Substrate and cofactor binding to fluorescently labeled cytoplasmic malate dehydrogenase

Cytoplasmic malate dehydrogenase (cMDH) is a key enzyme in several metabolic pathways. Though its activity has been examined extensively, there are lingering mechanistic uncertainties involving substrate and cofactor binding. To more completely understand this enzyme's interactions with cofactor and substrate ligands, a fluorescent reporter group was introduced into the enzyme's structure. This was accomplished by selective modification of Cys 110. The reaction placed an aminonaphthaline sulfonic acid group near the enzyme's active site. Substrate, inhibitor, and NAD binding activities were characterized using changes in this label's fluorescence. Results demonstrated that both substrate and cofactor molecules bound to the enzyme in the absence of their companion ligands. This is in contrast to strictly ordered cofactor then substrate binding as has been suggested by kinetic analyses of closely related enzymes. Binding results also indicated that the cofactor, NAD, bound to cMDH in a negatively cooperative manner, but substrates and the inhibitor, hydroxymalonate, bound non-cooperatively. Multiple substrate binding modes were identified and interactions between substrate and cofactor binding were found.     

Quantitative characterization of the binding of fluorescently labeled colchicine to tubulin in vitro using fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is a new technique that allows the determination of the diffusion constant of a fluorescent molecule in solution. Also, the binding of the fluorescent molecule to a target can be analyzed, if the difference in the diffusion coefficients of the free and bound ligand is sufficiently large. With FCS, the interaction between fluorescein-colchicine (FC) and tubulin has been studied in vitro. A fast and reversible binding is observed with an association constant at room temperature of (3.9 +/- 0.1) x 10(4) M-1. No competition with colchicine is seen, indicating that FCS reveals the existence of a new binding site on tubulin. FCS is not able to show the binding of FC to the original colchicine binding site, even though it exists, because the fluorescence of FC is strongly quenched upon binding to this site. This quenching is evident in spectrofluorometry experiments, revealing a slow binding of FC to tubulin that is subject to competition with colchicine. FCS allows the determination of the diffusion coefficients of both free and bound fluorescent colchicine which were found to be (2.6 +/- 0.2) x 10(-)10 and (2.0 +/- 0.2) x 10(-)11 m2 s-1, respectively. It can be concluded that fluorescent labeling, especially of small molecules, can interfere considerably with the binding behavior that is being studied. Although general qualitative effects in vivo are similar for colchicine and its fluorescein derivative, this quantitative study of the binding to tubulin presents a nuanced view, and the existence of a second binding site for FC can even explain some conflicting indications in the literature.     

Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative

The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.     

Flow cytometric competitive binding assay for determination of actinomycin-D concentrations

A single step, separation free competitive binding reaction between the fluorescent antibiotic mithramycin and actinomycin-D for common binding sites on DNA coated 10 microns diameter microspheres is described. The fluorescence of the microspheres is measured with a flowcytometer. In the presence of a constant amount of mithramycin, the microsphere fluorescence is inversely proportional to actinomycin-D concentration.     

Directed covalent immobilization of fluorescently labeled cytokines

Cytokines are important mediators coordinating inflammation and wound healing in response to tissue damage and infection. Therefore, immobilization of cytokines on the surface of biomaterials is a promising approach to improve biocompatibility. Soluble cytokines signal through receptors on the cell surface leading to cell differentiation, proliferation, or other effector functions. Random immobilization of cytokines on surfaces will result in a large fraction of inactive protein due to impaired cytokine--receptor interaction. We developed a strategy that combined (i) directed covalent coupling of cytokines, (ii) quantification of coupling efficiency through fluorescence detection, and (iii) a reliable protease cleavage assay to control orientation of coupling. For this purpose, fusion proteins of the SNAP-tag followed by an enterokinase recognition site, yellow fluorescent protein (YFP), and the cytokine of interest being either interleukin-6 (IL-6) or oncostatin M (OSM) were generated. The SNAP-tag is a derivative of O(6)-alkylguanine-DNA alkyltransferase that couples itself covalently to benzylguanine. Bioactivities of the SNAP-YFP-cytokines were shown to be comparable with the nontagged cytokines. Efficient coupling of SNAP-YFP-cytokines to benzylguanine-modified beads was demonstrated by flow cytometry. The fact that enterokinase treatment released most of the fluorescence from the beads is indicative for directed coupling and only marginal adsorptive binding. Cellular responses to SNAP-YFP-cytokine beads were analyzed in cellular lysates and by confocal microscopy indicating that the directionally immobilized cytokines are fully signaling competent with respect to the activation of ERK and STAT3. The strategy presented here is generally applicable for the directed covalent immobilization of fluorescently labeled proteins including the convenient and reliable control of coupling efficiency and orientation.     

