Synthesis of trilobatin from naringin via prunin as the key intermediate: acidic hydrolysis of the α-rhamnosidic linkage in naringin under improved conditions.

Trilobatin [4'-(ß-D-glucopyranosyloxy)-2',4",6'-trihydroxydihydrochalcone] was synthesized from commercially available naringin in three steps with an overall yield of 30%. The key step was the acid-catalyzed site-selective hydrolysis of terminal α-rhamnopyranosidic linkage in neohesperidose involved in naringin under controlled conditions, by applying a high-pressure steam sterilizer.

UPLC-PDA quantification of chemical constituents of two different varieties (golden and royal) of apple leaves and their antioxidant activity.

BACKGROUND: Malus domestica is the most widely cultivated fruit tree and is well known for its therapeutic value. Apple leaves are known to contain phenolic compounds but the nature of these has not been explored to the same extent as in apple fruit. A simple, rapid and sensitive ultra-performance liquid chromatography-diode array detection (UPLC-DAD) quantification method has been developed. Total polyphenol and flavonoid contents, as well as the antioxidant activity of golden and royal apple leaves were evaluated. RESULTS: Four compounds, namely rutin, 3-hydroxyphloridzin, phloridzin and quercetin-3-O-arabinoside were identified by UPLC. The separation was achieved in less than 7 min. Total polyphenol and flavonoid contents were found to be slightly higher in apple golden variety than royal variety. The IC50 values determined by the DPPH assay were 49.94 µg mL(-1) for golden apple leaves and 43.89 µg mL(-1) for royal apple leaves. IC50 values determined by the ABTS assay were 47.10 and 66.53 µg mL(-1) for golden and royal apple leaves, respectively. Antioxidant activity was determined as 24.45 and 21.15 mg ascorbic acid g(-1) for golden and royal apple leaves, respectively, by using the FRAP assay. CONCLUSION: This study showed that apple leaves (both varieties) contain considerable amounts of polyphenols and flavonoids and are also a promising source of phloridzin.

Efficient biosynthesis of prunin in methanol cosolvent system by an organic solvent-tolerant α-L-rhamnosidase from Spirochaeta thermophila.

Prunin of desirable bioactivity and bioavailability can be transformed from plant-derived naringin by the key enzyme α-L-rhamnosidase. However, the production was limited by unsatisfactory properties of α-L-rhamnosidase such as thermostability and organic solvent tolerance. In this study, biochemical characteristics, and hydrolysis capacity of a novel α-L-rhamnosidase from Spirochaeta thermophila (St-Rha) were investigated, which was the first characterized α-L-rhamnosidase for Spirochaeta genus. St-Rha showed a higher substrate specificity towards naringin and exhibited excellent thermostability and methanol tolerance. The Km of St-Rha in the methanol cosolvent system was decreased 7.2-fold comparing that in the aqueous phase system, while kcat/Km value of St-Rha was enhanced 9.3-fold. Meanwhile, a preliminary conformational study was implemented through comparative molecular dynamics simulation analysis to explore the mechanism underlying the methanol tolerance of St-Rha for the first time. Furthermore, the catalytic ability of St-Rha for prunin preparation in the 20% methanol cosolvent system was explored, and 200 g/L naringin was transformed into 125.5 g/L prunin for 24 h reaction with a corresponding space-time yield of 5.2 g/L/h. These results indicated that St-Rha was a novel α-L-rhamnosidase suitable for hydrolyzing naringin in the methanol cosolvent system and provided a better alternative for improving the efficient production yield of prunin.

Prunin from Poncirus trifoliata (L.) Rafin Inhibits Aldose Reductase and Glucose-Fructose-Mediated Protein Glycation and Oxidation of Human Serum Albumin.

Diabetes complications are associated with aldose reductase (AR) and advanced glycation end products (AGEs). Using bioassay-guided isolation by column chromatography, 10 flavonoids and one coumarin were isolated from Poncirus trifoliata Rafin and tested in vitro for an inhibitory effect against human recombinant AR (HRAR) and rat lens AR (RLAR). Prunin, narirutin, and naringin inhibited RLAR (IC50 0.48-2.84 µM) and HRAR (IC50 0.68-4.88 µM). Docking simulations predicted negative binding energies and interactions with the RLAR and HRAR binding pocket residues. Prunin (0.1 and 12.5 µM) prevented the formation of fluorescent AGEs and nonfluorescent Nε-(carboxymethyl) lysine (CML), as well as the fructose-glucose-mediated protein glycation and oxidation of human serum albumin (HSA). Prunin suppressed the formation of the ß-cross-amyloid structure of HSA. These results indicate that prunin inhibits oxidation-dependent protein damage, AGE formation, and AR, which may help prevent diabetes complications.

