Sodium-induced conformational changes in the glucose transporter of intestinal brush borders

Using brush-border membranes prepared from rabbit small intestine by Ca2+ precipitation and KSCN treatment, we have studied the kinetics and conformational changes of the glucose carrier. Na+ behaves as a competitive activator of glucose transport under zero-trans conditions. Phenyl isothiocyanate and fluorescein isothiocyanate (FITC) inhibit Na+-dependent transport in an irreversible but substrate-protectable manner. Vesicles pretreated with phenyl isothiocyanate in the presence of substrates were then selectively labeled at the glucose carrier with FITC. Competition experiments with Na+ and phlorizin or glucose indicated that FITC binds to the glucose site on the carrier. Carrier-bound FITC displays a saturable quenching of fluorescence in the presence of Na+. The K0.5 of the Na+-specific quench is 25 mM, which is similar to the apparent Km for Na+ activation of glucose transport. Two tyrosine group-specific reagents, N-acetylimidazole and tetranitromethane, inhibit glucose uptake and fluorescent quenching in a Na+-protectable fashion. FITC labeled a 75-kilodalton peptide on sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a substrate-sensitive manner. We conclude that Na+ binds to the glucose symporter of intestinal brush borders, a 75-kilodalton peptide, and this induces a rapid conformation change in the transporter which increases its affinity for D-glucose.     

Identification and conformational changes of the intestinal proline carrier

Fluorescein isothiocyanate (FITC) was used to selectively label the rabbit intestinal brush-border imino carrier, identify the binding protein on SDS-polyacrylamide gel electrophoresis, and monitor the effect of ions on fluorescein quenching. FITC inhibits Na+-dependent L-proline transport irreversibly, but transport is protected by physiological concentrations of Na+ and L-proline. About 1 nmol of FITC/mg of protein binds specifically to the transporter, which was identified by SDS-polyacrylamide gel electrophoresis as a 100 +/- 5-kDa peptide. Na+ produced a specific, saturable quench in the fluorescence of FITC bound to the proline carrier. Both transport and FITC quenching are inhibited by n-acetylimidazole, and membranes are protected from acetylation by Na+. We conclude that Na+ binds to the proline carrier (100-kDa peptide) to produce a change in conformation that results in an increase in the affinity of the carrier for proline.     

A bioresorbable, polylactide reservoir for diffusional and osmotically controlled drug delivery

The purpose of this study was to design and characterize a zero-order bioresorbable reservoir delivery system (BRDS) for diffusional or osmotically controlled delivery of model drugs including macromolecules. The BRDS was manufactured by casting hollow cylindrical poly (lactic acid) (PLA): polyethylene glycol (PEG) membranes (10 x 1.6 mm) on a stainless steel mold. Physical properties of the PLA:PEG membranes were characterized by solid-state thermal analysis. After filling with drug (5 fluorouracil [5FU] or fluorescein isothiocyanate [FITC]-dextran:mannitol, 5:95 wt/wt mixture) and sealing with viscous PLA solution, cumulative in vitro dissolution studies were performed and drug release monitored by ultraviolet (UV) or florescence spectroscopy. Statistical analysis was performed using Minitab (Version 12). Differential scanning calorimetry thermograms of PLA:PEG membranes dried at 25 degrees C lacked the crystallization exotherms, dual endothermal melting peaks, and endothermal glass transition observed in PLA membranes dried at -25 degrees C. In vitro release studies demonstrated zero-order release of 5FU for up to 6 weeks from BRDS manufactured with 50% wt/wt PEG (drying temperature, 25 degrees C). The release of FITC dextrans of molecular weights 4400, 42 000, 148 000, and 464 000 followed zero-order kinetics that were independent of the dextran molecular weight. When monitored under different concentrations of urea in the dissolution medium, the release rate of FITC dextran 42 000 showed a linear correlation with the calculated osmotic gradient(DeltaPi). This study concludes that PEG inclusion at 25 degrees C enables manufacture of uniform, cylindrical PLA membranes of controlled permeability. The absence of molecular weight effects and a linear dependence of FITC-dextran release rate on DeltaPi confirm that the BRDS can be modified to release model macromolecules by an osmotically controlled mechanism.     

