Regulation of estrogen receptor protein and messenger ribonucleic acid by estradiol and progesterone in rat uterus

The objective of this study was to examine the mechanisms of estrogen receptor (ER) processing and replenishment in the uterus of ovariectomized rats after estradiol and progesterone treatment. Uterine ER binding activity, ER protein and ER mRNA were measured by receptor binding exchange assay, Western blot and slot blot, respectively. The regulation of ER levels in rat uterus by estradiol and progesterone was very dramatic. Changes in ER protein were faithfully reflected by changes in binding activity. Estradiol caused receptor “processing” within 4 h of administration followed by recovery or “replenishment” of ER levels to the initial level by 20 h. The term “processing” has previously been used to describe the loss of ER binding activity in the early phase of estradiol-action, but it was never clear whether the ligand binding site was inactivated by processing or if the receptor molecule actually disappeared. This study shows that receptor “processing” constitutes disappearance of receptor protein and the later “replenishment” phase represents new ER protein rather than recycling of “processed” receptor. Progesterone-action, on the other hand, influenced only the “replenishment” phase by blocking recovery of ER protein. ER mRNA was suppressed by estradiol at 8 h, after the receptor was “processed” and “replenishment” already initiated. Progesterone, on the other hand, did not alter the steady state level of the message. Other mechanisms, such as regulation of translation rate of existing mRNA and changes in the rate of degradation of ER proteins are more likely involved in acute regulation of ER by these ovarian steroid hormones.     

Human mitochondrial NDUFS3 protein bearing Leigh syndrome mutation is more prone to aggregation than its wild-type

NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCl induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly.     

Kinetics of binding between thermolysin and Streptomyces metalloprotease inhibitor, talopeptin (MKI)

The mechanism of binding between thermolysin with its specific inhibitor, talopeptin (MKI), was studied kinetically with the stopped-flow method by monitoring the enhancement of tryptophan fluorescence caused by the complex formation. Only one relaxation obeying first-order kinetics was observed. The dependence of the apparent first-order constant, kapp, on the inhibitor concentration is consistent with a minimum two-step mechanism, including a fast bimolecular binding step followed by a slow unimolecular step. It was found that the increase in tryptophan fluorescence occurs solely in the slow unimolecular step. The apparent second-order rate constant, (kon)app, in the low inhibitor concentration range, was determined over the pH range between 5 and 8.5 and decreases with increasing pH. The activation parameters for the overall binding process were obtained from the temperature dependence of (kon)app.     

Visualization and quantification of endoplasmic reticulum Ca2+ in renal cells using confocal microscopy and Fluo5F

Sarcoplasmic/endoplasmic reticulum (ER) Ca(2+) is the most abundant store of intracellular Ca(2+), and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca(2+) in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca(2+) indicator, to directly monitor changes in RPTC ER Ca(2+). Fluo5F staining reflected ER Ca(2+), resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca(2+) pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca(2+) ionophore caused more rapid ER Ca(2+) release (55% and 75% decrease in fluorescence at 5 and 15 min). Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca(2+). In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca(2+) release. ER Ca(2+) release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca(2+) in live cells.     

Stimulation of microsecond-delayed fluorescence from spinach chloroplasts by uncouplers and by phosphorylation

Delayed fluorescence, as measured with a laser phosphoroscope, is stimulated not inhibited by uncouplers during the first 100 μs after the light is turned off. This is true only wen uncouplers cause an increase in the rate of electron transport. When ADP and P_i cause an increase in the electron transport rate, microsecond-delayed fluorescence is also increased. Indeed, there is a complex quantitative relationship between the rate of electron transport and the initial intensity of delayed fluorescence under a wide range of conditions.

Uncouplers or ADP and P_i also increase the rate of decay of delayed fluorescence so that after about 150 μs they become inhibitory, as already reported by many authors.

Microsecond-delayed fluorescence continues to rise with rising light intensities long after the rate of reduction of exogenous acceptor is light-saturated.

These observations suggest a correlation of the rate of electron transport both with the intensity of the 5–100 μs-delayed fluorescence and with the rate of decay in the intensity of delayed fluorescence. The data imply that the decrease in intensity of millisecond-delayed fluorescence which has often been noted with uncouplers is probably not due to the elimination of a membrane potential. It seems more likely that the decrease in millisecond-delayed fluorescence is a reflection of the rate of disappearance of some other electron transport-generated condition, a condition which is uncoupler-insensitive. Certainly stimulations of microsecond-delayed fluorescence by electron transport which has been uncoupled by gramicidin suggest that ion gradients are not an essential component of the conditions responsible for delayed fluorescence.     

