Gastric mucin hydrophobicity: effects of associated and covalently bound lipids, proteolysis, and reduction

The hydrophobic properties of gastric mucus glycoprotein were investigated using the fluorescent probe, bis(8-anilino-1-naphthalenesulfonate). The glycoprotein was subjected to removal of associated and covalently bound lipids, peptic degradation, and disulfide bridge reduction. Fluorescence titration data revealed the presence of 55 hydrophobic binding sites in the intact mucin molecule, 71 binding sites in the glycoprotein devoid of associated lipids, and 53 binding sites in the glycoprotein devoid of associated lipids and covalently bound fatty acids. Proteolytic digestion of the glycoprotein with pepsin essentially abolished the probe binding, while reduction of disulfide bridges resulted in glycoprotein subunits whose combined number of binding sites was about 3 times greater than that of the mucin polymer. The binding of the probe to mucus glycoprotein varied with the pH of the medium, being highest at pH 2.0 and lowest at pH 9.0. The results indicate that lipids contribute to the hydrophobic character of gastric mucin and that hydrophobic binding sites reside on the nonglycosylated regions of the glycoprotein polymer buried within its core.     

Role of salivary epidermal growth factor in the maintenance of physicochemical characteristics of oral and gastric mucosal mucus coat

The involvement of salivary epidermal growth factor (EGF) in the maintenance of oral and gastric mucosal mucus coat dimension and chemical characteristics was investigated using sialoadenectomized rats. Examination of the oral and gastric mucosal surface by phase contrast microscopy and Alcian blue uptake revealed that deprivation of salivary EGF caused a 31-36% reduction in mucus coat thickness and a 38-43% reduction in adherent mucin content. Chemical analyses indicated that the mucus coat of sialoadenectomized group exhibited a 21-28% increase in protein and a 67% decrease in covalently bound fatty acids, a 30% decrease in carbohydrates, and a 32-37% decrease in lipids. Sialoadenectomy also evoked changes in the chemical composition of mucus glycoprotein component of oral and gastric mucus coat reflected in the lower content of sulfate (25-26%), associated lipids (24-25%), and covalently bound fatty acids (67-75%). Intragastric supplementation of EGF had no effect on the physicochemical changes caused by sialoadenectomy in the oral mucosal mucus coat, while nearly complete restoration to normal characteristics occurred in the gastric mucosal mucus coat. The results suggest that salivary EGF is essential for the maintenance of mucus coat dimension and quality needed in the protection of alimentary tract epithelium.     

Mucus glycoprotein of human saliva: differences in the associated and covalently bound lipids with caries

A high molecular weigh mucus glycoprotein has been isolated from submandibular saliva of caries-resistant and caries-susceptible individual by a procedure involving fractionation on Bio-Gel P-100 and A-50 columns followed by equilibrium density-gradient centrifugation in CsCl. The purified caries-resistant mucus glycoprotein displayed a buoyant density of 1.50 and accounted for 9.5% of the dry weight of caries-resistant saliva. The caries-susceptible mucus glycoprotein representd 14.1% of the dry weight of caries-susceptible saliva and gave a buoyant density of 1.43. Both glycoproteins exhibited similar protein and carbohydrate content, but the caries-resistant mucus glycoprotein contained 28.7% less associated lipids and 3-times less covalently bound fatty acids than the caries-susceptible mucus glycoprotein. The associated lipids were represented by neutral lipids, glycolipids and phospholipids, whereas the covalently bound fatty acids consisted mainly of hexadecanoate, octadecanoate and docosanoate. Extraction of associated lipids caused the caries-resistant glycoprotein to band in CsCl gradient at the density of 1.54 and caused the caries-susceptible glycoprotein to band at the density of 1.52. A further shift in the buoyant densities occurred following removal of the covalently bound fatty acids, and both glycoproteins banded at the density of 1.57. While the intact caries-resistant and caries-susceptibel glycoproteins were susceptible to proteolysis by pronase, the lipid-rich caries-susceptible glycoprotein was degraded to a lesser extent. Extraction of associated lipids increased the degradation of both glycoproteins, but the caries-susceptible glycoprotein still remained 25% less susceptible. However, the susceptibility to pronase of the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins was essentially identical. The caries-resistant and caries-susceptible mucus glycoproteins also differed in susceptibility to peptic degradation. The apparent K_m values for intact caries-resistant and caries-susceptible glycoproteins were 10.5 · 10⁻⁷ M and 8.1 · 10⁻⁷ M, while the values for the delipidated and deacylated caries-resistant and caries-susceptible glycoproteins were 13.0 · 10⁻⁷ M and 12.4 · 10⁻⁷ M. The results suggest that the differences in the content of associated lipids and covalently bound fatty acids are responsible for the different physicochemical characteristics of caries-resistant and caries-susceptible salivary mucus glycoproteins, which may be determining falctors in the resistance to caries.     