Structure of the IgG-binding ligand of the T-gel

We examine the ligand requirements for the divinylsulphone (DVS) based T-gel to bind immunoglobulins. The original gel consisted of 2-mercaptoethanol coupled to a DVS activated support, with both the thioether and sulphone sulphurs thought necessary for protein binding. No differences in the capacity for human IgG were observed for a highly activated gel coupled with mercaptoethanol, or when the same activated gel was incubated at high pH to hydrolyse the majority of its reactive groups before the remainder were coupled with the thiol, indicating that the thioether S may be replaced with a hydroxyl O. Increasing the time of the DVS-activation results in gels with higher concentrations of immobilised sulphone but lower concentrations of active groups. The IgG capacities of the mercaptoethanol coupled gels were found to increase with the time of the activation reaction, which may be exploited to produce high capacity gels while minimising the concentration of DVS. Reducing the vinyl of the DVS-activated gel with borohydride was found to decrease the amount of protein binding, with residual binding being attributed to the presence of hydrolysed or cross-linked sulphones in the gel. Reacting the activated gels with amines decreased the capacity for IgG still further, suggesting that not only are these ligands unable to bind IgG, they also prevent its interacting with neighbouring sulphones, perhaps due to the small amount of positive charge they carry.     

Monoclonal antibodies that bind the renal Na+/glucose symport system. 2. Stabilization of an active conformation

Conformation-dependent fluorescein isothiocyanate (FITC) labeling of the pig renal Na+/glucose symporter was investigated with specific monoclonal antibodies (MAb's). When renal brush border membranes were pretreated with phenyl isothiocyanate (PITC), washed, and then treated at neutral pH with FITC in the presence of transporter substrates Na+ and glucose, most of the incorporated fluorescence was associated with a single peak after resolution by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular mass of the FITC-labeled species ranged from 79 to 92 kDa. Labeling of this peak was specifically reduced by 70% if Na+ and glucose were omitted. Na+ could not be replaced by K+, Rb+, or Li+. FITC labeling of this peak was also stimulated after incubation of membranes with MAb's known to influence high-affinity phlorizin binding, and stimulation was synergistically increased when MAb's were added in the presence of Na+ and glucose. Substrate-induced or MAb-induced labeling correlated with inactivation of Na+-dependent phlorizin binding. MAb's recognized an antigen of 75 kDa in the native membranes whereas substrate-induced FITC labeling was accompanied by loss of antigen recognition and protection from proteolysis. These findings are consistent with a model in which MAb's stabilize a Na+-induced active conformer of the Na+/glucose symport system.     

Fluorescence measurements of immune complexes of Mab 4-4-20 with isomeric haptens.

Relative differences in the active site environment of a monoclonal antibody when covalently bound to two isomeric haptens were studied using fluorescence quenching and lifetime measurements. Murine monoclonal antibody 4-4-20, a well-characterized high affinity antifluorescein antibody, served as the model IgG protein. Isomeric haptenic probes comparatively studied were fluorescein-5-isothiocyanate (FITC I, the immunogen) and fluorescein-6-isothiocyanate (FITC II). In kinetic binding studies, the association rate for the interaction of 4-4-20 with FITC I was greater than 2,000 times faster than the reaction with FITC II. Fluorescence lifetimes for FITC I covalently bound to 4-4-20 were 3.89 ns and 0.37 ns, indicative of hapten bound outside and inside the active site, respectively. Fluorescence lifetime for FITC II within the active site was indistinguishable from bound FITC I, indicating that interactions with active site residues which resulted in a decreased lifetime were similar for both isomers. A decreased lifetime for active site bound FITC I was consistent with the 90-95% quenching of fluorescein fluorescence. Dynamic fluorescence quenching experiments with iodide and FITC I in the active site showed no solvent accessibility, whereas bound FITC II showed significant accessibility. These results suggest that the difference in bond angle which accompanies binding of isomer II relative to isomer I within the active site probably leads to steric constraints resulting in a more open configuration of the 4-4-20 active site.     

