Labeling of human erythrocyte membranes with eosin probes used for protein diffusion measurements. Inhibition of anion transport and photo-oxidative inactivation of acetylcholinesterase

The binding of eosin-isothiocyanate (eosin-NCS) and iodoacetamido-eosin (IA-eosin) to band 3 proteins in the membrane of human erythrocytes is characterized by studying the effect of these probes on the anion transport system. Although the unbrominated fluorescein precursors do not affect anion transport, both eosin labels are strong inhibitors of sulphate exchange in intact erythrocytes. 50% inhibition is obtained by binding 4.7 · 10⁵ or 6.0 · 10⁵ molecules/cell for eosin-NCS and IA-eosin, respectively. Both eosin probes are irreversibly bound and occupy common binding sites with 4,4′-diisothiocyano-1,2-diphenyl-ethane-2,2′-disulfonic acid (H₂DIDS), although other sites are labeled as well. The inhibition of anion transport is light independent and can therefore not be attributed to a photosensitizing action of the eosin probes. Both eosin derivatives, however, inactivate acetylcholinesterase upon illumination of air-equilibrated samples of hemoglobin-free labeled ghosts. The inactivation of the enzyme is accompanied by the formation of protein aggregates as visualized by polyacrylamide gel electrophoresis. These effects are not observed when intact erythrocytes are illuminated in the presence of eosin probes suggesting a protective effect of hemoglobin during the labeling procedure. Protection of ghosts from photo-oxidation is achieved by displacing air with argon. These results are discussed in relation to the use of these and similar probes to measure protein diffusion in membranes.     

Interaction of p-aminobenzoic acid with erythrocyte membrane: photoaffinity labeling of the binding sites

p-Aminobenzoic acid (PABA) was found to prevent eichinocytosis of red cells in vitro. Equilibrium binding studies with right-side-out membrane vesicles revealed a similar number of binding sites and Kd values for both normal and sickle cell membranes. A [14C]Azide analog of PABA was synthesized as a photoaffinity label to probe its sites of interaction on the erythrocyte membranes. Competitive binding studies of PABA with its azide indicated that both the compounds share common binding sites on the membrane surface. The azide was found to covalently incorporate into the membrane components upon irradiation; 52-35% of the label was associated with the proteins and the remaining with the lipids. Electrophoretic analysis of photolabeled membranes revealed that the azide interacts mainly with Band 3 protein in the case of intact erythrocytes and right-side-out sealed vesicles; however, if unsealed ghosts are used, other membrane proteins besides Band 3 are photolabeled. PABA was found to inhibit both high and low affinity calcium-binding sites situated on either surface of the membrane apparently in a non-competitive manner. However, calcium binding stimulated by magnesium and ATP was only slightly affected. Calcium transport into inside-out vesicles was inhibited by PABA, but it did not affect the calcium ATPase activity.     

The effects of anion transport inhibitors on structural transitions in erythrocyte membranes

Red blood cell membranes have been labeled with several covalent and non-covalent inhibitors of anion transport and their heat capacity profiles determined as a function of temperature. Covalent inhibitors include the amino reactive agents 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid, 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid, pyridoxal phosphate and 1-fluoro-2,4-dinitro benzene. The non-covalent inhibitors include several well known local anesthetics. The study was undertaken in order to identify regions of the membrane involved in anion transport. Covalent modification in all cases resulted in a large upward shift of the C transition, which is believed to involved a localized phospholipid region. Evidence is presented which indicates that Band III protein and this phospholipid region are in close physical proximity on the membrane. Addition of non-covalent inhibitors affects the membrane in either or both of two ways. In some cases, a lowering and broadening of the C transition occurs; in other the B₁ and B₂ transitions are altered. These latter transitions are believed to involve both phospholipid and protein, including Band III. These results may indicate that the non-covalent inhibitors produce their inhibitory effect on anion transport at least in part by interacting with membrane phospholipid.     

Direct photoaffinity labeling of leukotriene binding sites

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.     

Photoaffinity labeling of adenosine 3',5'-cyclic monophosphate binding sites of human red cell membranes.