Mitochondrial membrane potential in lymphocytes as monitored by fluorescent cation diS-C3-(5)

A lipophilic fluorescent cation diS-C3-(5) and rotenone suppress the oxygen consumption rate of thymocytes in similar concentrations. Seventy percent inhibition corresponds to an inhibitor:cytochrome a molar ratio of about 1:1. Addition of uncouplers decreases the inhibition of respiration by diS-C3-(5) (but not rotenone). FCCP in similar concentrations increases O2 consumption in the absence of diS-C3-(5) and the diS-C3-(5) fluorescence intensity in the presence of TMPD in thymocyte suspensions. In most thymocyte preparations, oligomycin (0.05-0.1 microgram/mL) increases the fluorescence of diS-C3-(5) and further addition of TMPD (50-100 microM) decreases the fluorescence. Addition of NaCN (400 microM) after oligomycin leads to a fluorescence increase that is hardly affected by subsequent addition of 0.2 microM FCCP. Nigericin (10-50 nM) decreases the diS-C3-(5) fluorescence. The data indicate that the diS-C3-(5) fluorescence associated with mitochondrial transmembrane potential (delta psi m) may be an essential part of the diS-C3-(5) fluorescence in lymphocyte suspensions. The changes of the diS-C3-(5) fluorescence intensity in the presence of TMPD after FCCP addition reflect delta psi m.     

Visualization and quantification of endoplasmic reticulum Ca in renal cells using confocal microscopy and Fluo5F

Sarcoplasmic/endoplasmic reticulum (ER) Ca²⁺ is the most abundant store of intracellular Ca²⁺, and its release is an important trigger of physiological and cell death pathways. Previous work in our laboratory revealed the importance of ER Ca²⁺ in toxicant-induced renal proximal tubular cell (RPTC) death. The purpose of this study was to evaluate the use of confocal microscopy and Fluo5F, a low affinity Ca²⁺ indicator, to directly monitor changes in RPTC ER Ca²⁺. Fluo5F staining reflected ER Ca²⁺, resolved ER structure, and showed no colocalization with tetramethyl rhodamine methyl ester (TMRM), a marker of mitochondrial membrane potential. Thapsigargin, an ER Ca²⁺ pump inhibitor, decreased ER fluorescence by 30% and 55% at 5 and 15 min, respectively, whereas A23187, a Ca²⁺ ionophore caused more rapid ER Ca²⁺ release (55% and 75% decrease in fluorescence at 5 and 15 min).Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler, added at the end of the experiment, further decreased ER fluorescence after thapsigargin treatment, revealing that thapsigargin did not release all ER Ca²⁺. In contrast, FCCP did not decrease ER fluorescence after A23187 treatment, suggesting complete ER Ca²⁺ release. ER Ca²⁺ release in response to A23187 or thapsigargin resulted in a modest but significant decrease in mitochondrial membrane potential. These data provide evidence that confocal microscopy and Fluo5F are useful and effective tools for directly monitoring ER Ca²⁺ in live cells.     

Use of fluorescence enhancement technique to study bilirubin–albumin interaction

Bilirubin–albumin solution gave an emission spectrum in the wavelength range 500–600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 μM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin–albumin interaction and the value of binding constant was found to be 1.72×10⁷ l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin–albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin–albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin–albumin interaction.     

Bromobimanes — fluorescent labeling agents for histochemical detection of sulfur containing neuropeptides in semithin sections

Summary Sulfur containing neuropeptides could be demonstrated in semithin sections of invertebrate nervous tissue, especially of gastropods, by using the bromobimanes as fluorescent labeling agents for thiol groups. Semithin sections showed a brilliant fluorescence of labeled peptides and should be used if an excellent resolution is important. The three bromobimanes (MB: monobromobimane, DB: dibromobimane, MQ: monobromotrimethylammoniobimane) gave positive results under our experimental conditions. Dibromobimane (DB) was selected because the application is more convenient.In gastropods, the bromobimane technique seems to be the most specific and sensitive one compared to the classical neurosecretory staining methods. Neuropeptides with sulfur containing amino acids could be demonstrated in perikarya, axons, and axon swellings easily. We suppose that there are neurons—not stainable by the classical methods—which can be identified as peptidergic ones by the bromobimane technique.A slight reduction of fluorescence intensity (fading) was observed. So, the fading rate was determined for dibromobimane reaction products; a decrease in the fluorescence intensity of 50% was only reached after 1 h using a Neofluar objective 10/0.30. Nevertheless, we suppose that a comparative quantification of the labeled neuropeptides should be possible if special parameters are considered.     