Hydrogen ion diffusion in dog gastric mucus glycoprotein: effect of associated lipids and covalently bound fatty acids

The effect of neutral lipids, glycolipids and phospholipids associated with dog gastric mucus glycoprotein, and that of covalently bound fatty acids on the ability of glycoprotein to retard the diffusion of hydrogen ion was investigated. Purified mucus glycoprotein in its native form, placed between equimolar (0.155M) solutions of HCl and NaCl in a specially designed two-compartment chamber, caused a 90% reduction in permeability to hydrogen ion when compared with a layer of NaCl. Extraction of associated lipids lead to a 68% increase in permeability of the glycoprotein to hydrogen ion, while removal of the covalently bound fatty acids increased further the diffusion rate by 6%. Reassociation of the delipidated glycoprotein with its neutral lipids reduced the permeability to hydrogen ion by 34%, an 11% reduction was obtained with glycolipids, and 23% with phospholipids. Since neutral lipids account for 47% of the glycoprotein lipids, glycolipids 41.1% and phospholipids 11.9%, the quantitative decrease in permeability of the delipidated glycoprotein following its reassociation with phospholipids is 2.7 times greater than that of neutral lipids and 7.3 times greater than that of glycolipids.     

Effect of lipids and proteins on the viscosity of gastric mucus glycoprotein

The effect of associated lipids and covalently bound fatty acids, and the contribution of serum albumin and secretory IgA to the viscosity of dog gastric mucus glycoprotein was investigated. Using a cone/plate viscometer at shear rates between 1.15 - 230s -1, it was found that extraction of associated lipids from the glycoprotein lead to 80-85% decrease in the viscosity. Further loss (39%) in viscosity of the delipidated glycoprotein occurred following removal of covalently bound fatty acids. Reassociation of the delipidated glycoprotein with its neutral lipids increased the viscosity 3-fold, a 2.5-fold increase was obtained with glycolipids, and 2-fold with phospholipids. Preincubation of purified mucus glycoprotein with albumin or IgA resulted in the increase in viscosity. This increase in viscosity was proportional to albumin concentration up to 10%, and to IgA concentration up to 5%. The results show that interaction of lipids and proteins with mucus glycoprotein contributes significantly to the viscosity of gastric mucus.     

Hydrophobic interaction of fluorescent probes with fetuin, ovine submaxillary mucin, and canine tracheal mucins.

The presence of hydrophobic sites in fetuin, ovine submaxillary mucin and two homogeneous canine tracheal mucins was established by fluorescence probe techniques. The interaction between the above-mentioned glycoproteins and two hydrophobic fluorescent compounds, sodium mansate and mansylphenylalanine, was accompanied by an enhancement in fluorescence and a shift of the fluorescence maxima to shorter wavelengths. The introduction of a phenylalanine residue to the mansyl group enhanced the binding affinity of the probe for the hydrophobic sites of these glycoproteins as evidenced by lower values for the dissociation constants. The high molecular weight (581 600) tracheal mucin, which had the highest carbohydrate content (80%) of all the glycoproteins investigated, exhibited the highest fluorescence enhancement and the largest number of binding sites for these fluorescent probes.     

Only trace amounts of fatty acids are found in pure mucus glycoproteins

The presence of noncovalently associated lipids and covalently bound fatty acids was investigated in preparations of mucus glycoproteins obtained by using density-gradient centrifugation in CsCl/guanidinium chloride. No phospholipids, glycolipids, cholesterol, or triglycerides could be detected. However, small amounts of extractable fatty acids were consistently found, the sum of which ranged from 0.3 to 0.9 micrograms/mg of glycoprotein. The amount of fatty acid released after subsequent treatment with KOH ranged from 0 to 27 ng/mg of glycoprotein. We conclude that density-gradient centrifugation in CsCl/guanidinium chloride is very efficient in removing noncovalently associated lipids from mucus glycoproteins and that covalently bound fatty acids are probably not present in the macromolecules.     