To identify and investigate the cAMP binding sites of human red cell membranes a photoaffinity analog of cAMP, 8-azidoadenosine 3',5'-cyclic monophosphate (8-N3cAMP), has been synthesized. This analog activates cAMP-dependent protein kinase(s) in the red cell membrane. It exhibits tight, but reversible binding to the membranes which is competitive with cAMP. Photolysis of [32P]-8-N3cAMP with red cell membranes results in covalent incorporation of radioactive label onto two specific membrane proteins. This incorporation requires activating light and is reduced to background levels with addition of low levels of cAMP. Prephotolysis of 8-N3cAMP completely abolished its ability to photolabel membrane proteins. Both the reversible and photocatalyzed binding of 8-N3cAMP show saturation kinetics. The molecular weights of the two primarily labeled proteins are approximately 49,000 and 55,000. The differential effects of cAMP, ATP, and adenosine on the photocatalyzed incorporation of [32P]-8-N3cAMP onto these two proteins suggest that they have biochemically different properties. The potential usefulness of this compound for investigating various molecular aspects of cAMP action is discussed.     

Anion transport across the erythrocyte membrane,in situ proteolysis of band 3 protein,and cross-linking of proteolytic fragments by 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonate

Extracellular chymotrypsin cleaves the 95 000 dalton protein that migrates in band 3 of SDS-polyacrylamide gel electropherograms of the erythrocyte membrane into fragments of 60 000 and 35 000 daltons, but not further. Minor components of band 3 that remain at the original 95 000 dalton location may be eluted from the membrane by 0.1 N NaOH, indicating that, in contrast to the major component and the chymotryptic fragments, they are not integral membrane constituents.Incubation at neutral pH of chymotrypsinized erythrocytes with the bifunctional anion transport inhibitor 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid results in covalent binding of that inhibitor primarily to the 60 000 dalton fragment and some cross-linking of the 60 000 dalton fragment with the 35 000 dalton fragment. Increasing the pH to 9.5 leads to a crosslinking of virtually all of the pairs of chymotryptic fragments and thus to a reconstitution of band 3 with its typical diffuse appearance in the 95 000 dalton region of the SDS-polyacrylamide gels. This indicates that (1) each integral 95 000 dalton protein molecule is capable of binding at least one 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid molecule; (2) the 35 000 dalton fragment, though it is only weakly stained with Coomassie blue, is present in an amount that is equimolar with that of the 60 000 dalton fragment. Since the number of 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid binding sites on the protein in band 3/cell is known to be close to the number of band 3 molecules/cell, it is suggested that the cross-linking takes place at a region of the band 3 molecule that is involved in the control of anion transport.Like chymotrypsin, papain digests the band 3 protein from the outer membrane surface. Unlike chymotrypsin, however, papain digestion results in an inhibition of anion exchange. Papain produces a major fragment of 60 000 daltons that differs from the major chymotryptic fragment by at most six amino acid residues. The only detectable difference between the non-inhibitory action of chymotrypsin and the inhibitory action of papain on the band 3 protein is that papain is capable of partially digesting the 35000 dalton fragment. No reconstitution of band 3 by cross-linking of the fragments with 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid can be achieved. Since the 35 000 dalton fragment reacts with one of the two reactive groups of 4,4′-diisothiocyano dihydrostilbene-2,2′-disulfonic acid and is also susceptible to digestion by the inhibitory papain, we suggest that a portion of this peptide participates, together with a portion of the 60 000 dalton fragment, in the control of anion transport.     

Adenosine cyclic 3',5'-monophosphate uptake and regulation of membrane protein kinase in intact human erythrocytes

The uptake of adenosine cyclic 3',5'-monophosphate (cAMP) and stimulation of membrane-associated protein kinase in mature human erythrocytes were investigated. cAMP transport across the membrane was temperature dependent, and cAMP binding to the isolated membrane had less temperature dependence. More than 99% of the [3H]-cAMP taken up by erythrocytes was nonmembrane bound. Maximal stimulation of membrane protein kinase and maximal occupancy of membrane cAMP binding sites by extracellular cAMP cccurred at 30 degrees C within 30 min after initiation of the incubation of erythrocytes with cAMP. The concentration of extracellular cAMP that gave half-maximal stimulation of membrane protein kinase was 5.4 X 10-4 M, a value consistent with the concentrations of cAMP (5.2 X 10-4 M) found to occupy half-maximally the membrane cAMP binding sites in erythrocytes. Extracellular cAMP and to a lesser extent guanosine cyclic 3',5'-monophosphate and inosine cyclic 3',5'-monophosphate stimulated membrane protein kinase in erythrocytes. The cAMP uptake by human erythrocytes as well as cAMP binding to membranes in the erythrocyte was blocked by an inhibitor [4,4'-bis(isothiocyano)stilbene-2,2-disulfonate] of the anion channel. These studies indicate that cAMP can be transported across membranes into human erythrocytes and can bind to membranes to activate membrane protein kinase. It appears that there is a shared transport channel for cAMP and anion transport.     