[Quantitative analysis of six flavonoids of hongzhu capsule by QAMS].

OBJECTIVE: To establish a method of quality assurance for mass-screening (QAMS) for simultaneously determining six flavonoids in Hongzhu Capsules. METHODS: An HPLC (high performance liquid chromatography) method was developed as QAMS to determine neoeriocitrin, prunin, naringin, rhoifolin, miliditin and naringenin in Hongzhu Capsules. Using naringin as the internal reference substance, relative correction factors (RCF) of the five flavonoids were determined and given as follows (1.05, 0.782, 1.89, 1.27, 0.532). The contents of the flavonoids in Hongzhu Capsules were determined by QAMS and validated by the external standard method. RESULTS: RCF values were determined by HPLC with good reproducibility. No significant difference between the quantitative results of QAMS and external standard method was observed. CONCLUSION: The present-developed method is convenient and accurate to determine multiple components when some standard substances are unavailable, thus considered as a potential method for quality control of Hongzhu Capsules.

Synthesis, antioxidant and antimicrobial activity of a new phloridzin derivative for dermo-cosmetic applications.

The phenolic compound phloridzin (phloretin 2′-O-glucoside, variously named phlorizin, phlorrhizin, phlorhizin or phlorizoside) is a prominent member of the chemical class of dihydrochalcones, which are phenylpropanoids. Phloridzin is specifically found in apple and apple juice and known for its biological properties. In particular we were attracted by potential dermo-cosmetic applications. Here we report the synthesis, stability studies and antimicrobial activity of compound F2, a new semi-synthetic derivative of phloridzin. The new derivative was also included in finished formulations to evaluate its stability with a view to a potential topical use. Stability studies were performed by HPLC; PCL assay and ORAC tests were used to determine the antioxidant activity. F2 presented an antioxidant activity very close to that of the parent phloridzin, but, unlike the latter, was more stable in formulations. To further explore potential health claims, antifungal activity of phloridzin and its derivative F2 were determined; the results, however, were rather low; the highest value was 31,6% of inhibition reached by F2 on Microsporum canis at the highest dose.

Benzyl benzoates: New phlorizin analogs as mushroom tyrosinase inhibitors.

In our research, 14 benzyl benzoates with hydroxyl(s) (3-16) were synthesized and their inhibitory activity on mushroom tyrosinase was tested. Results indicated that among these compounds, 4-hydroxybenzyl 3,5-dihydroxybenzoate (3), 4-hydroxybenzyl 2,4-dihydroxybenzoate (5), 4-hydroxybenzyl 2,4,6-dihydroxybenzoate (7), 3-hydroxybenzyl 3,5-dihydroxybenzoate (8), 3-hydroxybenzyl 2,4-dihydroxybenzoate (10) exhibited inhibitory activity with their IC(50) less than 10µM. Further studies showed these five compounds were competitive inhibitors of tyrosinase and their structure-activity relationships were investigated in this article.

Antibacterial activity of naringin derivatives against pathogenic strains.

AIMS: To study the antimicrobial activity of naringin (NAR), a flavonoid extracted from citrus industry waste, and NAR derivatives [naringenin (NGE), prunin and alkyl prunin esters] against pathogenic bacteria such as L. monocytogenes, E. coli O157:H7 and S. aureus. The relationship between the structure of the chemical compounds and their antagonistic effect was also analysed. METHODS AND RESULTS: The agar dilution technique and direct contact assaying were applied. NGE, prunin and NAR showed no antimicrobial activity at a concentration of 0.25 mmol l(-1). Similarly, fatty acids with a chain length between C2 and C18 showed no antimicrobial activity at the same concentration. However, prunin-6″-O-acyl esters presented high antibacterial activity, mainly against Gram-positive strains. This activity increased with increasing chain length (up to 10-12 carbon atoms). Alkyl prunin esters with 10-12 carbon atoms diminished viability of L. monocytogenes by about 3 log orders and S. aureus by 6 log orders after 2 h of contact at 37°C and at a concentration of 0.25 mmol l(-1). The compounds examined were not effective against any of the Gram-negative strains assayed, even at the highest concentration. CONCLUSIONS: Addition of sugars to the aglycone did not enhance its antimicrobial activity. Attachment of a saturated aliphatic chain with 10-12 carbon atoms to the A ring of the flavonoid (or to sugars attached to this ring), seems to be the most promising modification. In conclusion, alkyl prunin esters with a chain length of C10-C12 have promising features as antimicrobial agents because of their high antilisterial and antistaphylococcal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that it is possible to obtain NAR derivatives with important antimicrobial activity, especially against Gram-positive pathogenic bacteria. It also provides guidelines on the structural modifications in similar molecules to enhance the antimicrobial activity.

