L-glucose absorption in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>) is nonmediated

We previously demonstrated in intact house sparrows substantial absorption in vivo of L-glucose, the stereoisomer of D-glucose that is assumed not to interact with the intestines D-glucose transporter. Results of some studies challenge this assumption for other species. Therefore, we tested it in vitro and in vivo, based on the principle that if absorption of a compound (L-glucose) is mediated, then absorption of its tracer will be competitively inhibited by high concentrations of either the compound itself or other compounds (e.g., D-glucose) whose absorption is mediated by the same mechanism. An alternative hypothesis that L-glucose absorption is primarily paracellular predicts that its absorption in vivo will be increased (not decreased) in the presence of D-glucose, because the permeability of this pathway is supposedly enhanced when Na⁺-coupled glucose absorption occurs. First, using intact tissue in vitro, we found that uptake of tracer-radiolabeled L-glucose was not significantly inhibited by high concentrations (100 mM) of either L-glucose or 3-O-methyl-D-glucose, a non-metabolizable but actively transported D-glucose analogue. Second, using intact house sparrows, we found that fractional absorption of the L-glucose tracer was significantly increased, not reduced, when gavaged along with 200 mM 3-O-methyl-D-glucose. This result was confirmed in another experiment where L-glucose fractional absorption was significantly higher in the presence vs. absence of food in the gut. The greater absorption was apparently not due simply to longer retention time of digesta, because no significant difference was found among retention times. Our results are consistent with the idea that L-glucose is absorbed in a non-mediated fashion, largely via the paracellular pathway in vivo.Abbreviations AUC area under the curve - 3OMD-glucose 3-O-methyl-D-glucose Communicated by I.D. Hume     

Paracellular absorption: a bat breaks the mammal paradigm

Bats tend to have less intestinal tissue than comparably sized nonflying mammals. The corresponding reduction in intestinal volume and hence mass of digesta carried is advantageous because the costs of flight increase with load carried and because take-off and maneuverability are diminished at heavier masses. Water soluble compounds, such as glucose and amino acids, are absorbed in the small intestine mainly via two pathways, the transporter-mediated transcellular and the passive, paracellular pathways. Using the microchiropteran bat Artibeus literatus (mean mass 80.6+/-3.7 g), we tested the predictions that absorption of water-soluble compounds that are not actively transported would be extensive as a compensatory mechanism for relatively less intestinal tissue, and would decline with increasing molecular mass in accord with sieve-like paracellular absorption. Using a standard pharmacokinetic technique, we fed, or injected intraperitoneally the metabolically inert carbohydrates L-rhamnose (molecular mass = 164 Da) and cellobiose (molecular mass = 342 Da) which are absorbed only by paracellular transport, and 3-O-methyl-D-glucose (3OMD-glucose) which is absorbed via both mediated (active) and paracellular transport. As predicted, the bioavailability of paracellular probes declined with increasing molecular mass (rhamnose, 90+/-11%; cellobiose, 10+/-3%, n = 8) and was significantly higher in bats than has been reported for laboratory rats and other mammals. In addition, absorption of 3OMD-glucose was high (96+/-11%). We estimated that the bats rely on passive, paracellular absorption for more than 70% of their total glucose absorption, much more than in non-flying mammals. Although possibly compensating for less intestinal tissue, a high intestinal permeability that permits passive absorption might be less selective than a carrier-mediated system for nutrient absorption and might permit toxins to be absorbed from plant and animal material in the intestinal lumen.     

Capacity for absorption of water-soluble secondary metabolites greater in birds than in rodents

