichia coli cells

The fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was used to measure rates of glucose uptake by single Escherichia coli cells. When cell populations were exposed to the glucose analog, 2-NBDG was actively transported and accumulated in single cells to a steady-state level that depended upon its extracellular concentration, the glucose transport capacity of the cells, and the intracellular degradation rate. The dependence upon substrate concentration could be described according to Michaelis-Menten kinetics with apparent saturation constant KM = 1.75 microM, and maximum 2-NBDG uptake rate= 197 molecules/cell-second. Specificity of glucose transporters to the analog was confirmed by inhibition of uptake of 2-NBDG by D-glucose, 3-o-methyl glucose, and D-glucosamine, and lack of inhibition by L-glucose. Inhibition of 2-NBDG uptake by D-glucose was competitive in nature. The assay for 2-NBDG uptake is extremely sensitive such that the presence of even trace amounts of D-glucose in the culture medium (approximately 0.2 microM) is detectable. The rates of single-cell analog uptake were found to increase proportionally with cell size as measured by microscopy or single-cell light scattering intensity. The assay was used to identify and isolate mutant cells with altered glucose uptake characteristics. A mathematical model was developed to provide a theoretical basis for estimating single-cell glucose uptake rates from single-cell 2-NBDG uptake rates. The assay provides a novel means of estimating the instantaneous rates of nutrient depletion in the growth environment during a batch cultivation.     

The structure of a fucose-containingO-glycosidic carbohydrate chain of human platelet glycocalicin

The hydrazinolysis procedure currently used for the release ofN-glycosidic carbohydrate chains was applied to glycocalicin. The resulting mixture of oligosaccharide-alditols was fractionated by high-voltage paper electrophoresis into a neutral (5%) and several acidic fractions. The neutral compounds were passed over Bio-Gel P-4. SomeN-glycosidic oligosaccharide-alditols, of theN-acetyllactosamine type as well as of the oligomannoside type, were found to be present. However, oligosaccharide-alditols derived fromO-glycosidic carbohydrate chains were also found, indicating a partial cleavage of GalNAc1-OSer/Thr linkages under the hydrazinolysis conditions applied. One of the neutralO-glycosidic components was characterized, by 500-MHz¹H-NMR spectroscopy in combination with sugar analysis, as the following pentasaccharidealditol: In addition the afuco analogue of this compound was obtained.     

Nature of the (Ca + Mg)-ATPase activator protein which associates with human erythrocyte membranes

(Ca²⁺ + Mg²⁺)-ATPase activator protein associated with human erythrocyte membranes could be extracted with EDTA under isotonic condition at pH 7.6. No activator was released, however, using isotonic buffer alone. Like calmodulin, the activator in the EDTA extract migrated as a fast moving band on polyacrylamide gel electrophoresis. It was also heat-stable, was capable of stimulating active calcium transport and could stimulate (Ca²⁺ + Mg²⁺)-ATPase to the same extent. When chromatographed on a Sephacryl S-200 column, it was eluted in the same position as calmodulin and a membrane associated (Ca²⁺ + Mg²⁺)-ATPase activator prepared according to Mauldin and Roufogalis (Mauldin, D. and Roufogalis, B.D. (1980) Biochem. J. 187, 507–513). Furthermore, both Mauldin and Roufogalis protein and the activator in the EDTA extract exhibited calcium-dependent binding to a fluphenazine-Sepharose affinity column. On the basis of these data, it is concluded that the activator protein released from erythrocyte membranes by EDTA is calmodulin. A further pool of the ATPase activator could be released by boiling but not by Triton X-100 treatment of the EDTA-extracted membranes. This pool amounted to 8.9% of the EDTA-extractable pool.     

Expression of glucose transporter isoform GLUT-2 and glucokinase genes in human brain

The glucose transporter isoform-2 (GLUT-2) and glucokinase are considered to be components of a glucose sensor system controlling several key processes, and hence may modulate feeding behaviour. We have found GLUT-2 and glucokinase mRNAs in several brain regions, including the ventromedial and arcuate nuclei of the hypothalamus. GLUT-2, glucokinase and glucokinase regulatory protein mRNAs and proteins were present in these areas as determined by biochemical approaches. In addition, glucose-phosphorylating activity with a high apparent Km for glucose that displayed no product inhibition by glucose-6-phosphate was observed. Increased glycaemia after meals may be recognized by specific hypothalamic neurones due to the high Km of GLUT-2 and glucokinase. This enzyme is considered to be the true glucose sensor because it catalyses the rate-limiting step of glucose catabolism its activity being regulated by interaction with glucokinase regulatory protein, that functions as a metabolic sensor.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

Glycosylation of the human erythrocyte glucose transporter is essential for glucose transport activity

The human erythrocyte glucose transporter is a fully integrated membrane glycoprotein having only one N-linked carbohydrate chain on the extracellular part of the molecule. Several authors have suggested the involvement of the carbohydrate moiety in glucose transport, but not definitive results have been published to date. Using transport glycoproteins reconstituted in proteoliposomes, kinetic studies of zero-trans influx were performed before and after N-glycanase treatment of the proteoliposomes: this enzymatic treatment results in a 50% decrease of the Vmax. The orientation of transport glycoproteins in the lipid bilayer of liposomes was investigated and it appears that about half of the reconstituted transporter molecules are oriented properly. Finally, it could be concluded that the release of the carbohydrate moiety from the transport glycoproteins leads to the loss of their transport activity.     

An effective automated glucose sensor for fermentation monitoring and control

An industrial glucose analyser was partnered to an automated injection system to evaluate glucose in the culture medium of a bioreactor. This sensor has been validated on continuous cultures ofSchizosaccharomyces pombe and continuous and fed-batch cultures ofSaccharomyces cerevisiae. In addition to the advantage of a more accurate process monitoring, the main interest of this sensor deals with the control of the substrate concentration to a prespecified reference signal. Several experiments have been carried out first to validate the sensor, then to control the process evolution.     

Aglycone exploration of C-arylglucoside inhibitors of renal sodium-dependent glucose transporter SGLT2

Inhibition of sodium-dependent glucose transporter 2 (SGLT2), the transporter that is responsible for renal re-uptake of glucose, leads to glucosuria in animals. SGLT-mediated glucosuria provides a mechanism to shed excess plasma glucose to ameliorate diabetes-related hyperglycemia and associated complications. The current study demonstrates that the proper relationship of a 4′-substituted benzyl group to a β-1C-phenylglucoside is important for potent and selective SGLT2 inhibition. The lead C-arylglucoside (7a) demonstrates superior metabolic stability to its O-arylglucoside counterpart (4) and it promotes glucosuria when administered in vivo.