SGLT inhibitors as new therapeutic tools in the treatment of diabetes.

Recently, the idea has been developed to lower blood glucose blood glucose levels in diabetes by inhibiting sugar reabsorption sugar reabsorption in the kidney kidney . The main target is thereby the early proximal tubule proximal tubule where secondary active transport secondary active transport of the sugar is mediated by the sodium-D: -glucose D-glucose cotransporter SGLT2 SGLT2 . A model substance for the inhibitors inhibitors is the O-glucoside O-glucoside phlorizin phlorizin which inhibits transport transport competitively. Its binding to the transporter involves at least two different domains: an aglucone binding aglucone binding site at the transporter surface, involving extramembranous loops extramembraneous loops , and the sugar binding sugar binding /translocation site buried in a hydrophilic pocket of the transporter. The properties of these binding sites differ between SGLT2 and SGLT1 SGLT1 , which mediates sugar absorption sugar absorption in the intestine intestine . Various O-, C-, N- and S-glucosides have been synthesized with high affinity affinity and high specificity specificity for SGLT2 SGLT2 . Some of these glucosides are in clinical trials clinical trials and have been proven to successfully increase urinary glucose excretion urinary glucose excretion and to decrease blood sugar blood sugar levels without the danger of hypoglycaemia hypoglycaemia during fasting fasting in type 2 diabetes type 2 diabetes .

Immobilization of GH78 α-L-Rhamnosidase from Thermotoga petrophilea with High-Temperature-Resistant Magnetic Particles Fe3O4-SiO2-NH2-Cellu-ZIF8 and Its Application in the Production of Prunin Form Naringin.

To efficiently recycle GH78 thermostable rhamnosidase (TpeRha) and easily separate it from the reaction mixture and furtherly improve the enzyme properties, the magnetic particle Fe3O4-SiO2-NH2-Cellu-ZIF8 (FSNcZ8) was prepared by modifying Fe3O4-NH2 with tetraethyl silicate (TEOS), microcrystalline cellulose and zinc nitrate hexahydrate. FSNcZ8 displayed better magnetic stability and higher-temperature stability than unmodified Fe3O4-NH2 (FN), and it was used to adsorb and immobilize TpeRha from Thermotoga petrophilea 13995. As for properties, FSNcZ8-TpeRha showed optimal reaction temperature and pH of 90°C and 5.0, while its highest activity approached 714 U/g. In addition, FSNcZ8-TpeRha had better higher-temperature stability than FN. After incubation at 80°C for 3 h, the residual enzyme activities of FSNcZ8-TpeRha, FN-TpeRha and free enzyme were 93.5%, 63.32%, and 62.77%, respectively. The organic solvent tolerance and the monosaccharides tolerance of FSNcZ8-TpeRha, compared with free TpeRha, were greatly improved. Using naringin (1 mmol/l) as the substrate, the optimal conversion conditions were as follows: FSNcZ8-TpeRha concentration was 6 U/ml; induction temperature was 80°C; the pH was 5.5; induction time was 30 min, and the yield of products was the same as free enzyme. After repeating the reaction 10 times, the conversion of naringin remained above 80%, showing great improvement of the catalytic efficiency and repeated utilization of the immobilized α-L-rhamnosidase.

Antimicrobial properties of prunin, a citric flavanone glucoside, and its prunin 6″-O-lauroyl ester.

AIMS: To determine the antimicrobial potential of prunin (P), a flavanone glucoside resulting from the hydrolysis of naringin present in grapefruit, and of its prunin 6″-O-lauroyl ester (PL), synthesized by enzymatic catalysis. METHODS AND RESULTS: P and its lauroyl ester were tested against Gram-negative and Gram-positive bacteria, yeasts and moulds. P showed no inhibitory effect against the micro-organisms assayed, but stimulated growth of Pseudomonas aeruginosa and different Bacilllus sp. However, 150 µg ml(-1) of PL inhibited Escherichia coli, Salmonella enterica serovar Enteritidis, Salmonella enterica serovar Typhimurium, many Bacillus sp., Staphylococcus aureus ATCC29213, Enterococcus avium DSMZ17511, and different Listeria monocytogenes strains. In the last case, L. monocytogenes, sensitive or bacteriocin-resistant cells, lost nearly 4-log reductions after 30 min of contact. A bactericidal mode of action was determined using both scanning and transmission electronic microscopies. CONCLUSIONS: PL could be used as a food additive, because at low concentration (150 µg ml(-1)) it exhibited antimicrobial activity against important food-borne pathogens. A bactericidal effect was also determined on L. monocytogenes sensitive and bacteriocin-resistant mutant strains. P did not show any antimicrobial property at all. SIGNIFICANCE AND IMPACT OF THE STUDY: PL is a potential antimicrobial compound with a high anti-Listeria property.