Hydroxyl radical footprinting of fluorescently labeled DNA

Footprinting is one of the simplest and most accurate approaches to investigate structure and interaction of biopolymers. It is based on the more difficult accessibility of intra- and intermolecular contacts for external damaging agents. According to this method, one end of polymer molecules is labeled before a sample is incubated with a damaging agent. The distribution of split products is used to conclude on the accessibility of different polymer regions under specific conditions. A variety of enzymatic and chemical splitting agents are used for footprinting. Currently, the highest temporal and spatial resolution without profound specificity to a nucleotide sequence can be reached with the use of hydroxyl radicals. A new variant of this approach, which suggests the use of DNA fluorescent labeling together with the present-day quantitative analysis, will allow extending the method’s boundaries.     

Microsatellite genotyping of post-PCR fluorescently labeled markers

We show that a post-PCR multicolor fluorescence-labeling technique is applicable to multiplex microsatellite genotyping. Forty-three dinucleotide microsatellite markers, which are located on 11q13-23, a candidate region for dominant familial exudative vitreoretinopathy (FEVR), were used to evaluate the quality of the marker profile produced by this technique. Thirty-eight people from six families with this disease were subjects for genotyping. The samples revealed clearly separated fragment peaks with signal intensities sufficient for analysis. All genotypes were consistent with Mendelian inheritance within each family. This method is cost effective because it does not use expensive fluorescently labeled primers. It also has the advantage of avoiding ambiguity in the analysis, which may arise from the addition of non-template nucleotides by Taq DNA polymerase.     

Synthesis and biophysical studies on fluorescently labeled oligodeoxyribonucleotides

Two highly fluorescent compounds, viz. 6-(6-isobutyrylamino-1,3-dioxo-1 H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid and 6-(6-dimethylamino-1,3-dioxo-1 H,3H-benzo[de]isoqu-inolin-2-yl)-hexanoic acid have been synthesized, characterized, and attached to 12-mer oligodeoxyribonucleotides at their 5'-end using suitable linker molecule. These labeled oligodeoxyribonucleotides have shown appreciable fluorescence even at 0.0019 microM concentrations. Thermal denaturation studies have shown comparatively higher Tm values when oligodeoxyribonucleotides are labeled. These labeled oligodeoxyribonucleotides have been purified on RP-HPLC utilizing their hydrophobicity and on polyacrylamide gel because of their easy detection due to fluorescence.     

Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

A fluorescently-labeled, conformationally-sensitive Bacillus stearothermophilus (Bs) dihydrofolate reductase (DHFR) (C73A/S131C_MDCC DHFR) was developed and used to investigate kinetics and protein conformational motions associated with methotrexate (MTX) binding. This construct bears a covalently-attached fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC) attached at a distal cysteine, introduced by mutagenesis. The probe is sensitive to the local molecular environment, reporting on changes in the protein structure associated with ligand binding. Intrinsic tryptophan fluorescence of the unlabeled Bs DHFR construct (C73A/S131C DHFR) also showed changes upon MTX association. Stopped-flow analysis of all data can be understood by invoking the presence of two native state DHFR conformers that bind to MTX at different rates (20.2 and 0.067 μM⁻¹ s⁻¹), similar to previously published findings for Escherichia coli DHFR. Probe fluorescence of C73A/S131C_MDCC DHFR predominantly reports on MTX binding to one of the conformers while intrinsic tryptophan fluorescence of C73A/S131C DHFR reports on binding to the other conformer. This study demonstrates the use of an extrinsic fluorophore attached to a distal region to investigate ligand binding interactions that are not experimentally accessible via intrinsic tryptophan fluorescence alone. The thermostability of C73A/S131C_MDCC DHFR provides an important new tool with applications for investigating the temperature dependence of DHFR conformational changes associated with binding and catalysis.     