Plant secondary metabolites (SMs) are pervasive in animal foods and potentially influence feeding behavior, interspecies interactions, and the distribution and abundance of animals. Some of the major classes of naturally occurring SMs in plants include many water-soluble compounds in the molecular size range that could cross the intestinal epithelium via the paracellular space by diffusion or solvent drag. There are differences among species in paracellular permeability. Using Middle Eastern rodent and avian consumers of fruits containing SMs, we tested the hypothesis that avian species would have significantly higher paracellular permeability than rodent species. Permeability in intact animals was assessed using standard pharmacological methodology to measure absorption of two radiolabeled, inert, neutral water-soluble probes that do not interact with intestinal nutrient transporters, L-arabinose (M_r = 150.1 Da) and lactulose (M_r = 342.3 Da). We also measured absorption of labeled 3-O-methyl-D-glucose (3OMD-glucose; M_r = 194.2 Da), which is a nonmetabolized analogue of D-glucose that is passively absorbed through the paracellular space but also transported across the enterocyte membranes. Most glucose was absorbed by all species, but arabinose fractional absorption (f) was nearly three times higher in birds (1.03±0.17, n = 15 in two species) compared to rodents (0.37±0.06, n = 10 in two species) (P<0.001). Surprisingly, the apparent rates of absorption in birds of arabinose exceeded those of 3OMD-glucose. Our findings are in agreement with previous work showing that the paracellular pathway is more prominent in birds relative to nonflying mammals, and suggests that birds may be challenged by greater absorption of water-soluble, dietary SMs. The increased expression of the paracellular pathway in birds hints at a tradeoff: the free energy birds gain by absorbing water-soluble nutrients passively may be offset by the metabolic demands placed on them to eliminate concomitantly absorbed SMs.     

Fluorescent rhodol derivatives: versatile, photostable labels and tracers.

A series of chemically reactive, fluorescent rhodol derivatives was prepared and evaluated. Reactive functional groups included activated esters, amines, haloacetamides, fixable hydrazide derivatives, acrylamides, and photoaffinity reagents. Depending on the choice of substituents, absorption maxima of the dyes varied from 490 to 550 nm with extinction coefficients that were generally greater than 50,000 M-1 cm-1 in aqueous solution and emission maxima from 520 to 580 nm. Most of the compounds investigated exhibited fluorescence lifetimes between 3 and 4 ns. Individual derivatives were suitable for excitation with the 488 and 514-nm lines of the argon ion laser and the 546-nm line of the mercury arc lamp and were compatible for use with standard fluorescein and rhodamine filter sets. The rhodol dyes were more photostable and less sensitive to pH changes in the physiological range than fluorescein derivatives. Some examples show absorption maxima at or near 514 nm, an excitation wavelength that is useful for multicolor fluorescence microscopy, flow cytometry, and DNA sequencing. Derivatives were also prepared that exhibit absorption and emission maxima similar to those of tetramethylrhodamine (TMR) analogs but with higher quantum yields in aqueous solution. A number of the dyes had higher solubilities in aqueous systems and were less quenched on conjugation to proteins than TMR derivatives. Appropriate substitution results in a wider range of solubilities in hydrophilic or lipophilic solvents than is easily accomplished with fluorescein or TMR derivatives. Conjugates of a number of the rhodol fluorophores were generally more photostable and less pH sensitive than fluorescein conjugates and more fluorescent than TMR conjugates.     

Intestinal absorption of lysozyme,an egg-white allergen,in rats: Kinetics and effect of NSAIDs

The absorption pathway(s) of a representative food allergen, lysozyme, and the mechanisms of lysozyme absorption facilitated by non-steroidal anti-inflammatory drugs were examined by intestinal closed-loop and re-circulating perfusion methods in rats. The absorption rate of fluorescein isothiocyanate (FITC)-labeled lysozyme in the proximal intestine was higher than that for a marker of non-specific absorption, FD-10, and was suppressed by colchicine (endocytosis inhibitor). Aspirin increased the absorption of FITC-lysozyme in the proximal intestine with no effects on tissue accumulation. Diclofenac facilitated FITC-lysozyme absorption, but meloxicam and loxoprofen exerted no effects on absorption. Co-administration of misoprostol (synthetic prostaglandin-E1 analog) with aspirin significantly ameliorated the aspirin-facilitated absorption of FITC-lysozyme to the same level as that seen with controls. Thus, lysozyme absorption was mediated by endocytic and paracellular pathways in the proximal intestine, and was facilitated by aspirin and diclofenac after impairment of the paracellular pathway. Misoprostol may suppress the allergen absorption facilitated by aspirin.     