Functional interactions between subunits of aspartate aminotransferase: Formation of monoliganded dimers during titration of the apoenzyme by pyridoxal 5′-phosphate

The interaction between apoaspartate aminotransferase and pyridoxal 5′-phosphate at either pH 8.3 (active form of holoenzyme) or pH 5.0 (inactive form) corresponds to a strong quenching of tryptophan fluorescence. The hybrid molecule containing one pyridoxal 5′-phosphate bound per dimer has been prepared both by electrofocusing and by ion exchange chromatography. At both pH values, the fluorescence of the hybrid is 80 to 85% of the arithmetic mean between the fluorescence of the symmetrical holoenzyme and apoenzyme. This is direct evidence of energy transfer from tryptophan residues of the subunit of apoenzyme to the coenzyme of the other subunit.Fluorescence intensity was used to determine the quantity of hybrid holoapoenzyme formed during titration of the apoenzyme by pyridoxal 5′-phosphate. At pH 8.3 a non-linear decrease in the fluorescence is observed, corresponding to 60% of hybrid for the point of half reactivation; this value corresponds to the percentage obtained by electrofocusing (Schlegel & Christen, 1974). At pH 5.0, the decrease in fluorescence is linear during pyridoxal binding; this indicates that at this pH the hybrid is never obtained at detectable concentrations. These results indicate strong interactions between subunits of aspartate aminotransferase corresponding to a weakly negative co-operativity at alkaline pH and a positive cooperativity at acidic pH for the binding of the coenzyme.     

A kinetic study of the subunit dissociation and reassembly of rabbit muscle phosphofructokinase.

The kinetics of dissociation and reassembly of rabbit skeletal muscle phosphofructokinase has been studied using fluorescence, stopped-flow fluorescence and enzyme activity measurements. The dissociation of the fully active tetramer in 0.8 M guanidine hydrochloride (0.1 M potassium phosphate, pH 8.0) occurs in three kinetic phases as measured by changes in the protein fluorescence emission intensity: dissociation of tetramer to dimer with a relaxation time of a few milliseconds; dissociation of dimer to monomer with a relaxation time of a few seconds; and a conformational change of the monomer with a relaxation time of a few minutes. All three phases exhibit first-order kinetics; ATP (0.05 mM) retards the second step but does not influence the rate of the other two processes. The rate of the second process increases with decreasing temperature; this may be due to the involvement of hydrophobic interactions in the stabilization of the dimeric enzyme. A further unfolding of the monomer polypeptide chain occurs at higher guanidine concentrations, and the relaxation time associated with this process was found to be 83 ms in 2.5 M guanidine, 0.1 M potassium phosphate (pH 8.0) at 23 degrees C. The phosphofructokinase monomers were reassembled from 0.8 M guanidine chloride by 1:10 dilution of the guanidine hydrochloride concentration and yielded a protein with 70-94% of the original activity, depending on the protein concentration. The reactivation process follows second-order kinetics; ATP (5 mM) increases the rate of reactivation without altering the reaction order, while fructose 6-phosphate does not influence the rate of reaction. The rate-determining step is probably the association of monomers to form the dimer.     

Homeostasis of the protonmotive force in phosphorylating mitochondria

The relationship between the respiration rate and the magnitude of the electrochemical proton potential (Δμ_H⁺) in rat liver mitochondria was investigated. (1) Under the active-state conditions, the action of inhibitors of either phosphorylation (oligomycin) or respiration (rotenone, malonate) on the respiration and Δμ_H⁺ was measured. Both inhibitors diminished the respiration, whereas rotenone resulted in a decrease of Δμ_H⁺, and oligomycin produced an increase of this potential. The effect of the inhibitors was much more pronounced on the respiration rate than on Δμ_H⁺; for example, the excess of oligomycin produced a 90% inhibition of the respiration while Δμ_H⁺ was changed only by 9%. (2) Under the resting-state conditions, small concentrations of the uncoupler stimulated the respiration while changing Δμ_H⁺ to a relatively small extent. The uncoupler concentrations which doubled and tripled the respiration rate produced only 5 and 9% decrease of Δμ_H⁺, respectively. (3) The present results enabled us to propose a model describing the interrelationship between respiration and Δμ_H⁺.     