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 (Ørskov 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.     

Anion transport in relation to proteolytic dissection of band 3 protein

Sulfate efflux was measured in inside-out vesicles obtained from human red cells. Inhibition was observed in vesicles derived from cells pretreated with DIDS (4,4′-diisothiocyano-2,2′-stilbene disulfonate) or after addition of dipyridamole to the vesicles, both agents being specific and potent inhibitors of anion transport in cells. Trypsinization of the cytoplasmic side of the membrane in order to release a 40 000 dalton fragment from band 3 (the purported anion transport protein) had no effect on sulfate efflux. Further degradation of band 3 to a 17 000 dalton segment, by trypsinization of inside-out vesicles derived from cells that had been pretreated with chymotrypsin, also showed little reduction in transport activity. Furthermore, such vesicles derived from DIDS pretreated cells were inhibited by over 90%. In DIDS-treated cells, the agent is highly localized in band 3. In trypsinized inside-out vesicles, it is largely found in a 55 000 fragment and in trypsinized vesicles derived from cells pretreated with chymotrypsin it is largely located in the 17 000 fragment. The data suggest that both the anion transport and inhibitor binding sites are located in a 17 000 transmembrane segment of band 3.     

Localization of the tight ADP-binding site on the membrane-bound chloroplast coupling factor one

The photoaffinity analog 2-azido-ADP (2-azidoadenosine 5'-diphosphate) was used as a probe of the spinach chloroplast ATP synthase. The analog acted as a substrate for photophosphorylation. Several observations suggested that 2-azido-ADP and ADP bound to the same class of tight nucleotide binding sites: (a) 2-azido-ADP competitively inhibited ADP tight binding (Ki = 1.4 microM); (b) the concentration giving 50% maximum binding, K0.5 for analog tight binding (1 microM) was similar to that observed for ADP (2 microM); (c) nucleotide tight binding required prior membrane energization and was completely reversed by re-energization; (d) the tight binding of 2-azido-[beta-32P]ADP was completely prevented by ADP; (e) the analog inhibited the light-triggered ATPase activity at micromolar concentrations. Ultraviolet irradiation of washed thylakoid membranes containing tightly bound 2-azido-[beta-32P]ADP resulted in the covalent incorporation of the label into the membranes. Denaturing polyacrylamide gel electrophoresis of the labeled membranes demonstrated that the beta subunit of the coupling factor one complex was the only polypeptide in the thylakoid membranes which was labeled. These results identify the beta subunit of the coupling factor as the location of the tightly bound ADP on the thylakoid membranes.     

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.     

Identification of the erythrocyte nucleoside transporter as a band 4.5 polypeptide. Photoaffinity labeling studies using nitrobenzylthioinosine

Nitrobenzylthioinosine (NBMPR) was employed as a covalent probe of the erythrocyte nucleoside transporter. This nucleoside analogue, a potent inhibitor of nucleoside transport, binds tightly (KD = 10(-10) - 10(-9) M) but reversibly to specific sites on the carrier mechanism. High intensity UV irradiation of intact human erythrocytes, isolated "ghosts," and "protein-depleted" membranes in the presence of [3H]NBMPR and dithiothreitol (as a free radical scavenger) under nonequilibrium and equilibrium binding conditions resulted in selective covalent incorporation of 3H into the band 4.5 region of sodium dodecyl sulfate-polyacrylamide gels (Mr = 45,000-65,000). Covalent labeling of band 4.5 protein(s) under equilibrium binding conditions was inhibited by nitrobenzylthioguanosine, dipyridamole, uridine, and adenosine. A similar photolabeling pattern was observed using membranes from pig erythrocytes. In contrast, no incorporation of radioactivity into band 4.5 was observed under equilibrium binding conditions with membranes from nucleoside-impermeable sheep erythrocytes. These experiments suggest that the human and pig erythrocyte nucleoside transporters are band 4.5 polypeptides, a conclusion supported by previous isolation studies based on the assay of reversible [3H]NBMPR binding activity.     