Ratiometric fluorescence imaging of nuclear pH in living cells using Hoechst-tagged fluorescein

Small-molecule fluorescent sensors that allow specific measurement of nuclear pH in living cells will be valuable for biological research. Here we report that Hoechst-tagged fluorescein (hoeFL), which we previously developed as a green fluorescent DNA-staining probe, can be used for this purpose. Upon excitation at 405 nm, the hoeFL–DNA complex displayed two fluorescence bands around 460 nm and 520 nm corresponding to the Hoechst and fluorescein fluorescence, respectively. When pH was changed from 8.3 to 5.5, the fluorescence intensity ratio (F₅₂₀/F₄₆₀) significantly decreased, which allowed reliable pH measurement. Moreover, because hoeFL binds specifically to the genomic DNA in cells, it was applicable to visualize the intranuclear pH of nigericin-treated and intact living human cells by ratiometric fluorescence imaging.     

Benzoxazinone derivatives: new fluorescent probes for two-color flow cytometry analysis using one excitation wavelength

A new class of fluorescent dye which upon excitation at 488 nm turns red is shown to be probe-suitable for using in flow cytometry alone or in conjunction with fluorescein derivatives. 7-dimethylamino 3-(p-formylstyryl) 1,4 benzoxazin 2-one is suitable for rendering microorganisms, such as Plasmodium merozoites and cells detectable by flow cytometry, allowing a dual fluorescence analysis when the cells are labelled with suitable fluoresceinylated ligands such as fluorescein labeled neoglycoproteins or antibodies. The synthesis of the new benzoxazinone derivatives is described: p-[beta-(7-dimethylamino 1,4 benzoxazin 2-one 3-yl)-vinyl]-phenylpropenoic acid can be easily activated as a hydroxysuccinimide derivative and linked to amino groups of polypeptides. Hydrophilic polypeptides such as poly-L-lysine or glycosylated polymers combined with this new fluorescent dye are shown to be helpful in analyzing cell surface receptors, in dual fluorescence flow cytometry analysis, using a single excitation wavelength and two sets of compounds labeled with the new benzoxazinone derivative and with fluorescein isothiocyanate, respectively. The new benzoxazinone derivative has a high molar absorbance, a good quantum yield fluorescence when it is bound to hydrophilic polypeptides and its fluorescence intensity is not dependent on pH in the physiological pH range.     

Fatty acids increase paracellular absorption of aluminium across Caco-2 cell monolayers

Passive paracellular absorption, regulated by tight junctions (TJs), is the main route for absorption of poorly absorbed hydrophilic substances. Surface active substances, such as fatty acids, may enhance absorption of these substances by affecting the integrity of TJ and increasing the permeability. It has been suggested that aluminium (Al) absorption occurs mainly by the paracellular route. Herein, we investigated if physiologically relevant exposures of fully differentiated Caco-2 cell monolayers to oleic acid and docosahexaenoic acid (DHA), which are fatty acids common in food, increase absorption of Al and the paracellular marker mannitol. In an Al toxicity test, mannitol and Al absorption through Caco-2 cell monolayers were similarly modulated by Al concentrations between 1 and 30 mM, suggesting that absorption of the two compounds occurred via the same pathways. Exposure of Caco-2 cell monolayers to non-toxic concentrations of Al (2 mM) and ¹⁴C-mannitol in fatty acid emulsions (15 and 30 mM oleic acid, 5 and 10 mM DHA) caused a decreased transepithelial electrical resistance (TEER). Concomitantly, fractional absorption of Al and mannitol, expressed as percentage of apical Al and mannitol retrieved at the basolateral side, increased with increasing dose of fatty acids. Transmission electron microscopy was applied to assess the effect of oleic acid on the morphology of TJ. It was shown that oleic acid caused a less structured morphology of TJ in Caco-2 cell monolayers. Taken together our findings indicate that fatty acids common in food increase the paracellular intestinal absorption of Al. These findings may influence future risk assessment of human Al exposure.     