Modulation of the binding characteristics of a fluorescent nucleotide derivative to the sarcoplasmic reticulum adenosinetriphosphatase

Trinitrophenyladenosine monophosphate (TNP-AMP) binding to the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum results in manyfold higher fluorescence intensity and longer lifetimes of the nucleotide analogue, as compared to TNP-AMP binding to the nonphosphorylated enzyme. This is observed when the phosphoenzyme intermediate is formed either from ATP or from inorganic phosphate (Pi). An important question is whether the TNP-AMP fluorescence properties can also reflect the kinetically defined interconversions of different phosphoenzyme species during catalysis. We have approached this question by manipulating the phosphorylation conditions in a manner which is known to result in accumulation of different species of the phosphoenzyme, i.e., by variations in pH, substrates, and K+ and Ca2+ concentrations. Decreasing pH or increasing [K+] caused large decreases in fluorescence intensity at a given concentration of TNP-AMP under conditions of phosphorylation with either ATP or Pi. In contrast, low to high intravesicular Ca2+ concentrations had no effect on fluorescence during steady-state turnover. TNP-AMP titrations of the phosphorylated enzyme stabilized in different states revealed that H+ and K+ caused a shift in TNP-AMP binding affinity to the site responsible for high fluorescence enhancement, while maintaining approximately the same maximal fluorescence yield at saturation. The fluorescence lifetimes of TNP-AMP bound to phosphoenzyme did not change with variations in pH, [K+], and substrates. We conclude that the environment of that part of the TNP-AMP binding site which binds the trinitrophenyl moiety undergoes a change upon enzyme phosphorylation resulting in enhanced fluorescence yield; this change is invariant between different phosphoenzyme species.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of fluorescent 4,6,8(14)-triene-3-one steroids to cyclodextrins as a model for steroid-protein interactions

The 4,6,8(14)-triene-3-one steroids, highly fluorescent in aqueous solutions, lose their fluorescence power when binding occurs to hydrophobic regions of other molecules, such as the hydrophobic cavity in the ring system of cyclodextrins. The fluorescence intensity decreases almost completely when beta- and gamma-cyclodextrins are present in the solution. Scatchard plots derived from fluorescence titrations show that one or two molecules of steroid bind to one cyclodextrin molecule with KD,F-values of about 10(-4)-10(-5) mol/liter. Temperature-jump experiments show a single relaxation process, with rate constants for the decay of the beta-cyclodextrin-steroid complexes of about 10(4)-10(5) per s. For alpha- and gamma-cyclodextrins such relaxation processes are not observed.     

Binding of fluorescent lanthanides to rat liver mitochondrial membranes and calcium ion-binding proteins.

(1) Tb3+ binding to mitochondrial membranes can be monitored by enhanced ion fluorescence at 545 nm with excitation at 285 nm. At low protein concentrations (less than 30 mug/ml) no inner filter effects are observed. (2) This binding is localized at the external surface of the inner membrane and is unaffected by inhibitors of respiration or oxidative phosphorylation. (3) A soluble Ca2+ binding protein isolated according to Lehninger, A.L. ((1971) Biochem. Biophys. Res. Commun. 42, 312-317) also binds Tb3+ with enhanced ion fluorescence upon excitation at 285 nm. The excitation spectrum of the isolated protein and of the intact mitochondria are indicative of an aromatic amino acid at the cation binding site. (4) Further characterization of the Tb3+-protein interaction revealed that there is more than one binding site per protein molecule and that these sites are clustered (less than 20 A). Neuraminidase treatment or organic solvent extraction of the protein did not affect fluorescent Tb3+ binding. (5) pH dependency studies of Tb3+ binding to the isolated protein or intact mitochondria demonstrated the importance of an ionizable group of pK greater than 6. At pH less than 7.5 the amount of Tb3+ bound to the isolated protein decreased with increase in pH as monitored by Tb3+ fluorescence. With intact mitochondria the opposite occurred with a large increase in Tb3+ fluorescence at higher pH. This increase was not observed when the mitochondria were preincubated with antimycin A and rotenone.     