Striated myofibrils in anti-myosin stained, isolated chicken gizzard smooth muscle cells.

Highly purified chicken gizzard myosin was used to induce antibody production in rabbits. The IgG fraction was separated from the antisera and coupled to fluorescein isothiocyanate (FITC). Specific antibody (AGM) was isolated from the IgG fraction by affinity purification. Comparisons of the specificity of IgG and AGM for chicken smooth muscle myosin revealed a much greater specificity by AGM. Staining with IgG led to an apparent cross-reactivity with guinea pig smooth muscles which was not seen with AGM staining. Therefore, staining of cells for localization of myosin was performed with AGM. Isolated cells were obtained from chicken gizzards either by collagenase digestion or by agitation of glycerinated pieces. Stained cells and cell fragments revealed the presence of myofibrils as structural units with diameters of about 1.0 micrometer. Stained myofibrils occasionally displayed regular banding patterns with a repeating period of about 1.5 +/- 0.2 micrometer. The presence of banded myofibrils in non-cultured cells shows that the organization of the contractile material is similar to that previously reported for cultured cells by Gr?schel-Stewart.     

Flow cytometric quantification of electroporation-mediated uptake of macromolecules into plant protoplasts

Summary Flow cytometry was used to provide a rapid and accurate assessment of electroporation-induced uptake of macromolecules into plant protoplasts. Rice protoplasts were electroporated in the presence of fluorescein isothiocyanate-conjugated dextran (FITC-dextran). After washing, the protoplasts were resuspended in a solution containing propidium iodide which intercalates with DNA, but which is excluded by an intact plasma membrane. Electroporation in the presence of FITC-dextran gave rise to populations of protoplasts that fluoresced green or yellow due to the presence of non-conjugated FITC. Non-viable protoplasts fluoresced red because of their inability to exclude propidium iodide molecules. Flow cytometry was used to resolve and quantify these protoplast populations and thus identify optimal conditions for macromolecule uptake. A direct relationship was observed between FITC-dextran uptake and transient gene expression following plasmid uptake. Thus, simultaneous electroporation of protoplasts with foreign DNA and FITC-dextran followed by fluorescence activated cell sorting may permit partial selection of transformed cells and so reduce the need for a selectable marker.Abbreviations ADC analogue to digital converter - CAT chloramphenicol acetyl transferase (enzyme) - cat chloramphenicol acetyl transferase (gene) - CPW solution cell and protoplast wash solution - DC direct current - EF electrofusion - FALS forward angle light scatter - FITC fluorescein isothiocyanate - FITC-dextran fluorescein isothiocyanate conjugated dextran - PI propidium iodide - PMT photomultipliertube - TLC thin layer chromatography     

Enhanced chemiluminescence ELISA for Listeria specific antigen in cerebrospinal fluid using an FITC-anti-FITC system

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELISA an anti-Listeria monoclonal antibody, immobilized onto assay wells, was used to capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody to FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients.     

Generation and evaluation of anti-mouse IgG IgY as secondary antibody

Abstract

In order to evaluate the possibility of using IgY as the secondary antibody in immunoassay, specific IgY (1: 128,000) was generated by immunizing hens with mouse serum IgG purified by protein A column. IgY was extracted from egg yolk by polyethylene glycol 6000 (PEG-6000), and further purified using protein M affinity chromatography column. The purified IgY was conjugated with horseradish peroxidase (HRP) and fluorescein?isothiocyanate (FITC), in that order. The reactivity of conjugated antibodies was evaluated by ELISA, Western blot and Immunofluorescence, demonstrating that the obtained IgY was able to conjugate with enzymes, react with mouse primary IgG antibody, and subsequently amplify the antigen-antibody signals in different immune reaction conditions, in a comparable secondary effect to conventional goat anti-mouse IgG antibody. The obtained conjugated antibodies showed high stability in broad pH ranges (4–10; >70%) and high thermostability at 37?°C for 84?h (>85%). Despite the need to further consider and evaluate the industrial standardization and production process, our data provided the primary evidence that conjugated IgY antibodies can be used as a secondary antibody for broad immunological analysis.     

