The anion transporter and a 28 kDa protein are selectively photolabeled by p-azidobenzylphlorizin under conditions that alter RBC morphology, flexibility, and volume.

Tritiated p-azidobenzylphlorizin (p-AzBPhz) was photoactivated in the presence of red blood cells under conditions previously found to alter morphology, flexibility and volume. When less than 0.25 million molecules were added per cell, only a 28 kDa peptide was photolabeled: at 1-2 million molecules added, band 3 also incorporated significant radioactivity. When using leaky ghosts, other proteins became labeled, including those limited to the cytoplasm. Protein N-deglycosylation caused a shift of radiolabeled band 3 to higher Rf values on SDS-PAGE gels but not for the 28 kDa band; the latter was, however, susceptible to enzymatic digestion by NANase (N-acetylneuraminidase) III but not by NANase II. Inhibition of photoincorporation into both receptors by unlabeled p-AzBPhz was dose-dependent. Mercuric chloride and p-CMBS selectively blocked 28 kDa peptide labeling. DIDS partially blocked at band 3; after 15% inhibition, greater DIDS concentrations caused increased incorporation into the 28 kDa peptide. These results, and a temperature-dependent labeling pattern, suggest that: (i) cellular changes occur when p-AzBPhz binds to the exofacial sides of the anion transporter and 28 kDa peptide; (ii) these proteins may be physically associated in the native membrane; (iii) they mediate ligand-induced changes in morphology, flexibility, and volume.

Band 3 antagonists, p-azidobenzylphlorizin and DIDS, mediate erythrocyte shape and flexibility changes as characterized by digital image morphometry and microfiltration.

Two nonpenetrating membrane probes, p-azidobenzylphlorizin (p-AzBPhz) and 4,4'-diisothiocyano-2,2'-stilbene disulfonate (DIDS), have been shown in earlier studies to induce dose-dependent changes in red blood cell (RBC) shape and volume at the same low concentrations that inhibit anion transport. In the present work, these ligand-induced morphology and rheology changes were studied using video digital image morphometry (VDIM) and microfiltration techniques. The results of these experiments corroborate our earlier investigation. RBCs were filmed using a Nomarski optics microscope with video camera attachment and cell size and shape changes were computer analyzed using VDIM. Low microM p-AzBPhz or DIDS levels caused collapse of the cell's biconcave structure and cell flattening occurred within 1-2 sec after drug exposure. Higher doses of either agent converted cells to a new steady-state in which a concurrent limited increase in erythrocyte volume and blunt membrane protrusions were produced. These changes were reversed in less than 2 sec by washing the drug from the membrane. Both ligands increased the deformability of RBCs in a dose-dependent manner as determined by filtration through Nuclepore polycarbonate filters (3 microns pore diameter). The improvement in deformability of drug-treated sickle cells was much more dramatic than for normal cells at low p-AzBPhz concentrations. These results support our earlier conclusions that the ligands, through a common interaction with band 3, induce volume-associated cytoskeletal alterations which lead to changes in morphology and flexibility.

Morphology and volume alterations of human erythrocytes caused by the anion transporter inhibitors, DIDS and p-azidobenzylphlorizin.

p-Azidobenzylphlorizin (p-AzBPhz) is a potential photoaffinity labeling agent for the anion and glucose transporters in human RBCs. In the absence of light and at the same low concentrations which block these transport processes (only 1-2 million molecules bound/cell), this impermeable membrane probe produces rapid morphological and volume alterations. This high-affinity activity, called phase 1, can be rapidly and completely reversed by simply diluting the azide-treated cell suspension. Phase 2 effects, including formation of cells with multiple, long spicules (stage 3/4 echinocytes), followed by an influx of salt and water with eventual lysis, occur at two log units higher concentration by a different mechanism, probably by intercalating into and selectively expanding the outer lipid monolayer. Light scattering, electronic cell sizing, microhematocrit measurements and scanning electron microscopy have been employed to compare the effects of the azide and the anion transport inhibitor, DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate), on red cells. DIDS produced only those changes analogous to the azide's low dose phase 1 action; cells swell, lose the ability to scatter 800 nm light and undergo a limited shape change (comparable to stage 1 echinocytosis). The mechanism by which the two ligands perturb the membrane is additive, suggesting that a Band 3-mediated transmembrane signaling is involved which leads to altered cytoskeleton dynamics.

Interaction of phlorizin, a potent inhibitor of the Na+/D-glucose cotransporter, with the NADPH-binding site of mammalian catalases.

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.

Erythrocyte shape and volume changes caused by an inhibitor of the glucose and anion transporters.