Quantitation of the binding, uptake, and degradation of fluoresceinylated neoglycoproteins by flow cytometry

The fluorescence properties of the fluorescein residues bound to a protein are used to analyze by flow cytometry the neoglycoproteins' endocytosis mediated by membrane lectins of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein is dependent on the number of fluorophore molecules bound to a protein molecule and the pH of the environmental medium. The mean fluorescence intensity of a fluorescein molecule bound to a protein decreases when the number of fluorescein residues per protein molecule increases. However, after proteolytic digestion, the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The binding of fluorescein-labeled alpha-glucosylated serum albumin to 3LL cells at 4 degrees C can easily be determined by flow cytometry because under these conditions the environmental pH is neutral, and the neoglycoprotein is not degraded. When the cells are incubated at 37 degrees C in the presence of a fluorescein-labeled neoglycoprotein, the fluorescence intensity of a cell is low because of the low pH of endosomes and lysosomes but is increased upon a postincubation at 4 degrees C in the presence of monensin, a proton/sodium ionophore. The extent of the proteolytic digestion of an endocytosed neoglycoprotein can be assessed by comparing, upon a monensin postincubation at 4 degrees C, the high cell-associated fluorescence of cells incubated in the absence of leupeptin (an inhibitor of lysosomal proteases) and the relatively low fluorescence intensity of cells incubated in the presence of leupeptin.     

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease,and 1 phosphatase

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.     

The sweet life: diet sugar concentration influences paracellular glucose absorption

Small birds and bats face strong selection pressure to digest food rapidly in order to reduce digesta mass carried during flight. One mechanism is rapid absorption of a high proportion of glucose via the paracellular pathway (transfer between epithelial cells, not mediated by transporter proteins). Intestinal paracellular permeability to glucose was assessed for two nectarivorous passerines, the Australian New Holland honeyeater (Phylidonyris novaehollandiae) and African white-bellied sunbird (Cinnyris talatala) by measuring the bioavailability of radiolabelled, passively absorbed l -glucose. Bioavailability was high in both species and increased with diet sugar concentration (honeyeaters, 37 and 81% and sunbirds, 53 and 71% for 250 and 1000mmoll-1 sucrose diets, respectively). We conclude that the relative contribution of paracellular to total glucose absorption increases with greater digesta retention time in the intestine, and paracellular absorption may also be modulated by factors such as intestinal lumen osmolality and interaction with mediated glucose uptake. The dynamic state of paracellular absorption should be taken into account in future studies.     

Two-photon thermal bleaching of single fluorescent molecules

We have studied the fluorescence emission by two-photon excitation of four dyes widely used for bioimaging studies, rhodamine 6G, fluorescein, pyrene and indo-1 at the single molecule level. The single dye molecules, spread on a glass substrate by spin coating, show a constant fluorescence output until a sudden transition to a dark state very close to the background. The bleaching time that is found to vary in the series pyrene, indo-1, fluorescein and rhodamine 6G from the fastest to the slowest one respectively, has a Gaussian distribution indicating that the observed behavior is not due to photobleaching. Moreover, the bleaching time decreases with the glass substrate temperature reaching a vanishing nonmeasurable value for a limiting temperature whose value is found in the same series as for the bleaching time, from the lowest to the highest temperature respectively. The observed bleaching shows a clear correlation to the amount of absorbed power not reirradiated as fluorescence and to the complexity of the molecule. These observations are interpreted as thermal bleaching where the temperature increase is induced by the two-photon absorption of the single dyes as confirmed also by numerical simulations.     

The capacity for paracellular absorption in the insectivorous bat Tadarida brasiliensis

Water-soluble nutrients are absorbed by the small intestine via transcellular and paracellular processes. The capacity for paracellular absorption seems greater in fliers than in nonfliers, although that conclusion rests mainly on a comparison of flying birds and nonflying mammals because only two frugivorous bat species have been studied. Furthermore, the bats studied so far were relatively large (>85 g, compared with most bat species which are <20 g) and were not insectivores (like about 70 % of bat species). We studied the small (11 g) insectivorous bat Tadarida brasiliensis and tested the prediction that the capacity for paracellular absorption would be as high as in the other bat and avian species studied so far, well above that in terrestrial, nonflying mammals. Using standard pharmacokinetic technique, we measured the extent of absorption (fractional absorption = f) of inert carbohydrate probes: L-arabinose (MM = 150.13) absorbed exclusively by paracellular route and 3OMD-glucose (MM = 194) absorbed both paracellularly and transcellularly. As predicted, the capacity of paracellular absorption in this insectivorous bat was high (L-arabinose f = 1.03 ± 0.14) as in other frugivorous bats and small birds. Absorption of 3OMD-glucose was also complete (f = 1.09 ± 0.17), but >80 % was accounted for by paracellular absorption. We conclude that passive paracellular absorption of molecules of the size of amino acids and glucose is extensive in this bat and, generally in bats, significantly higher than that in nonflying mammals, although the exact extent can be somewhat lower or higher depending on molecule size, polarity and charge.     