Antibody variable region glycosylation: position effects on antigen binding and carbohydrate structure.

The presence of N-linked carbohydrate at Asn58 in the VH of the antigen binding site of an antibody specific for alpha(1----6)dextran (TKC3.2.2) increases its affinity for dextran 10- to 50-fold. Site-directed mutagenesis has now been used to create novel carbohydrate addition sequences in the CDR2 of a non-glycosylated anti-dextran at Asn54 (TST2) and Asn60 (TSU7). These antibodies are glycosylated and the carbohydrates are accessible for lectin binding. The amino acid change in TSU7 (Lys62----Thr62) decreases the affinity for antigen; however, glycosylation of TSU7 increased its affinity for antigen 3-fold, less than the greater than 10-fold increase in affinity seen for glycosylated TKC3.2.2. The difference in impact of glycosylation could result either from the position of the carbohydrate or from its structure; unlike the other antibodies, TSU7 attaches a high mannose, rather than complex, carbohydrate in CDR2. In contrast, glycosylation of TST2 at amino acid 54 inhibits dextran binding. Thus slight changes in the position of the N-linked carbohydrate in the CDR2 of this antibody result in substantially different effects on antigen binding. Unlike what was observed for the anti-dextrans, a carbohydrate addition site placed in a similar position in an anti-dansyl is not utilized.     

Flow cytometric detection of activated mouse integrin alphaIIbbeta3 with a novel monoclonal antibody

BACKGROUND: Integrin alphaIIbbeta3 mediates platelet adhesion and aggregation and plays a crucial role in thrombosis and hemostasis. alphaIIbbeta3 is expressed in a low affinity state on resting platelets. Upon platelet activation, alphaIIbbeta3 shifts to a high affinity conformation that efficiently binds its ligands. On human platelets, the high affinity conformation of alphaIIbbeta3 is detected by the monoclonal antibody (mAb), PAC-1. However, a reagent with binding specificity to high affinity mouse alphaIIbbeta3 has not been described so far. METHODS: A novel rat mAb directed against mouse alphaIIbbeta3 (JON/A) was generated and characterized. JON/A was conjugated with fluorescein isothiocyanate (JON/A(FITC)) or with R-phycoerythrin (JON/A(PE)) and used for flow cytometric analysis of mouse platelets. RESULTS: Although JON/A(FITC) bound to resting and activated platelets, virtually no binding of the larger JON/A(PE) to resting platelets was detectable. However, strong binding of JON/A(PE) occurred on platelet activation in a dose-dependent manner. Binding of JON/A(PE) required extracellular free calcium and was irreversible, thereby stabilizing the high affinity conformation of alphaIIbbeta3. CONCLUSION: JON/A(PE) is the first tool for direct assessment of integrin alphaIIbbeta3 activation in mice. Furthermore, JON/A(FITC) and JON/A(PE) provide the first examples of fluorescent antibody derivatives with identical antigenic specificity that allow the discrimination between the resting and the activated state of an integrin.     

Permeability of the Peritrophic Envelopes of Herbivorous Insects to Dextran Sulfate: a Test of the Polyanion Exclusion Hypothesis

We tested the hypothesis that the permeability of the peritrophic envelope in herbivorous insects is greatly reduced for polyanions as a result of an extensive network of anionic sites in the proteoglycans of the matrix. 14C-Dextran sulfate (polyanionic, 8000 M(w)) and fluorescein isothiocyanate-labeled (FITC) dextran (monoanionic, 9400 M(w)) were introduced together into the endoperitrophic space of the midguts of Orgyia leucostigma (Lepidoptera) larvae and Melanoplus sanguinipes (Orthoptera) adults. In all cases more of the 14C-dextran sulfate permeated the peritrophic envelope than the FITC-dextran, the opposite of the result predicted by the polyanion exclusion hypothesis. We conclude that polyanion exclusion is not a mechanism that contributes significantly to the permeability properties of the peritrophic envelopes of these two species, or that explains the failure of tannic acid to cross the peritrophic envelopes of lepidopteran larvae. Copyright 1997 Elsevier Science Ltd. All rights reserved     