Light scattering measurements and scanning electron microscopy show that p-azidobenzylphlorizin (p-AzBPhz) causes changes in the shape and volume of human erythrocytes by at least two, dose-dependent mechanisms: At nominal concentrations above 5 microM, the azide induces cell swelling by either enlarging a pre-existent channel or by creating pores between phase boundaries of the membrane through which salt and water enter, but sucrose remains excluded. However, over the range 0.03 to 0.3 microM, in either isosmotic NaCl or KCl media, when fewer than 1 million molecules of azide are bound per cell, the ligand causes membrane deformations that convert discocytes into cells resembling stage 2 echinocytes. Whereas a cell volume increase of about 10% accompanies these shape changes, (microhematocrit and electronic cell sizing measurements), no net influx of either Na+ or K+ during this stage of swelling was detectable. These cell alterations take place at p-AzBPhz concentrations which concurrently inhibit both chloride and 3-methoxyglucose equilibrium exchange transport. The results may indicate that when the membrane impermeable p-AzBPhz interacts with the anion and/or sugar transporter, some trans-membrane perturbation occurs which alters the cytoskeleton.

Computer analysis reveals changes in renal Na+-glucose cotransporter in diabetic rats.

A novel, computer-assisted program was developed to analyze the time course of Na+-glucose cotransport by rat renal cortical brush-border membrane vesicles (BBMV). Transporter characteristics can be measured, which routine kinetic analyses fail to distinguish: cotransporter membrane density is derived from the picomoles of D-glucose bound per milligram of protein. Binding is stereospecific, blocked by phlorizin, and supported equally well by Na+ or K+ (but not Cs+). Quasi-first-order influx and efflux rate constants for the composite Na+-driven influx and the (presumed) Na+-independent efflux processes were highly dependent on glucose concentration. Either two Na+-glucose transporters exist in proximal tubules or a single mechanism abruptly changes rate when glucose falls to low levels. The major operation mode is slow, has a high capacity but low affinity, and may have a 2 Na+:2 glucose stoichiometry (Hill coefficient is unity). The minor system is a fast, smaller-capacity, higher-affinity operation with a 2 Na+:1 glucose stoichiometry that was not distinguishable when the same data were analyzed in conventional kinetic plots. Results with streptozocin-induced diabetic rats illustrate the method's utility. Low-glucose-affinity cotransporters were upregulated in hyperglycemic, but not in cachectic, ketoacidotic animals. Rate constants, especially for efflux, were decreased in diabetes.

Electron spin resonance investigation of the interaction of the anion and glucose transport inhibitor, p-azidobenzylphlorizin, with the human red cell membrane.

The membrane perturbations caused by the interaction of p-azidobenzylphlorizin (p-AzBPhz), a potential photoaffinity labeling agent of the anion and D-glucose transporters in the human erythrocyte, have been studied using electron spin resonance (ESR) spectrometry. Two lipid-specific spin labels have been employed; one of these agents, a hexadecyl-quarternary amine with the nitroxide reporter group covalently attached to the cationic nitrogen, (CAT-16), has been used to monitor changes in the physical state of the membrane's extracellular phospholipid/water interface. The other spin label, 5-doxylstearic acid (5-NS), is designed to examine the order and motion of the lipid bilayer near the cell surface. In separate experiments, intact human red cells labeled with these lipid-specific spin labels were exposed to small amounts of the phlorizin azide. A dose-dependent alteration in CAT-16 motion was observed, but the p-AzBPhz interaction with the membrane had no effect on the spectrum of 5-NS. The half-maximal effect of the phlorizin derivative on the CAT-16 spectrum occurred when about 2 million molecules were bound to each cell. This is also the combined amount of band 3 and band 4.5 present in the red cell membrane and represents the concentration necessary to inhibit both anion and glucose transport. Our results suggest that the first p-AzBPhz molecules binding to the red cell membrane interact with the anion and sugar transporters, and not with the bulk lipid bilayer.

Membrane action of phlorizinyl 5'-benzylazide induces volume and shape change of human erythrocytes and sickle cells.

In the absence of light, phlorizinyl benzylazide is a potent competitive inhibitor of sugar transport (app. Ki about 1.5 uM) and a reversible inhibitor of chloride exchange (app. Ki about 2 uM) in the normal red cell. It causes additional membrane alterations at about 10-fold higher concentrations which lead to a time-, pH-, temperature-, and cell storage time-dependent increase in cell volume in isosmotic phosphate-buffered saline. This drug effect may be related to its interaction with Bands 3 and 4.5 (both become covalently labeled when the azide is light activated) such that cation conductance is increased. Apparently, a channel is created or widened through which salt and water (but not sucrose) can enter causing cell swelling and eventually lysis. The membrane probe is capable of inhibiting deoxygenation-induced sickling of SS and SC cells; under conditions that cause normal cell bursting, sickle cells swell but do not lyse.