The role of covalently bound fatty acids in the degradation of human gastric mucus glycoprotein

The undegraded high-molecular-weight glycoprotein of human gastric mucus has been isolated free of noncovalently bound proteins and lipids, as judged by gel filtration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cesium chloride density gradient centrifugation, and lipid analysis. Mild alkaline methanolysis of the thoroughly delipidated glycoprotein revealed that, on the average, the native undegraded glycoprotein contains 2.9 mol of acyl linked fatty acids/mg glycoprotein. The low-molecular-weight glycoprotein subunits, obtained after pepsin digestion, contain 2 nmol of acyl linked fatty acids/mg glycopeptide. The highest content of covalently bound fatty acids was found in the fraction of glycoprotein which remained undegraded after pepsin digestion. On the average, 10.2 mol of fatty acids/mg was substituted on this pepsin-resistant glycoprotein. After deacylation with hydroxylamine, the undegraded pepsin-resistant glycoprotein became susceptible to proteolytic cleavage. The obtained results suggest that fatty acids covalently bound to gastric mucus glycoprotein are involved in the regulation of proteolytic digestion of mucus glycoprotein in the stomach.     

Calcium binding to biliary mucins is dependent on sodium ion concentration: relevance to cystic fibrosis

Because hypersecretion of gallbladder (GB) mucus occurs in gallstone formation and because binding of Ca(2+) to biliary lipids only accounts for 50% of the total Ca(2+) in GB bile, we investigated the binding of Ca(2+) to human biliary mucin. Biliary mucin was purified from GB bile and binding to Ca(2+) studied. Scatchard plot analysis suggested two binding sites. Removal of sialic acid by neuraminidase resulted in 10% reduction of Ca(2+) binding, whereas, sulfatase treatment reduced Ca(2+) binding by 30%. Using a hypotonic NaCl solution, Ca(2+) binding to mucin increased curvilinearly with mucin concentration. However, binding decreased with increasing ionic strength of the NaCl solution. We conclude that binding of Ca(2+) to mucin is effected mainly through sulfate. Binding to Ca(2+) can be displaced by Na(+). Ca(2+) binding to mucins is enhanced in the setting of low Na(+) concentrations. This phenomenon has pathophysiologic implications for the formation of thick mucus in cystic fibrosis epithelia.     

Mucus glycoprotein secretion by duodenal mucosa in response to luminal arachidonic acid

The effect of luminal application of arachidonic acid on the alkaline secretion, prostaglandin generation, and mucus glycoprotein output and composition was studied in proximal and distal duodenum of conscious dogs. Surgically prepared duodenal loops were instilled in vivo for up to 2 h with saline (control) followed by various concentrations (12.5–100 μg/ml) of arachidonic acid. The experiments were conducted with and without intravenous pretreatment with indomethacin. The recovered instillates were assayed for the content of prostaglandin and HCO₃⁻, and used for the isolation of mucus glycoprotein. Exposure of duodenal mucosa to arachidonic acid led to concentration-dependent increase in the output of HCO₃⁻ and prostaglandin generation. In both cases this response was greater in the proximal duodenum. Pretreatment with indomethacin caused reduction in the basal HCO₃⁻ and prostaglandin output, and prevented the increments evoked by arachidonic acid. The proximal and distal duodenum displayed similar basal output and composition of mucus glycoprotein. Comparable increases in these glycoproteins were also obtained with arachidonic acid, the effect of which was abolished by indomethacin. Compared to basal conditions, mucus glycoproteins elaborated in response to arachidonic acid exhibited higher contents of associated lipids and covalently bound fatty acids, and contained less protein. The associated lipids of mucus glycoproteins elaborated in the presence of arachidonic acid showed enrichment in phospholipids and decrease in neutral lipids. The carbohydrate components in these glycoproteins also exhibited higher proportions of sialic acid and sulfate. The changes brought about by arachidonic acid were prevented by indomethacin pretreatment, and in both cases the glycoprotein composition returned to that obtained under basal conditions. The enrichment of mucus glycoprotein in lipids, sialic acid and sulfate in response to endogenous prostaglandin may be of significance to the function of this glycoprotein in the hostile environment of the duodenum.     