Phase separation in dextran/locust bean gum mixtures

Dextran/locust bean gum (LBG) mixtures have been prepared and investigated with respect to their phase separation behaviour. These systems exhibited phase separation at 20 °C, the upper phase, itself biphasic, being enriched with locust bean gum but also containing dextran, whereas the lower phase contained only dextran. This lower phase was a liquid. The upper phase, which did not flow, was characterized by means of rheological dynamic measurements. Clearly, its behaviour was typical of a gel, the three-dimensional structure of which can be ascribed to self-association of LBG chains owing to the very high concentration of the galactomannan in this upper phase. The self-association of the galactomannan was confirmed by fluorescence microscopy carried out on mixtures containing fluorescein isothiocyanate (FITC)-labelled dextran. The rheological behaviour of a concentrated LBG solution was also investigated as a function of time, clearly showing progressive formation of a weak gel structure.     

Integrated protein microchip assay with dual fluorescent- and MALDI read-out

A pore chip protein array (PCPA) concept based on a dual readout configuration, fluorescence imaging, and MALDI-TOF MS has been developed. Highly packed, (>4000 spots/cm2), antibody arrays were dispensed on the porous chip by using a piezo-electric microdispenser. Sandwich assay was made after blocking by addition of a secondary antibody either IgG-FITC-labeled or anti-Ang II. The antigen in the first system was a large protein (IgG), and in the other system, a FITC marked peptide Angiotensin II (Ang II) was used. Ang II antibodies showed specificity for Ang II, while the Ang I antibodies showed binding properties for Ang I, II, and Renin. Fluorescence and MALDI TOF MS read-out was made for IgG and Ang II. A major advantage of the dual read-out PCPA approach is that both affinity binding and mass identity are derived. Detection limits for Ang II on the chip is as low as 500 zmol (Ang II).     

The inhibitory monoclonal antibody M7-PB-E9 stabilizes E2 conformational states of Na+,K(+)-ATPase.

Monoclonal antibody M7-PB-E9 binds the sheep kidney Na+,K(+)-ATPase alpha-subunit with high affinity (Kd = 3 nM) and inhibits enzyme turnover in competition with ATP, and, like ATP, in the presence of Mg2+, it stimulates the rate of ouabain binding [Ball, W. J. (1984) Biochemistry 23, 2275-2281]. In this study, covalent attachment of fluorescein 5'-isothiocyanate (FITC) at (or near) the enzyme's ATP binding site did not alter the antibody's affinity for alpha nor did bound antibody alter the anisotropy of (r = 0.36) or the solvent accessibility of iodide to bound FITC. Further, in its E1Na+ conformation (4 mM NaCl), the enzyme's affinity for the ATP congener eosin was unaltered by the bound antibody (Kd = 9 nM). In contrast, partial E2 conformations induced by KCl lowered eosin affinities (0.2 mM KCl, Kd = 28 nM; 0.4 mM, Kd = 86 nM), and M7-PB-E9 reduced these affinities further (Kd = 66 and 130 nM, respectively). By monitoring the fluorescence changes of the FITC-labeled enzyme, the antibody was found to assist several ligand-induced conformational transitions from E1 (E1Na+ or E1Tris) to E2 (E2K+, E2-P(i)Mg2+, or E2Mg2+.ouabain) states, and inhibit the E2K(+)-->E1Na+ transition. Antibody binding alone, however, did not appear to significantly alter enzyme conformation. The antibody therefore is not directed against the ATP site but binds to a region of alpha distinct from any ligand binding site and which plays an important role in the E1<-->E2 transitions.     