An evaluation of N-phenyl-1-naphthylamine as a probe of membrane energy state in Escherichia coli

Colicin E1 and the uncoupler of oxidative phosphorylation, trifluoromethoxy-carbonylcyanidephenylhydrazone (FCCP), cause an increase in the fluorescence intensity of N-phenyl-1-naphthylamine bound to whole cells of Escherichia coli. It has been shown elsewhere that this fluorescence increase correlates well with de-energization. Addition of glucose causes a large cyanide-sensitive decrease of intensity, tentatively associated with energization, with the emission spectrum almost returning to the original trace with a peak at 417 nm. These data suggest that there may be a measurable competition between de-energization and energization of the cell membrane, and that the probe fluorescence intensity may be a general indicator of membrane energy level.

The conclusions reached about cellular energy level from measurements of the probe fluorescence intensity correlate partly (a, b below, not c) with the energy level assayed physiologically through rates of active transport: (a) FCCP is found to be a poor inhibitor of proline transport if cells are first incubated with glucose, showing either competition between the processes of energization and de-energization or an increase in the envelope permeability barrier to FCCP caused by glucose addition. (b) Cyanide blocks the fluorescence decrease caused by glucose and inhibits proline and serine transport, consistent with the decrease in probe fluorescence intensity indicating an increase in membrane energization. However, (c) it appears that the amplitude of the fluorescence intensity decrease caused by glucose addition in the presence of FCCP and colicin E1 greatly exaggerates the extent of real membrane energization. Glucose added after uncoupler can cause only a small increase, and after colicin, a negligible increase in the proline transport rate, indicating that the magnitude of the fluorescence intensity decrease after glucose addition is not a true measure of membrane energization, but rather seems to amplify this energization greatly. Glucose addition does not cause a decrease in fluorescence intensity in cells treated with EDTA to remove lipopolysaccharide and an apparent barrier to the probe.

The rotational relaxation time of the probe in intact cells appears to correlate somewhat better with the cellular energy level than does intensity.     

Mechanism of protein-induced membrane fusion: fusion of phospholipid vesicles by clathrin associated with its membrane binding and conformational change

The clathrin-induced fusion of liposome membranes, the membrane binding of clathrin, and the conformational states of clathrin were investigated over a wide pH range using large unilamellar and multilamellar vesicles composed of phosphatidylserine (PS), phosphatidylcholine (PC), PS/PC (2:1), PS/PC (1:1), or PS/PC (1:2). The pH profiles of clathrin-induced fusion of all types of liposomes containing PS showed biphasic patterns. Their pH thresholds were found in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH range of 5-6 and shifted to lower pH values with decrease in the PS content. Similar shifts were observed in the pH profiles of clathrin binding to these vesicles, but the pH profiles of binding were different from the biphasic fusion patterns. With PC vesicles, only small degrees of fusion and clathrin binding were observed at pH 2-4. The pH dependences of the conformation and hydrophobicity of clathrin were determined by measuring the extent of the blue shift of the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate in the presence of the protein, the fluorescence intensity of N-(1-anilinonaphthyl-4)maleimide bound to the clathrin molecule, the resonance energy transfer from its tryptophan to anilinonaphthyl residues, the partitioning of the protein in Triton X-114 solution, and the hydrophobicity index of clathrin using cis-parinaric acid. These measurements indicated that conformational change and exposure of hydrophobic regions occur below pH 6 and suggested that clathrin may adopt different conformational states in the pH region where it induced membrane fusion.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differentiation Between Alterations in Plasma and Mitochondrial Membrane Potentials in Synaptosomes Using a Carbocyanine Dye