Development of a fluorescently labeled thermostable DHFR for studying conformational changes associated with inhibitor binding

The tissue kallikrein (KLK) family contains 15 genes (KLK1–KLK15) tandemly arranged on chromosome 19q13.4 that forms the largest cluster of contiguous protease genes in the human genome. Here, we provide mechanistic evidence showing that the expression of KLK13, one of the most recently identified family members, is significantly up-regulated in metastatic lung adenocarcinoma. Whilst overexpression of KLK13 resulted in an increase in malignant cell behavior, knockdown of its endogenous gene expression caused a significant decrease in cell migratory and invasive properties. Functional studies further demonstrated that KLK13 is activated via demethylation of its upstream region. The elevated KLK13 protein then enhances the ability of tumor cells to degrade extracellular laminin that, subsequently, facilitates cell metastatic potential in the in vivo SCID mouse xenograft model. KLK13 was also found to induce the expression of N-cadherin to help promote tumor cell motility. Together, these results reveal the enhancing effects of KLK13 on tumor cell invasion and migration, and that it may serve as a diagnostic/prognostic marker and a potential therapeutic target for lung cancer.     

Nanosecond study of fluorescently labeled troponin C.

The time-resolved extrinsic fluorescence of rabbit skeletal troponin C was studied with the protein labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Both the intensity and anisotropy decays followed a biexponential decay law, regardless of the ionic condition, pH, viscosity or temperature. The lifetimes and their fractional amplitudes were insensitive to Mg2+, and the lifetimes were also insensitive to Ca2+. In response to Ca2+ binding to all four sites, the fractional amplitude (alpha 1) associated with the short lifetime (tau 1) decreased by a factor of two, thus increasing the ratio of the two amplitudes alpha 2/alpha 1 from 1.6 to 4.3. These amplitude changes suggest the existence of two conformational states of TnC-IAEDANS, with the conformation associated with the long-decay component (tau 2) being promoted by saturation of the two Ca(2+)-specific sites. At pH 5.2 the ratio alpha 2/alpha 1 for the apo-protein was 3.5 indicating different relative populations of the two decay components when compared with pH 7.2. In the presence of Ca2+ at the lower pH, alpha 2/alpha 1 decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca2+ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molecular motions that were not resolved in the present experiments, and some of these motions were sensitive to Ca2+ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of the central helix of troponin C.     

Sensitive detection of histamine using fluorescently labeled oxido-reductases

A detection scheme is described by which the histamine contents of biological samples can be established. The scheme is based on the use of methylamine dehydrogenase (MADH) which converts primary amines into the corresponding aldehydes and ammonia. The generated reducing equivalents are subsequently transferred to the physiological partner of MADH, amicyanin, which thereby is converted from the oxidized blue-colored form into the reduced colorless form. The change in absorption is detected by monitoring the fluorescence of a covalently attached Cy5 dye label whose fluorescence is (partly) quenched by Förster resonance energy transfer (FRET) to the Cu-site of the amicyanin. The quenching efficiency and, thereby, the label fluorescence, depends on the oxidation state of the amicyanin. When adding histamine to the assay mixture the proportionality between the substrate concentration and the observed rate of the fluorescence increase has enabled this assay as a sensor method with high sensitivity. The MADH and amicyanin composition can be tuned so that the sensor can be adapted over a broad range of histamine concentrations (13 nM–225 μM). The lowest concentration detected so far is 13 nM of histamine. The sensor retained its linearity up to 225 μM with a coefficient of variation of 11% for 10 measurements of 100 nM histamine in a 100 μL sample volume. The use of a label fluorescing around 660 nm helps circumventing the interference from background fluorescence in biological samples. The sensor has been tested to detect histamine in biological fluids such as fish extracts and blood serum.     

A filter assay for binding of labeled ligands to membrane-bound receptors

A filter assay was developed for the specific binding of labeled ligands to the acetylcholine receptor in an unpurified suspension of membrane fragments from Torpedo californica. Binding to the filter membrane and the standard deviation of replicates were studied, and it was possible to reduce relative standard deviation to about 3% by accounting for the variable weight of the particles dispensed, stopping the loss of particles to the filter apparatus, regulating the transmembrane pressure during filtration, controlling the time the filter remains subject to suction after all the liquid has passed through the filter, and correcting for variable counting efficiency. A method for dispensing equal aliquots of suspended particulates is also described.     