Impedimetric immunosensors based on the conjugated polymer PPV

In the work reported here, we investigated the interaction between the semiconducting polymer MDMO-PPV and antibodies against the fluorescent dyes fluorescein isothiocyanate (FITC) and Cy5. The antibodies are adsorbed physically onto thin polymer films on gold electrodes, as seen in AFM images of these films. By tuning the antibody concentration, the contact angle of distilled water with the film can be made to vary between 95 degrees and 50 degrees, showing that different surface densities of antibody can be obtained. That these biosensor films specifically bind their antigenic fluorescent molecules from PBS buffer solution is demonstrated by confocal fluorescence microscopy. Specific antigen-antibody recognition is demonstrated by lack of cross-sensitivity between the two antibodies and their antigens. In a biosensor prototype based on differential impedance spectroscopy, these polymer films show a clear response to 1 ppb antigen solution, with a time constant of 2-3 min.     

Isolation, partial chemical and biological characterization of thymone B

A new approach to the study of the molecular arrangements of proteins in membranes is described. Irradiation with visible light of native erythrocytes or washed erythrocyte membranes suspended in buffers containing a) riboflavin, fluorescein or fluorescein coupled to dextran and b) ³H-labelled tryptophan resulted in incorporation of radioactivity into the membrane proteins. Polyacrylamide gel electrophoresis of solubilized membranes followed by radioactivity measurements of the separated membrane proteins revealed that in native erythrocytes the protein components known to be located at the exterior cell surface, Band 3 and the major sialoglycoproteins became specifically labelled, whereas in washed lysed cells all of the major membrane proteins were labelled.     

Complement protein C9 labeled with fluorescein isothiocyanate can be used to monitor C9 polymerization and formation of the cytolytic membrane lesion

Human complement protein C9 was covalently labeled with the fluorescent chromophore fluorescein isothiocyanate (FITC) with only a small reduction in the cytolytic activity of the protein. Polymerization of the labeled protein--either by incubating with lipid vesicles treated with complement proteins C5b-8 (activating the C5b-9 membrane lesion) or by heating the protein [Tschopp, J., Muller-Eberhard, H.J., & Podack, E.R. (1982) Nature (London) 298, 534]--resulted in a 40-60% decrease in the fluorescence emission from FITC. The decrease in total fluorescence was accompanied by an increase in the steady-state anisotropy following activation and polymerization of FITC-C9 by C5b-8 membranes, while heat-induced aggregation of the protein resulted in a dramatic depolarization of fluorescence. Only small changes in either the absorbance spectrum or fluorescence lifetime of the chromophore were detected upon FITC-C9 polymerization. Evidence is presented that the measured changes in FITC fluorescence upon C9 activation are due to self energy transfer between closely apposed fluorescein chromophores which occur in the polymerized form of the protein. The significance of these observations to the molecular structure of the assembled C5b-9 complex is discussed, as are the potential applications of this fluorescent derivative of C9.     

Specific labeling of the bovine heart mitochondrial phosphate carrier with fluorescein 5-isothiocyanate: roles of Lys185 and putative adenine nucleotide recognition site in phosphate transport

The amine/SH-modifying fluorescein 5-isothiocyanate (FITC) specifically labeled Lys(185) in the putative membrane-spanning region of the phosphate carrier from both the cytosolic and matrix sides of bovine heart mitochondria at 0 degrees C and pH 7.2, and the labeling inhibited the phosphate transport. Nonmodifying fluorescein derivatives having similar structural features to those of ADP and ATP (Majima, E., Yamaguchi, N., Chuman, H., Shinohara, Y., Ishida, M., Goto, S., and Terada, H. (1998) Biochemistry 37, 424-432) inhibited the specific FITC labeling and phosphate transport, but the nonfluorescein phenylisothiocyanate did not inhibit FITC labeling, suggesting that there is a region recognizing the adenine nucleotides in the phosphate carrier and that this region is closely associated with the transport activity. The phosphate transport inhibitor pyridoxal 5'-phosphate inhibited the specific FITC labeling, possibly due to competitive modification of Lys(185). In addition, FITC inhibited the ADP transport and specific labeling of the ADP/ATP carrier with the fluorescein SH reagent eosin 5-maleimide. Based on these results, we discuss the structural features of the phosphate carrier in relation to its transport activity.