The cationic potentiometric fluorescent probe 3,3'-diethylthiadicarbocyanine iodide [DiS-C2(5)] was used in synaptosomes to assess the relative contributions of plasma and mitochondrial membrane potentials (psi p and psi m, respectively) to overall fluorescence. Addition of synaptosomes to media containing 0.5 microM dye caused a decrease in fluorescence intensity due to dye accumulation, which equilibrated usually within 5 min. Depolarization of mitochondria by combined treatment with cyanide and oligomycin increased fluorescence by 42%, indicating significant prior accumulation of dye into intrasynaptosomal mitochondria. psi p was calculated to be -54 mV and was not altered significantly by prior depolarization of psi m with cyanide and oligomycin (hereafter referred to as "poisoned" synaptosomes). Similarly, the linear relationship between dye fluorescence and psi p was not altered by depolarization of psi m. Valinomycin, a K+ ionophore, caused a psi p-dependent increase in fluorescence in control (nonpoisoned) synaptosomes, but did not alter fluorescence of poisoned synaptosomes except when the extracellular concentration of K+ ([K+]e) was 2 mM, in which case valinomycin hyperpolarized psi p by about 5 mV. The pore-forming antibiotic gramicidin depolarized both psi p and psi m maximally. Under these conditions, Triton X-100 further increased fluorescence by 40%, indicating significant dye binding to synaptosomal components. In poisoned synaptosomes depolarized by 75 mM K+, gramicidin caused a decrease in fluorescence intensity (hyperpolarization of psi p). The organic solvent dimethyl sulfoxide, used as a vehicle for the hydrophobic ionophores, had voltage-dependent effects on psi p and psi m.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ionic strength on the binding of ascorbate to albumin

A fluorophore-nitroxide free radical dual-functional probe (FN) was utilized to study the kinetics of ascorbate (AH(-)) binding to Bovine Serum Albumin (BSA). Since the free radical fragment in the FN probe intramolecularly quenches fluorescence, ascorbate reduction of the nitroxide function is accompanied by a concomitant fluorescence intensity increase from the fluorophore. Thus, both fluorescence and the EPR techniques could be utilized to measure the reaction rate. In the presence of BSA protein, the observed rate of the overall process is the sum of that from at least two reactions: the reaction between free ascorbate and free probe, and the reaction between bound ascorbate and bound probe. Our findings show that the observed rate is strongly dependent on the ionic strength of the medium. A corollary of this observation is the indication of a purely electrostatic interaction between ascorbate and the BSA protein. This conclusion was further corroborated by 1H NMR measurement of the transverse relaxation time, T(2), of ascorbate protons in BSA solutions. Ascorbate ion was released from the ascorbate/BSA ensemble in the presence of increasing concentrations of NaCl. Binding constants of AH(-) to BSA were calculated at different ionic strengths at pH 7.4. Furthermore, an increase in ionic strength did not affect the ability of albumin to protect ascorbate against autoxidation. This suggests that the protein's protective antioxidant effect may be attributed to BSA binding of trace quantities of transition-metal cations (rather than ascorbate binding to BSA). This conclusion is supported by ascorbate UV-absorption measurements in the presence of albumin and Cu(2+) ions as a function of ionic strength.     

Direct measure of fluorescence intensity for efficient receptor-binding assay: conjugates of ethinylcarboxyestradiol and 5(and 6)-carboxyfluorescein via alpha, omega-diaminoalkanes as a tracer for estrogen receptor

Steroidal nuclear receptors (NRs) have been acknowledged as a target binding protein of so-called endocrine disruptors. It is therefore necessary to develop an efficient assay system for screening these endocrine-disrupting chemicals. We here describe the first exemplification of a direct measure of fluorescence intensity for a binding assay of NRs. We designed and synthesized a series of conjugates of 17alpha-ethinylcarboxyestradiol with carboxyfluorescein, both carboxyl groups of which were cross-linked with alpha,omega-diaminoalkanes. The resulting fluorescein-linked estradiol derivatives E2(n)cF (n=2, 4, 6, 8, 10 and 12) were evaluated for their fluorescence and receptor-binding characteristics. E2(4)cF and E2(8)cF exhibited the sufficient binding affinity to the recombinant estrogen receptor (ER) in the radiolabel binding assay using [(3)H]17beta-estradiol, and showed excellent fluorescent characteristics in the fluorescence measurements with and without ER. They exhibited sufficiently large specific binding characteristics with adequate K(d)- and B(max)-values. When these fluorescent ligands were used as a tracer for the binding assay against the ER, assay data of various compounds were shown to be compatible with those obtained from the ordinary binding assay using [(3)H]17beta-estradiol. The present study clearly shows that measurement of fluorescence intensity, instead of fluorescence polarization, affords an adequate receptor-binding assay system.