The interaction of human erythrocyte band 3 with cytoskeletal components

Band 3 of the human erythrocyte is involved in anion transport and binding of the cytoskeleton to the membrane bilayer. Human erythrocytes were treated to incorporate varying concentrations of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) a non-penetrating, irreversible inhibitor of anion transport, and both functions of Band 3 were analyzed. The rate of efflux of ³⁵SO₄. was measured and the binding of cytoskeletal components to the membrane was evaluated by extracting the membranes with 0.1 n NaOH and analyzing for the peptides remaining with the membrane. It was found that 0.1 n NaOH extracts all the extrinsic proteins from membranes of untreated cells, while, in the case of the membranes from cells treated with DIDS, a portion of the cytoskeletal components, spectrin (Bands 1 and 2) and Band 2.1 (ankyrin, syndein) remain with the membrane. The amount of these cytoskeletal components remaining with the membrane depends on the concentrations of DIDS incorporated. The effect of DIDS on the extractability of the spectrin-Band 2.1 complex correlates well with DIDS inhibition of anion transport (r = 0.91). At DIDS concentrations which completely inhibit anion transport, about 10% of total spectrin-Band 2.1 complex remains unextracted. Another anion-transport inhibitor, pyridoxal phosphate, has no effect on binding of the cytoskeleton to the membrane. On the other hand, digestion of DIDS-pretreated intact erythrocytes with Pronase, chymotrypsin, or trypsin releases the tight binding of Band 3 to cytoskeleton on the inside of the membrane. Since trypsin does not hydrolyze Band 3 the data suggest that a second membrane protein which is trypsin sensitive may be involved with Band 3 in cytoskeletal binding.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Identification of the Cl− transport site of human red blood cells by a kinetic analysis of the inhibitory effects of a chemical probe

H₂DIDS, the dihydro analog of DIDS (4,4′-diisothiocyanostilbene-2,2′-disulfonic acid) can interact covalently with membrane sites, resulting in an irreversible inhibition of anion exchange. At low temperatures (0°C) and for relatively short times, however, its interaction is largely reversible, so that a kinetic analysis of the nature of its inhibitory effect on Cl^? self exchange can be performed. The effects of variations in the chloride concentration on the inhibitory potency of H₂DIDS are consistent with the concept that Cl^? and H₂DIDS compete for the transport site of the anion exchange system. The value of K_i for H₂DIDS is 0.046 μM, indicating that H₂DIDS has a higher affinity for the transport system than any other inhibitor so far examined. If, as seems probable, the covalent labelling of H₂DIDS occurs at the same site as the reversible binding, H₂DIDS can be used as a covalent label for the transport site. The specific localization of H₂DIDS in the band-3 protein thus indicates that this protein participates directly in anion exchange.     

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling

Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.     

Photoaffinity labeling of the beta-adrenergic receptor with azide derivatives of iodoccyanopindolol

Two photosensitive iodocyanopindolol derivatives, 1-(4-azidobenzimidyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-1) and 1-(4-azidobenzoyl)-3,3-dimethyl-6-hydroxy-7-(2-cyano-3-iodoindol-4-yloxy)-1,4-diazaheptane (ICYP-azide-2) have been prepared. [125I]ICYP-azide-1 and -2 (specific radioactivity up to 2.2 Ci/mumol) bind specifically and with very high affinity (KD = 40-45 pM) to beta-adrenergic receptors of turkey erythrocyte membranes. When [125I]ICYP-azide-1 or -2 were incubated with membranes and UV-irradiated, two polypeptides (Mr = 40,000 and 50,000) were specifically photolabeled as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides may represent subunits of the beta-adrenergic receptor. The yield of specific covalent label incorporation into both polypeptides was up to 17.2% with [125I]ICYP-azide-2 when expressed as fraction of total beta-receptor binding sites. Since the Mr = 40,000 polypeptide was labeled predominantly and since covalent incorporation had the same concentration dependence as reversible specific binding, this polypeptide could contain a beta-adrenergic ligand binding site. Due to the low working concentration (10-100 pM) of [125I]ICYP-azide-1 and -2, nonspecific labeling of membrane proteins was extremely low. The new photoaffinity labels should therefore become valuable tools for probing beta-receptor structure.     