Electroaffinity in paracellular absorption of hydrophilic d-dipeptides by sparrow intestine

We previously demonstrated size selectivity in the absorption of nonelectrolyte hydrosoluble probes in birds, presumably by the paracellular pathway. Our goal in this study was to determine the charge selectivity in the absorption of hydrosoluble d-dipeptides, because there have been no studies of the electroaffinity of this absorption pathway in birds. For this purpose isosmotic solutions with two hydrophilic d-dipeptides: serine-lysine (positive at pH 7.4) and serine-aspartic (negative at pH 7.4) were gavaged into the stomach in nonanesthetized house sparrows (Passer domesticus), and injected into the pectoralis with a syringe in different trials. Fractional absorption was calculated as F = [AUC by gavage)]/[AUC by injection] (AUC = area under the curve of plasma probe concentration vs. time). Fractional absorption was significantly higher for the positively charged than negatively charged dipeptide (respectively, F=0.30±0.05 vs. F=0.17±0.03). These findings give the first evidence of cation selectivity by the paracellular route in the absorption of hydrosoluble solutes in the small intestine in birds.     

Intestinal perfusion indicates high reliance on paracellular nutrient absorption in an insectivorous bat Tadarida brasiliensis

Flying vertebrates have been hypothesized to have a high capacity for paracellular absorption of nutrients. This could be due to high permeability of the intestines to nutrient-sized molecules (i.e., in the size range of amino acids and glucose, MW 75–180 Da). We performed intestinal luminal perfusions of an insectivorous bat, Tadarida brasiliensis. Using radio-labeled molecules, we measured the uptake of two nutrients absorbed by paracellular and transporter-mediated mechanisms (l-proline, MW 115 Da, and d-glucose, MW 180 Da) and two carbohydrates that have no mediated transport (l-arabinose, MW 150 Da, and lactulose, MW 342 Da). Absorption of lactulose (0.61 ± 0.06 nmol min^? 1 cm^? 1) was significantly lower than that of the smaller arabinose (1.09 ± 0.04 nmol min^? 1 cm^? 1). Glucose absorption was significantly lower than that of proline at both nutrient concentrations (10 mM and 75 mM). Using the absorption of arabinose to estimate the portion of proline absorption that is paracellular, we calculated that 25.1 ± 3.0% to 66.2 ± 7.8% of proline absorption is not transporter-mediated (varying proline from 1 mM to 75 mM). These results confirm our predictions that 1) paracellular absorption is molecule size selective, 2) absorption of proline would be greater than glucose absorption in an insectivore, and 3) paracellular absorption represents a large fraction of total nutrient absorption in bats.     

Visualization of 'water secretion' by confocal microscopy in rat salivary glands: possible distinction of para- and transcellular pathway

Visualization of water transport in cells, tissues and organs is an important, yet still difficult, task in morphological science. By using confocal microscopy and the fluid-phase fluorescent tracer technique, we visualized water secretion and estimated the routes of water transport across the acinar epithelia in rat parotid and submandibular glands. Confocal microscopy of whole glands perfused arterially with Lucifer yellow revealed a bright fluorescence at the basolateral space of acini. Luminal space was devoid of fluorescence, but revealed it after isoproterenol pretreatment, ductal infusion of fluorescent dextrans into the lumen, or tissue dissociation by collagenase. Under these conditions, stimulation of fluid secretion with carbachol caused a rapid decline of the luminal fluorescence intensity, indicating that the secreted water washed out the fluorescent probes in the acinar lumen. In the stimulated dissociated acini, the luminal fluorescence disappeared by 15 sec, but reappeared at 30-45 sec to maintain a low plateau level. By assuming that the tight junction was 'paralyzed' by the collagenase digestion and that the paracellular fluid transport could not influence the dilution of Lucifer yellow, we estimated that the initial water secretion by CCh occurs via the transcellular pathway, while later than 30-45 sec the additional water permeates through the paracellular pathway.     

