Fall In Fluorescence Research Articles

The residence of synaptically released dopamine on D2 autoreceptors.

SUMMARYNeuromodulation mediated by synaptically released endogenous transmitters acting in G-protein-coupled receptors (GPCRs) is slow primarily because of multistep downstream signaling. What is less well understood is the spatial and temporal kinetics of transmitter and receptor interaction. The present work uses the combination of the dopamine sensor, dLight, to detect the spatial release and diffusion of dopamine and a caged form of a D2-dopamine receptor antagonist, CyHQ-sulpiride, to rapidly block the D2 autoreceptors. Photoactivation of the CyHQ-sulpiride blocks receptors in milliseconds such that the time course of dopamine/receptor interaction is mapped onto the downstream signaling. The results show that highly localized release, but not dopamine diffusion, defines the time course of the functional interaction between dopamine and D2 autoreceptors, which determines downstream inhibition.

Changes in the antioxidant properties of protein solutions in the presence of epigallocatechin gallate

β-Casein and α-casein showed radical-scavenging activities in aqueous solution, whereas bovine serum albumin (BSA), α-lactalbumin and β-lactoglobulin showed much weaker antioxidant activity, when assessed by the 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical-scavenging assay. However, β-casein and α-casein showed reduced antioxidant activity after storage at 30 °C. An increase in radical-scavenging activity and a fall in fluorescence of the protein component were evident after 6 h, when BSA, β-lactoglobulin or casein were mixed with EGCG, and excess EGCG was removed, indicating the formation of a complex with this protein on mixing. Storage of all the proteins with EGCG at 30 °C caused an increase in the antioxidant activity of the isolated protein component after separation from excess EGCG. This showed that EGCG was reacting with the proteins and that the protein-bound catechin had antioxidant properties. The reaction of EGCG with BSA, casein and β-lactoglobulin was confirmed by the loss of fluorescence of the protein on storage, and the increase in UV absorbance between 250 and 400 nm. The increase in antioxidant activity of BSA after storage with EGCG was confirmed by the ferric reducing antioxidant potential (FRAP) and the oxygen radical antioxidant capacity (ORAC) assays.

Measurement of cytoplasmic calcium concentration in the rods of wild-type and transducin knock-out mice.

A 10 microm spot of argon laser light was focused onto the outer segments of intact mouse rods loaded with fluo-3, fluo-4 or fluo-5F, to estimate dark, resting free Ca(2+) concentration ([Ca(2+)](i)) and changes in [Ca(2+)](i) upon illumination. Dye concentration was adjusted to preserve the normal physiology of the rod, and the laser intensity was selected to minimise bleaching of the fluorescent dye. Wild-type mouse rods illuminated continuously with laser light showed a progressive decrease in fluorescence well fitted by two exponentials with mean time constants of 154 and 540 ms. Rods from transducin alpha-subunit knock-out (Tralpha-/-) animals showed no light-dependent decline in fluorescence but exhibited an initial rapid component of fluorescence increase which could be fitted with a single exponential (tau~1-4 ms). This fluorescence increase was triggered by rhodopsin bleaching, since its amplitude was reduced by pre-exposure to bright bleaching light and its time constant decreased with increasing laser intensity. The rapid component was however unaffected by incorporation of the calcium chelator BAPTA and seemed therefore not to reflect an actual increase in [Ca(2+)](i). A similar rapid increase in fluorescence was also seen in the rods of wild-type mice just preceding the fall in fluorescence produced by the light-dependent decrease in [Ca(2+)](i). Dissociation constants were measured in vitro for fluo-3, fluo-4 and fluo-5F with and without 1 mM Mg(2+) from 20 to 37 degrees C. All three dyes showed a strong temperature dependence, with the dissociation constant changing by a factor of 3-4 over this range. Values at 37 degrees C were used to estimate absolute levels of rod [Ca(2+)](i). All three dyes gave similar values for [Ca(2+)](i) in wild-type rods of 250 +/- 20 nM in darkness and 23 +/- 2 nM after exposure to saturating light. There was no significant difference in dark [Ca(2+)](i) between wild-type and Tralpha-/- animals.

Integration of calcium signals by calmodulin in rat sensory neurons.

We have used the fluorescently labelled calmodulin TA-CaM to follow calmodulin activation during depolarization of adult rat sensory neurons. Calcium concentration was measured simultaneously using the low affinity indicator Oregon Green BAPTA 5N. TA-CaM fluorescence increased during a 200-ms depolarization but then continued to increase during the subsequent 500 ms, even though total cell calcium was falling at this time. In the next few seconds TA-CaM fluorescence fell, but to a new elevated level that was then maintained for several tens of seconds. During a train of depolarizations that evoked a series of largely independent calcium changes TA-CaM fluorescence was in contrast raised for the duration of the train and for many tens of seconds afterwards. The presence of a peptide corresponding to the calmodulin binding domain of myosin light chain kinase significantly increased the depolarization-induced TA-CaM fluorescence increase and slowed the subsequent fall of fluorescence. We interpret the slow recovery component of the TA-CaM signal as reflecting the slow dissociation of calcium--calmodulin--calmodulin binding protein complexes. Our results show that after brief electrical activity calmodulin's interaction with calmodulin binding proteins persists for approximately one minute.

Poor mixing of microdrops with the tear fluid reduces the accuracy of tear flow estimates by fluorometry.