Separate sites of low and high affinity for agonists on Torpedo californica acetylcholine receptor

We have studied alkylation of the membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electric organ by the cholinergic agonist bromo-acetylcholine (BrAcCh). Following reduction of the AcChR with dithiothreitol (DTT) under strictly controlled conditions, a single class of binding sites was covalently labeled by BrAcCh. The extent of alkylation was dependent on the concentration of both DTT and BrAcCh and reached a maximum when a number of sites equivalent to the number of alpha-bungarotoxin (alpha-BTx) binding sites were labeled. The reaction with BrAcCh was completely inhibited by saturating concentrations of alpha-BTx. On the contrary, complete alkylation of the AcChR with [3H]BrAcCh consistently inhibited only approximately 50% of alpha-BTx binding. The effects of DTT reduction and subsequent BrAcCh alkylation on the cation-gating properties of the AcChR were investigated in rapid kinetic experiments. DTT reduction resulted in a slight decrease in the maximum cation flux and a small shift in the effective dissociation constant to higher acetylcholine (AcCh) concentration. The flux response was completely inhibited by maximal alkylation of the membrane vesicles by BrAcCh. A low-affinity binding site for AcCh, which is likely to be important in AcChR activation, has been revealed for T. californica AcChR by studying the effects of cholinergic ligands on the fluorescence of a probe, 4-[(iodoacetoxy)ethylmethylamino]-7-nitro-2,1,3-benzoxadiazole (IANBD), covalently bound to the AcChR protein. Maximal labeling by BrAcCh did not affect the binding of AcCh to the low-affinity binding site, as monitored by changes in the fluorescence of this probe. This low-affinity binding site must therefore be distinct from the site labeled by BrAcCh. The results strongly support the notion that the nicotinic AcChR contains multiple binding sites for cholinergic ligands.     

Sulfation in vitro of mucus glycoprotein by submandibular salivary gland: effects of prostaglandin and acetylsalicylic acid

Enzymatic sulfation of mucus glycoprotein by rat submandibular salivary gland and the effect of prostaglandin and acetylsalicylic acid on this process were investigated in vitro. The sulfotransferase enzyme which catalyzes the transfer of sulfate ester group from 3'-phosphoadenosine-5'-phosphosulfate to submandibular gland mucus glycoprotein has been located in the detergent extracts of Golgi-rich membrane fraction of the gland. Optimum enzyme activity was obtained at pH 6.8 with 0.5% Triton X-100, 25 mM NaF and 4 mM MgCl2, using the desulfated glycoprotein. The enzyme was also capable of sulfation of the intact mucus glycoprotein, but the acceptor capacity of such glycoprotein was 68% lower. The apparent Km of the submandibular gland sulfotransferase for salivary mucus glycoprotein was 11.1 microM. The 35S-labeled glycoprotein product of the enzyme reaction gave in CsCl density gradient a 35S-labeled peak which coincided with that of the glycoprotein. This glycoprotein upon reductive beta-elimination yielded several acidic 35S-labeled oligosaccharide alditols which accounted for 75% of the 35S-labeled glycoprotein label. Based on the analytical data, the two most abundant oligosaccharides were identified as sulfated tri- and pentasaccharides. The submandibular gland sulfotransferase activity was stimulated by 16,16-dimethyl prostaglandin E2 and inhibited by acetylsalicylic acid. The rate of enhancement of the glycoprotein sulfation was proportional to the concentration of prostaglandin up to 2.10(-5) M, at which point a 31% increase in sulfation was attained. The inhibition of the glycoprotein sulfation by acetylsalicylic acid was proportional to the drug concentration up to 2.5.10(-4) M at which concentration a 48% reduction in the sulfotransferase activity occurred. The apparent Ki value for sulfation of salivary mucus glycoprotein in presence of acetylsalicylic acid was 58.9 microM. The results suggest that prostaglandins may play a role in salivary mucin sulfation and that this process is sensitive to such nonsteroidal anti-inflammatory agents as acetylsalicylic acid.     

Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva

A high-molecular-weight mucin-glycoprotein (MG1) was isolated from human submandibular-sublingual saliva and was comprised of 14.9% protein, 29.0% N-acetylglucosamine, 9.4% N-acetylgalactosamine, 10.5% fucose, 24.2% galactose, 0.9% mannose, 4.0% N-acetylneuraminic acid, and 7.0% sulfate. Carbohydrate units were O-glycosidically linked and ranged in size from 4 to 16 residues. The biophysical properties of MG1 were compared to those of a smaller mucin (MG2) also isolated from submandibular-sublingual saliva. Fluorescence spectroscopy demonstrated that MG1 bound both 1-anilino-8-naphthalenesulfonate (ANS) and N-phenyl-1-naphthylamine (NPNA) in stable hydrophobic binding sites (melting temperature, 47 +/- 2 degrees C), whereas MG2 did not bind these hydrophobic probes. These hydrophobic domains occurred on nonglycosylated or naked portions of MG1 since Pronase treatment eliminated ANS binding. Reduction of disulfide bridges in MG1 increased the number of available hydrophobic binding sites. High ionic strength (0 to 2 M NaCl) had no effect on ligand binding, whereas lowering pH (9 to 2) increased ANS binding without affecting NPNA complexation. Circular dichroism (CD) data suggested that MG1's carbohydrate chains dominated its spectrum. In contrast, the peptide backbone dominated the CD spectrum of MG2. Collectively, the results of this study indicate that human submandibular-sublingual saliva contains two structurally distinct mucins.     

Investigation of spectrin binding to phospholipid vesicles using isoindole fluorescent probe. Thermal properties of the bound and unbound protein

Fluorescence of isoindole probe covalently bound to spectrin from pig erythrocytes, and fluorescence of tryptophanyl residues were used to study spectrin interaction with phospholipid bilayers. Evidence would be provided for conformational changes of spectrin occurring upon its binding to lipid bilayers. Fluorescence quenching experiments allowed to determine thermal stability of the protein in bound and unbound state. Spectrin binding to lipids was shown to protect the protein against thermal denaturation.     

Intestinal release of mucin in response to HCl and taurocholate: effect of indomethacin

1. Alkaline secretion and mucin output were analyzed along the gastrointestinal tract of a dog in response to luminal application of HCl and taurocholate with and without pretreatment with indomethacin. 2. Mucins derived from the different areas displayed similar contents of protein and carbohydrate but differed with respect to associated and covalently bound lipids. 3. Application of HCl or taurocholate in all the regions caused an increase in the output of mucins and HCO3-. However, mucins elaborated in response to HCl exhibited higher total lipid content and were richer in phospholipids. 4. Pretreatment with indomethacin prior to HCl application led to a reduction in HCO3- and caused a decrease in mucin phospholipid content, but had no effect on HCO3- secretion and the lipid content of mucins elaborated in response to taurocholate. 5. The results indicate that mucins elaborated along the gastrointestinal tract differ with respect to lipids, and that their output in response to HCl is mediated by prostaglandins.     

Role of carbohydrates in the viscosity and permeability of gastric mucin to hydrogen ion

The effect of carbohydrate removal on the viscosity of gastric mucin and its ability to impede the diffusion of hydrogen ion was investigated. The mucin, purified from dog gastric mucus, was subjected to partial or extensive deglycosylation with specific exoglycosidases and then used in the measurements. The obtained results revealed that removal of peripheral fucose or N-acetylglucosamine caused in each case only about 5% reduction of the glyco-protein viscosity. An 18% drop in the viscosity, however, occurred following removal of sialic acid, while extensive deglycosylation (removal of 86% carbohydrate) reduced the glycoprotein viscosity by 40%. The ability of mucin to retard the diffusion of hydrogen ion increased by 7% following removal of fucose or N-acetylgalactosamine, a 28% increase was obtained following removal of sialic acid, while the permeability to hydrogen ion of the extensively deglycosylated glycoprotein decreased by 42%. The results suggest that carbohydrates contribute significantly to the viscoelastic and permselective properties of gastric mucin.     

Interaction between calcofluor white and carbohydrates of alpha 1-acid glycoprotein.