Kinetic studies of D-glucose transport in renal brush-border membrane vesicles of streptozotocin-induced diabetic rats.

Renal brush-border membrane vesicles prepared from streptozotocin-induced 4-day-diabetic rats possessed a Na+-dependent D-glucose transport system that exhibited apparent Kt and Vmax values about 2-fold greater than normal. Apparently, hyperglycemia and probably other stimuli cause the induction and membrane incorporation of a low-affinity transporter in these membranes; this increased sugar-transport capacity is retained for at least 4 weeks so long as the animals maintained or increased their body weight. Membranes prepared from 28-day-diabetic, severely ill ketoacidotic animals lose this enhanced transport ability and the decrease in Vmax was found to correlate directly with the weight loss. Furthermore, the transporter in brush-border membranes prepared from these cachectic animals had an even lower affinity for glucose than those from the acute hyperglycemic animals. That these changes in the diabetic animals represent major alterations in renal brush-border membrane construction is further supported by our observation that the specific activity of the marker enzymes, alkaline phosphatase and neutral alpha-glucosidase, are profoundly increased and decreased, respectively, in this condition.

Solubilization, reconstitution, and attempted affinity chromatography of the sugar transporter of the erythrocyte membrane.

Reconstitution of the sugar transport system of human erythrocytes into artificial liposomes was achieved by freezing, thawing, and sonicating preformed phospholipid vesicles in the presence of intact ghosts, protein-depleted ghosts, or detergent-treated ghosts. D-glucose equilibrium exchange activities and affinity constants in the range of the reported erythrocyte values were reached in the best experiments. Whereas the extraction of peripheral membrane proteins did not depress the transport function crucially after reconstituting these protein-depleted ghosts, the selective solubilization of integral membrane proteins by a variety of nonionic detergents resulted in an uncontrollable, continuously increasing inactivation of the carrier. However, Emulphogene BC-720 extracts could be prepared in which the glucose transporter retained activity for days at 4 degrees C. These extracts were applied to affinity chromatography matrices of phloretin-Agarose, prepared by coupling phloretinyl-3'-benzylamine (PBA) to CH-Sepharose 4B and to Affigel 202. Although the solubilized sugar transporter appeared to be selectively adsorbed to both PBA matrices, it could not be eluted by specific counter ligands or gentle eluants in a biologically active form. However, chaotropic agents could be used to elute intrinsic proteins, including bands 3 and 4.5, from the Affigel affinity medium.

4-Azidophlorizin, a high affinity probe and photoaffinity label for the glucose transporter in brush border membranes.

A new phlorizin derivative (2'-O-(beta-D-glucopyranosyl)-4-azidophloretin, 4-azidophlorizin) has been synthesized and its affinity for the D-glucose, Na+ co-transport system in brush border vesicles from intestinal and renal membranes has been compared with that of phlorizin. The extent of the reversible interaction of the ligand with the transporter in dim light has been evaluated from three separate measurements: (1) Ki', the constant for fully-competitive inhibition of (Na+, delta psi)-dependent D-glucose uptake, (2) Kd', the dissociation constant of 4-azido[3H]phlorizin binding in the presence of an NaSCN inward gradient, and (3) Ki", the constant for fully-competitive inhibition of the specific ((Na+, delta psi)-dependent, D-glucose protectable) high-affinity [3H]phlorizin binding. In experiments with vesicles derived from rat kidney, all three constants (Ki', Kd' and Ki") were essentially equal and ranged between 3.2 and 5.2 microM, that is, the azide derivative has almost the same affinity for this transporter as phlorizin itself. On the other hand, compared to phlorizin, the 4-azidophlorizin has a lower affinity for the transporter in vesicles prepared from rabbit; its Ki' values are some 15-20-times larger than those determined with rat membranes. However, the affinity of the azide for the sugar transporter in membranes from either the intestine or kidney of the same animal species (rabbit or rat) was essentially the same. In spite of the lower affinity for the transporter in either membrane system from the rabbit, results described elsewhere (Hosang, M., Gibbs, E.M., Diedrich, D.F. and Semenza, G. (1981) FEBS Lett., 130, 244-248) indicate that 4-azidophlorizin is an effective photoaffinity label in this species also. Photolysis of the azide yields a reactive intermediate which reacts with a 72 kDa protein in rabbit intestine brush borders. Covalent labeling of this protein occurred under conditions which suggests that it is (a component of) the glucose transporter.

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 microM, 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 degrees 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 microM 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.

Phloretinyl-3'-benzylazide: a high affinity probe for the sugar transporter in human erythrocytes. I. Hexose transport inhibition and photolabeling of mutarotase.