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 °C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.     

Chemical modification of lysine residues in cytochrome P450LM2 (P450IIB4): influence on heme liganding of arylamines.

Treatment of cytochrome P450LM2 with fluorescein isothiocyanate to introduce up to two equivalents of fluorophore per polypeptide chain resulted in the selective derivatization of lysine residues. CD-spectral measurements revealed the overall conformation as well as the immediate heme environment of the hemoprotein to remain unaffected by attachment of the label. Modification caused decreased affinity of p-phenylenediamine and other 4-substituted anilines for the heme site, whereas there was a rise in the extent of substrate interaction. Experiments with pigment containing acetylated lysines gave analogous results, suggesting that the observed phenomenon was due to charge neutralization. There was linear correlation between the Hammett sigma P values and both the optical dissociation constants for arylamine binding to intact enzyme and the dipole moments of the anilines, indicating that basicity along with electronic factors controlled heme liganding; lipophilicity appeared to be of minor importance. Introduction of fluorescein isothiocyanate into the oxygenase was found to influence the bond-making process through modulating basicity of the nitrogenous compounds, but perturbation of optimal spacial orientation of the amine nitrogen toward the heme iron also might have been operative. The lysines studied seem to represent metabolically inactive elements of the substrate channel located on the cytosolic surface of the aggregates, as evidenced by steady-state fluorescence measurements. A hydrophilic segment in the cytochrome P450LM2 molecule that would accommodate the critical residues is discussed.     

Estimation of the degradation of endocytosed material by flow cytofluorometry using two neoglycoproteins containing different numbers of fluorescein molecules

The fluorescence properties of fluorescein bound to protein are used to quantitate by flow cytofluorometry the degradation of fluorescein-labeled alpha-glucosylated serum albumin (fluorescein-labeled neoglycoprotein) after endocytosis by the membrane lectin of Lewis lung carcinoma cells (3LL cells). The quantum yield of fluorescein bound to a protein decreases when the number of fluorescein residues per protein molecule increases; however, after proteolytic digestion the mean fluorescence intensity of a fluorescein molecule is constant and equal to that of free fluorescein. The extent of the degradation of the endocytosed neoglycoprotein was determined with a flow cytofluorometer by using two neoglycoproteins containing either a small or a large number of fluorescein residues per neoglycoprotein molecule. At 4 degrees C, 3LL cells bind 750,000 molecules of fluorescein-labeled alpha-glucosylated serum albumin with an apparent binding constant of 2 X 10(6) 1 X mole-1. At 37 degrees C, after 4 hr incubation 2.2 X 10(6) molecules of fluorescent alpha-glucosylated serum albumin were cell-associated, and of these at least one third were degraded.     

Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process

The photophysical properties of synthetic compounds derived from the imidazolidinone chromophore of the green fluorescent protein were determined. Various electron-withdrawing or electron-donating substituents were introduced to mimic the effect of the chromophore surroundings in the protein. The absorption and emission spectra as well as the fluorescence quantum yields in dioxane and glycerol were shown to be highly dependent on the electronic properties of the substituents. We propose a kinetic scheme that takes into account the temperature-dependent twisting of the excited molecule. If the activation energy is low, the molecule most often undergoes an excited-state intramolecular twisting that leads it to the ground state through an avoided crossing between the S(1) and S(0) energy surfaces. For a high activation energy, the torsional motion within the compounds is limited and the ground-state recovery will occur preferentially by fluorescence emission. The excellent correlation between the fluorescence quantum yields and the calculated activation energies to torsion points to the above-mentioned avoided crossing as the main nonradiative deactivation channel in these compounds. Finally, our results are discussed with regard to the chromophore in green fluorescent protein and some of its mutants.