Interactions between the fluorescent probe, calcofluor white, and human serum albumin (HSA) and alpha 1-acid glycoprotein (orosomucoid) are compared. The two proteins have comparable isoelectric points, but alpha 1-acid glycoprotein is highly glycosylated (40% of glycans by weight), while the serum albumin is not. Binding of calcofluor to the proteins induces an increase in both the fluorescence anisotropy and the fluorescence intensity of the fluorophore. Also, we found that the calcofluor exhibits a fluorescence emission with a maximum located at 432, 415 or 445 nm, respectively, in the absence of proteins, in the presence of HSA, and in the presence of alpha 1-acid glycoprotein. The stoichiometries of the calcofluor-serum albumin and calcofluor-alpha 1-acid glycoprotein complexes are 2:1 and 1:1, respectively. The association constants are 0.04 and 0.15 microM-1, respectively. The calcofluor does not interact with Lens culinaris agglutinin (LCA), although the protein has a hydrophobic site. Nevertheless, one cannot exclude that the binding of the fluorophore to the HSA is nonspecific. Our results, when compared with those obtained with calcofluor dissolved in the hydrophobic solvent isobutanol, and with the fluorescent probe, potassium 6-(p-toluidino)-2-naphthalenesulfonate (TNS), bound to alpha 1-acid glycoprotein, indicate that the emission of calcofluor bound to HSA occurs from a hydrophobic state, while that of calcofluor bound to alpha 1-acid glycoprotein occurs from a hydrophilic state. The fluorescence intensity of calcofluor decreases in the presence of carbohydrates isolated from alpha 1-acid glycoprotein, while it increases in the presence of alpha 1-cellulose. Thus, calcofluor interacts mainly with the glycan moiety of alpha 1-acid glycoprotein, and its fluorescence is sensitive to the secondary structure of the glycans.     

Clusterin, a binding protein with a molten globule-like region

Clusterin is a heterodimeric glycoprotein found in many tissues of the body and is the most abundant protein secreted by cultured rat Sertoli cells. The function of clusterin is unknown, but it has been associated with cellular injury, lipid transport, apoptosis, and it may be involved in the clearance of cellular debris caused by cell injury or death. Consistent with this last idea, clusterin has been shown to bind to a variety of molecules with high affinity including lipids, peptides, and proteins and the hydrophobic probe 1-anilino-8-naphthalenesulfonate (ANS). Given this variety of ligands, clusterin must have specific structural features that provide the protein with its promiscuous binding activity. Using sequence analyses, we show that clusterin likely contains three long regions of natively disordered or molten globule-like structures containing putative amphipathic alpha-helices. These disordered regions were highly sensitive to trypsin digestion, indicating a flexible nature. The effects of denaturation on the fluorescence of the clusterin-ANS complex were compared between proteins with structured binding pockets and molten globular forms of proteins. Clusterin bound ANS in a manner that was very similar to that of molten globular proteins. Furthermore, we found that, when bound to ANS, at least one cleavage site within the protease-sensitive disordered regions of clusterin was protected from trypsin digestion. In addition, we show that clusterin can function as a biological detergent that can solubilize bacteriorhodopsin. We propose that natively disordered regions with amphipathic helices form a dynamic, molten globule-like binding site and provide clusterin the ability to bind to a variety of molecules.     

Steady-state fluorescence emission from the fluorescent probe, 5-iodoacetamidofluorescein, bound to hemoglobin

In the past, fluorescence emission from an extrinsic fluorophore bound to heme-proteins would only be studied with the removal of the heme since fluorescence from the fluorophore could not be detected using right-angle optics. Using front-face fluorometry, a significant steady state emission signal originating from the probe bound to hemoglobin is detected. This is the first report of the detection of extrinsic fluorescence of a probe bound to a heme-protein. We also demonstrate that the extrinsic probe, 5-iodoacetamidofluorescein, is covalently bound to hemoglobin, specifically at beta 93 Cysteine. Ligand binding results in a change in the fluorophore fluorescence intensity as predicted by hemoglobin crystallographic studies. Efficiency of energy transfer measurements are made.     

Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet

The P-glycoprotein multidrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic drugs, natural products, and peptides. Multiple binding sites for substrates appear to exist, most likely within the hydrophobic membrane spanning regions of the protein. Since ATP hydrolysis is coupled to drug transport, the spatial relationship of the drug binding sites relative to the ATPase catalytic sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the drug binding site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 binding site is likely located 10-14 A below the membrane surface, within the cytoplasmic leaflet of the membrane, in both resting-state and transition-state P-glycoprotein.