A new phloretin derivative, phloretinyl-3'-benzylazide (PBAz), has been synthesized and compared with phloretin for its ability to inhibit the hexose transporter in human erythrocyte membranes in subdued light. Transport measurements were made using the light scattering (Orskov optical) method and a Millipore filtration technique with isotopically labeled sugars. Initial rates of sugar flux were measured under four different conditions to test for inhibition asymmetry. In each experimental condition, PBAz is from 6-20-times more potent than phloretin, making it one of the most effective reversible inhibitors known. Although both agents penetrate the cell membrane, they apparently fail to reach inhibitory levels at the inner surface over the time course of our nonequilibrated experiments, because of extensive binding to hemoglobin. The mechanism by which PBAz and its parent phloretin inhibit transport is pure competition with hexose for the carrier which faces the exterior of the membrane. If given time to equilibrate with the cells, the inhibition by both agents converts to a mixed type, i.e., both competitive and noncompetitive. The noncompetitive component could be due to inhibition of those transporter units oriented internally. Alternatively pre-equilibration with the inhibitors may cause them to attain high levels in the lipid membrane and produce nonspecific effects. PBAz and its precursor amine, phloretinyl-3'-benzylamine (PBA), compete with glucose for the sugar binding site on mutarotase at least as well as phloretin. When exposed to long wavelength ultraviolet radiation, PBAz is converted to a reactive intermediate which becomes covalently bound to the enzyme. Both irreversible ligand attachment and mutarotase inhibition are related to dose of the azide and irradiation time, but inactivation is from 5 to 6-times greater than label incorporation. We conclude that PBAz is a potentially useful photoaffinity labeling agent capable of covalently interacting with the transporter site facing the exterior of the red cell.

The hexose transport system in the human K-562 chronic myelogenous leukemia-derived cell.

Kinetics of glucose transport in K-562 cells was studied using 3-O-methylglucose, a nonmetabolizable analog of glucose. A Km of 3.7 mM and Vmax of 32.0 nmoles/minute/10(6) cells was found for the process. D-Glucose, phloretin, and phlorizin competitively inhibit the transport of 3-O-methylglucose with Ki values of 4.1 mM, 4.1 muM and 225 muM, respectively, whereas L-glucose did not inhibit transport at all. The results indicate that K-562 cells, which are known to have erythropoietic characteristics, possess a glucose carrier system similar to the one in adult human erythrocytes. However, the Vmax data suggest that more copies of the carrier are present in the malignant cell, presumably to support the high rate of anaerobic glycolysis.

Kinetic advantage for transport into hamster intestine of glucose generated from phlorizin by brush border beta-glucosidase.

Phlorizin, labeled with tritium only in the glucose moiety, was used as substrate for the beta-glucosidase present in brush border membranes from hamster intestine in order to study, simultaneously, the kinetics of hydrolysis and the fate of the [3H]glucose liberated by the enzyme. The [3H]glucose seems to experience the same hydrolase related transport into the intestinal villi as the hexoses liberated from the common disaccharides byu their respective hydrolases. The released [3H]glucose accumulation rate is only partially inhibited by unlabelled glucose added to the medium as either the free sugar or as the precursors sucrose, lactose or glucose 1-phosphate, and then only when these sugars are present at very high levels. Furthermore, glucose oxidase, added to the medium as a glucose scavenger, has no effect on the uptake rate of the phlorizin hydrolase-liberated sugar. These and other findings are presented as evidence that, under conditions where the Na+-dependent glucose carrier is more than 97% inhibited by phlorizin, the glucose derived from the inhibitor, like the hexoses from disaccharides, has a kinetic advantage for transfer into the intestinal tissue.

A hydrolase-related transport system is not required to explain the intestinal uptke of glucose liberated from phlorizin.

The fate of [3H]glucose released from a wide range of [3H]phlorizin concentrations by phlorizin hydrolase has been studied under conditions where the Na+-dependent glucose transport system in hamster intestine is profoundly inhibited by the glucoside. At 0.2-2.0 mM phlorizin, the [3H]glucose uptake was a constant 11-12% of that generated by the enzyme and at the highest level, it was reduced to that of passive diffusion. Glucose liberated from 0.2 mM [3H]phlorizin is accumulated at a rate nearly equal to that found for 0.2 mM [14C]glucose when this free sugar uptake is measured in a medium containing 0.2 mM unlabeled phlorizin. Furthermore, without sodium, the accumulation rates of hydrolase-derived or exogenous glucose are both reduced to the rate of [14C]mannitol. Our results indicate that the glucose released from phlorizin enters the tissue via the small fraction of the Na+-dependent glucose carriers which escape phlorizin blockade together with a mannitol-like passive diffusion. It enjoys a kinetic advantage for tissue entry over free glucose in the medum by virtue of the position of the site where it is formed, i.e inside the unstirred water layer and near normal entry portals. No special hydrolase-related transport system, like the one proposed for disaccharides, needs to be considered to account for our findings.