Synthesis and biological evaluation of phloridzin analogs as human concentrative nucleoside transporter 3 (hCNT3) inhibitors.

Nucleoside transporter inhibitors have potential therapeutic applications as anticancer, antiviral, cardioprotective and neuroprotective agents. Although quite a few potent inhibitors of the equilibrative nucleoside transporters are known, largely missing are the concentrative nucleoside transporter inhibitors. Phloridzin (3, K(i)=16.00 microM) is a known moderate inhibitor of the concentrative nucleoside transporters. We have synthesized and evaluated analogs of phloridzin at the hCNT3 nucleoside transporter. Within the series of synthesized analogs compound 16 (K(i)=2.88 microM), possessing a ribofuranose sugar unit instead of a glucopyranose as present in phloridzin, exhibited the highest binding affinity at the hCNT3 transporter. Phloridzin and compound 16 have also been shown to be selective for the hCNT3 transporter as compared with the hENT1 transporter. Compound 16 can serve as a new lead which after further modifications could yield selective and potent hCNT3 inhibitors.

Novel natural and synthetic inhibitors of solute carriers SGLT1 and SGLT2.

Selective analogs of the natural glycoside phloridzin are marketed drugs that reduce hyperglycemia in diabetes by inhibiting the active sodium glucose cotransporter SGLT2 in the kidneys. In addition, intestinal SGLT1 is now recognized as a target for glycemic control. To expand available type 2 diabetes remedies, we aimed to find novel SGLT1 inhibitors beyond the chemical space of glycosides. We screened a bioactive compound library for SGLT1 inhibitors and tested primary hits and additional structurally similar molecules on SGLT1 and SGLT2 (SGLT1/2). Novel SGLT1/2 inhibitors were discovered in separate chemical clusters of natural and synthetic compounds. These have IC50-values in the 10-100 µmol/L range. The most potent identified novel inhibitors from different chemical clusters are (SGLT1-IC50 Mean ± SD, SGLT2-IC50 Mean ± SD): (+)-pteryxin (12 ± 2 µmol/L, 9 ± 4 µmol/L), (+)-ε-viniferin (58 ± 18 µmol/L, 110 µmol/L), quinidine (62 µmol/L, 56 µmol/L), cloperastine (9 ± 3 µmol/L, 9 ± 7 µmol/L), bepridil (10 ± 5 µmol/L, 14 ± 12 µmol/L), trihexyphenidyl (12 ± 1 µmol/L, 20 ± 13 µmol/L) and bupivacaine (23 ± 14 µmol/L, 43 ± 29 µmol/L). The discovered natural inhibitors may be further investigated as new potential (prophylactic) agents for controlling dietary glucose uptake. The new diverse structure activity data can provide a starting point for the optimization of novel SGLT1/2 inhibitors and support the development of virtual SGLT1/2 inhibitor screening models.

Prunin suppresses viral IRES activity and is a potential candidate for treating enterovirus A71 infection.

Human enterovirus A71 (HEVA71) causes hand, foot, and mouth disease (HFMD) in young children and is considered a major neurotropic pathogen but lacks effective antivirals. To identify potential therapeutic agents against HFMD, we screened a 502-compound flavonoid library for compounds targeting the HEVA71 internal ribosome entry site (IRES) that facilitates translation of the HEVA71 genome and is vital for the production of HEVA71 viral particles. We validated hits using cell viability and viral plaque assays and found that prunin was the most potent inhibitor of HEVA71. Downstream assays affirmed that prunin disrupted viral protein and RNA synthesis and acted as a narrow-spectrum antiviral against enteroviruses A and B, but not enterovirus C, rhinovirus A, herpes simplex 1, or chikungunya virus. Continuous HEVA71 passaging with prunin yielded HEVA71-resistant mutants with five mutations that mapped to the viral IRES. Knockdown studies showed that the mutations allowed HEVA71 to overcome treatment-induced suppression by differentially regulating recruitment of the IRES trans-acting factors Sam68 and hnRNPK without affecting the hnRNPA1-IRES interaction required for IRES translation. Furthermore, prunin effectively reduced HEVA71-associated clinical symptoms and mortality in HEVA71-infected BALB/c mice and suppressed hepatitis C virus at higher concentrations, suggesting a similar mechanism of prunin-mediated IRES inhibition for both viruses. These studies establish prunin as a candidate for further development as a HEVA71 therapeutic agent.