Inhibition of anion transport in the red blood cell by anionic amphiphilic compounds I. Determination of the flufenamate-binding site by proteolytic dissection of the band 3 protein

Flufenamate, a non-steroidal anti-inflammatory drug, is a powerful inhibitor of anion transport in the human erythrocyte ($\text{I}{}_{50}\text{= 6·10}{}^{\mathrm{?7}}\text{M}$). The concentration dependence of the binding to ghosts reveals two saturable components. [¹⁴C]Flufenamate binds with high affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_1\text{= 1.2·10}{}^{\mathrm{?7}}\text{M}$) to 8.5·10⁵ sites per cell (the same value as the number of band 3 protein per cell); it also binds, with lower affinity ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}{}_2\text{= 10}{}^{\mathrm{?4}}\text{M}$) to a second set of sites (4.6·10⁷ per cell). Pretreatment of cells with 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonic acid (SITS), a specific inhibitor of anion transport, prevents [¹⁴C]flufenamate binding only to high affinity sites. These results suggest that high affinity sites are located on the band 3 protein involved in anion transport. Extracellular chymotrypsin and pronase at low concentration cleave the 95 kDa band 3 into 60 kDa and 35 kDa fragments without affecting either anion transport or [¹⁴C]flufenamate binding. Splitting by trypsin at the inner membrane surface of the 60 kDa chymotryptic fragment into 17 kDa transmembrane fragment and 40 kDa water-soluble fragment does not affect [¹⁴C]flufenamate binding. In contrast degradation at the outer membrane surface of the 35 kDa fragment by high concentration of pronase or papain decreases both anion transport capacity and number of high affinity binding sites for [¹⁴C]flufenamate. Thus it appears that 35 kDa peptide is necessary for both anion transport and binding of the inhibitors and that the binding site is located in the membrane-associated domain of the band 3 protein.     

The location of a disulfonic stilbene binding site in band 3, the anion transport protein of the red blood cell membrane

The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid, a specific, potent, irreversible inhibitor of anion transport in red blood cells is located in a 15 000 dalton transmembrane segment of band 3, produced by chymotrypsin treatment of ghosts stripped of extrinsic proteins. The segment was cleaved into three fragments of 7000, 4000 and 4000 daltons by CNBr. The C-terminus of the segment is located in the 7000 dalton fragment; the N-terminus in one of the 4000 dalton fragments; and the binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid in the middle 4000 dalton fragment. The latter was cleaved by $\text{N-}\text{bromosuccinimide}$ into two fragments of 2000 daltons. The binding site for 4,4′-diisothiocyano-2,2′-stilbenedisulfonic acid was located on the fragment containing the newly formed N-terminus. It is concluded that the binding site is located about 9000 daltons from the C-terminus (at the outside face of the membrane) and 6000 daltons from the N-terminus (at the cytoplasmic face). In view of the existing evidence that the binding site may be located near the outside face of the membrane, it is suggested that the 15 000 dalton segment is folded, so that it crosses the bilayer three times.     

Photoaffinity labeling of the adenosine transport system in adipocyte plasma membranes

A membrane component involved in the transport of adenosine in adipocytes has been identified utilizing the techniques of photoaffinity labeling with the adenosine derivative, 8-azidoadenosine. In the absence of light, adenosine and 8-azidoadenosine exhibited similar transport characteristics. In addition, adenosine was shown to be a competitive inhibitor of 8-azidoadenosine uptake, and the photoprobe, a competitive inhibitor of adenosine uptake. Analysis of the nucleotide metabolites indicated that the photoprobe was metabolized in a similar fashion to that observed for adenosine. Several nucleoside transport inhibitors were also equally effective in inhibiting the uptake of both nucleosides. These results suggest that 8-azidoadenosine is transported by the same membrane system as adenosine. Photolysis of 8-azido[2-³H]adenosine in the presence of adipocytes resulted in the covalent incorporation of the photoprobe into the plasma membrane fraction. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that essentially all of the radioactivity was incorporated into a glycoprotein with a molecular weight of 56,000. This labeling was inhibited by greater than 90% when the photolysis was carried out in the presence of excess adenosine or the transport inhibitors, persantin or theophylline. Fractionation of the labeled plasma membranes by dialysis against water (pH 9.5) indicated that approximately 75% of the radioactivity was associated with a glycoprotein which resisted solubilization by this procedure. These results suggest that the major labeled species is a 56,000 M_r intrinsic membrane glycoprotein which may function as a component of a transmembrane assembly involved in the transport of adenosine.