Validation of a flow cytometric acridine orange micronuclei methodology in rats

Our laboratory has previously reported a flow cytometric acridine orange method for detection of micronucleus (MN) in the rat using cyclophosphamide as a test compound. To replace the manual method of scoring and satisfy Good Laboratory Practice (GLP) requirements, an extensive validation of the flow method was required. Therefore, manual scoring and flow cytometric determination of MN were compared using vincristine, chlorambucil, methotrexate, and doxorubicin compounds known to induce MN formation with various mechanisms of action. 1,2-Dimethylhydrazine (1,2-DH), a compound with negative or equivocal MN findings also was evaluated. The flow method consistently demonstrated dose- and time-dependent responses for MN production at all concentrations of vincristine, methotrexate, clorambucil, and doxorubicin. In contrast, manual scoring of slides failed to detect an increase in MN at the lowest doses of doxorubicin (1mg/kg) at 24 or 48 h, and methotrexate at 48 h, or any dose of methotrexate (50, 100, or 250 mg/kg) at 24h. Additionally, a dose-response for methotrexate at 48 h, and chlorambucil at 24 h were missed using manual scoring. For 1,2-DH, the flow method showed a low level (< 1.4-fold) increase in MN at all doses and times. In contrast, the manual method showed five-seven-fold increases at 24 h, but a < two-fold increase at 48 h in the highest dose only. These data may suggest that the flow method has a greater sensitivity and possibly accuracy than manual scoring. Significant decreases in polychromatic erythrocytes (PCE) were seen using both methods at approximately the same dose for all compounds. However, absolute flow cytometric PCE values were consistently higher than manual. Additional cytotoxicity parameters obtained by the flow method allowed a more complete assessment of cytotoxicity than PCE alone. Furthermore, data reported here combined with improved throughput, shortened data turnaround and reporting times, and possibly better precision due to evaluation of much larger numbers of cells clearly demonstrate the usefulness of flow cytometry method in the routine micronucleus evaluation.     

Binding of protein kinase substrates by fluorescently labeled calmodulin

A synthetic protein kinase substrate, PRO-LEU-SER-ARG-THR-LEU-SER-VAL-SER-SER-NH₂, undergoes calcium-dependent binding by calmodulin. Phosphorylation of the peptide decreases its affinity for calmodulin with the dissociation constant increasing from 2.4 to $\text{ca}$. 7 mM. The results are consistent with the suggestion that calmodulin and the cAMP-dependent protein kinase can act on common recognition sequences.     

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.     

Phagotrophy of fluorescently labeled bacteria by an oceanic phytoplankter

Using fluorescently-labeled bacteria and detection by flow cytometry and epifluorescence microscopy, we demonstrate inducible mixotrophy in a marine photosynthetic flagellate, Ochromonas sp. (class Chrysophyceae). Phagotrophic uptake of bacteria increases under conditions of low or limiting light and nutrients, but deceases in periods of prolonged darkness; sustained phagotrophy may require light. In addition, this alga appears to discriminate between and preferentially ingest different types of bacteria. Although this clone is primarily photosynthetic, phagotrophy contributes to its nutrition, especially when light or nutrients limit photosynthesis.Correspondence to: M.D. Keller     

Reduction of lipofuscin-like autofluorescence in fluorescently labeled tissue.

The fluorescent pigment lipofuscin accumulates with age in the cytoplasm of cells of the CNS. Because of its broad excitation and emission spectra, the presence of lipofuscin-like autofluorescence complicates the use of fluorescence microscopy (e.g., fluorescent retrograde tract tracing and fluorescence immunocytochemistry). In this study we examined several chemical treatments of tissue sections for their ability to reduce or eliminate lipofuscin-like autofluorescence without adversely affecting other fluorescent labels. We found that 1-10 mM CuSO4 in 50 mM ammonium acetate buffer (pH 5) or 1% Sudan Black B (SB) in 70% ethanol reduced or eliminated lipofuscin autofluorescence in sections of monkey, human, or rat neural tissue. These treatments also slightly reduced the intensity of immunofluorescent labeling and fluorescent retrograde tract tracers. However, the reduction of these fluorophores was far less dramatic than that for the lipofuscin-like compound. We conclude that treatment of tissue with CuSO4 or SB provides a reasonable compromise between reduction of lipofuscin-like fluorescence and maintenance of specific fluorescent labels.