Prunin is a highly potent flavonoid from Prunus davidiana stems that inhibits protein tyrosine phosphatase 1B and stimulates glucose uptake in insulin-resistant HepG2 cells.

Prunin is the main flavonoid in Prunus davidiana stems and improves hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats. The aim of this study was to investigate the in vitro anti-diabetic potential of prunin via the inhibition of protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, peroxynitrite (ONOO-)-mediated tyrosine nitration, and stimulation of glucose uptake in insulin-resistant hepatocytes. In addition, a molecular docking simulation was performed to predict specific prunin binding modes during PTP1B inhibition. Prunin showed strong inhibitory activity against PTP1B, with an IC50 value of 5.5 ± 0.29 µM, and significant inhibitory activity against α-glucosidase, with an IC50 value of 317 ± 2.12 µM. Moreover, a kinetics study revealed that prunin inhibited PTP1B (K i = 8.66) and α-glucosidase (K i = 189.56) with characteristics typical of competitive and mixed type inhibitors, respectively. Docking simulations showed that prunin selectively inhibited PTP1B by targeting its active site and exhibited good binding affinity, with a docking score of -9 kcal/mol. Furthermore, prunin exhibited dose-dependent inhibitory activity against ONOO--mediated tyrosine nitration and stimulated glucose uptake by decreasing PTP1B expression level in insulin-resistant HepG2 cells. These results indicate that prunin has significant potential as a selective PTP1B inhibitor and may possess anti-diabetic properties by improving insulin resistance.

Photo-activated inhibition of sulfate equilibrium exchange in human erythrocyte ghosts by a 4-azido-2-nitrobenzoate derivative of phlorizin.

Like phlorizin, two glycosidic esters of phlorizin, the 4-azido-2-nitrobenzoate (ANB-phlorizin) and the 2-nitrobenzoate (NB-phlorizin) were found to be effective inhibitors of SO42- equilibrium exchange at the outer but not at the inner membrane surface of the human erythrocyte ghost. After photolysis of ghost suspensions in the presence of extracellular ANB-phlorizin an irreversible inhibition of SO42- exchange was observed, while photolysis of intracellular ANB-phlorizin was without effect. After photolysis in the presence of extracellular or intracellular tritiated ANB-phlorizin gel electrophoresis of the labelled membranes revealed similar locations of binding. These findings suggest that the sidedness of action of ANB-phlorizin could not be related to inaccessibility of the inner membrane surface for the agent but that inhibition occurs via binding to fixed sites at the outer membrane surface that are not associated with a mobile carrier which crosses the membrane.

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.

Synthesis of phlorizin derivatives and their inhibitory effect on the renal sodium/D-glucose cotransport system.

To characterize further the Na+/D-glucose cotransport system in renal brush border membranes, phlorizin - a potent inhibitor of D-glucose transport - has been chemically modified without affecting the D-glucose moiety or changing the side groups that are essential for the binding of phlorizin to the Na+/D-glucose cotransport system. One series of chemical modifications involved the preparation of 3-nitrophlorizin and the subsequent catalytic reduction of the nitro compound to 3-aminophlorizin. From 3-aminophlorizin, 3-bromoacetamido-, 3-dansyl- and 3-azidophlorizin have been synthesized. In another approach, 3'-mercuryphlorizin was obtained by reaction of phlorizin with Hg(II) acetate. The phlorizin derivatives inhibit sodium-dependent but not sodium-independent D-glucose uptake by hog renal brush border membrane vesicles in the following order of potency: 3'-mercuryphlorizin = phlorizin greater than 3-aminophlorizin greater than 3-bromoacetamidophlorizin greater than 3-azidophlorizin greater than 3-nitrophlorizin greater than 3-dansylphlorizin. 3-Bromoacetamidophlorizin - a potential affinity label - also inhibits sodium-dependent but not sodium-independent phlorizin binding to brush border membranes. In addition, sodium-dependent phosphate and sodium-dependent alanine uptake are not affected by 3-bromoacetamidophlorizin. The results described above indicate that specific modifications of the phlorizin molecule at the A-ring or B-ring are possible that yield phlorizin derivatives with a high affinity and high specificity for the renal Na+/D-glucose cotransport system. Such compounds should be useful in future studies using affinity labeling (3-bromoacetamido- and 3-azidophlorizin) or fluorescent probes (3-dansylphlorizin).

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.

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.