The initial rapid fall in tear film fluorescence after instilling a 1 microliter drop of fluorophore could be a result of stimulated tear flow or of distribution of the dye throughout the tear fluid present in the conjunctival sac. An attempt was made to decide this by comparing the fall in fluorescence of two different fluorophores instilled at an interval of several minutes. In fact, very few patterns of initial rapid loss were found, but it was noted that there was usually a strong gradient of fluorescence from side to side across the cornea that took several minutes to dissipate. It seems that lateral mixing of a microdrop with the tear fluid is not instantaneous and that caution should be exercised in interpreting such measurements to estimate the total volume of tear fluid.

A continuous fluorescence-displacement assay for triacylglycerol lipase and phospholipase C that also allows the measurement of acylglycerols

A new continuous fluorescence-displacement assay for enzymes that release long-chain fatty acids [Wilton (1990) Biochem. J. 266, 435-439] is described in detail for pig pancreatic triacylglycerol lipase. The assay involves the displacement of the highly fluorescent fatty acid probe 11-(dansylamino)undecanoic acid from rat liver fatty acid-binding protein by long-chain fatty acids released as a result of enzyme activity. The assay is surprisingly effective for triacylglycerol lipase, allowing the expression of full activity with low concentrations of substrates in the absence of detergents. The initial rate of decrease in fluorescence is linearly related to enzyme concentration, and activity can be detected in the assay down to concentrations of 10 pg of pure enzyme/ml. The assays demonstrated the quantitative conversion of limiting amounts of substrate into the monacylglycerol. This observation allowed the assay to be used to measure substrates such as triacylglycerols and particularly 1,2-diacylglycerols at concentrations down to about 0.1 microM. Because phospholipase C releases 1,2-diacylglycerols, the coupling of this enzyme to excess lipase allowed the measurement of pure phospholipase C from Bacillus cereus at concentrations down to about 2 ng/ml, and the initial rate of fall in fluorescence in the assay was linearly related to enzyme activity.

Monitoring receptor mediated regulation of cytosolic calcium in single pituitary cells by dual excitation microfluorimetry.

Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca2+]i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluorimetry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, lambda 1 and lambda 2, at which an increase in [Ca2+]i causes either a rise or a fall in fluorescence; the ratio of fluorescence at lambda 1 and lambda 2 (R = F lambda 1/F lambda 2) is a measure of [Ca2+]i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca2+]i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca2+]i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca2+]i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.

Direct evidence for the modulation of human platelet cytosolic free Ca 2+ by intracellular cyclic AMP produced with a photoactivatable derivative

We have previously reported that intraplatelet “cyclic AMP jumps” produced with newly synthesized photoactivatable cyclic AMP analogue, inhibited washed rat platelet aggregation and serotonin release as induced by thrombin. Using the same approach on human platelets, thrombin-induced platelet aggregation was dose-dependently inhibited only when a flash was delivered. The mechanism of action of intraplatelet cyclic AMP as resulting from photolysis could be by controlling the level of cytosolic Ca 2+. In order to test this hypothesis, the same protocol was used on human platelets preloaded with the internal Ca 2+ fluorescent indicator, Quin 2, we found that the extent and the rate of the rise of the cytosolic Ca 2+ induced by thrombin were dramatically decreased, in the presence of the photoactivatable cyclic AMP, only following photoirradiation. In addition, the flashes were produced, in the presence of photoactivatable cyclic AMP, after the thrombin-induced rise of internal Ca 2+ had reached its peak. In these conditions, photoirradiation caused a rapid fall in fluorescence. These experiments provide the first direct evidence that intracellular cyclic AMP is involved in the control of platelet cytosolic Ca 2+ by inhibition of its mobilization and by stimulation of its sequestration.

Glutathione localization by a novel o-phthalaldehyde histofluorescence method.

Glutathione in tissues forms an intense fluorophore with a solution of o-phthalaldehyde at room temperature. We have studied the loss of glutathione from tissue sections and find that it is not measurable from thick sections. The fluorescence spectra of the induced fluorophore between glutathione and o-phthalaldehyde are identical in model and tissue sections, while depletion of hepatic glutathione by diethyl maleate produces a comparable fall in fluorescence measured biochemically or histochemically. This simple method is specific as interfering substances, such as spermine and spermidine, produce very weak fluorescence under the conditions employed.

Simultaneous Measurement of Oscillations in Oxygen Evolution and Chlorophyll a Fluorescence in Leaf Pieces

In spinach (Spinacia oleracea) and barley (Hordeum vulgare) leaves, chlorophyll a fluorescence and O(2) evolution have been measured simultaneously following re-illumination after a dark interval or when steady state photosynthesis has been perturbed by changes in the gas phase. In high CO(2) concentrations, both O(2) and fluorescence can display marked dampening oscillations that are antiparallel but slightly out of phase (a rise or fall in fluorescence anticipating a corresponding fall or rise in O(2) by about 10 to 15 seconds). Infrared gas analysis measurements showed that CO(2) uptake behaved like O(2) evolution both in the period of oscillation (about 1 minute) and in its relation to fluorescence. In the steady state, oscillations were initiated by increases in CO(2) or by increases or decreases in O(2). Oscillations in O(2) or CO(2) did not occur without associated oscillations in fluorescence and the latter were a sensitive indicator of the former. The relationship between such oscillations in photosynthetic carbon assimilation and chlorophyl a fluorescence is discussed in the context of the effect of ATP or NADPH consumption on known quenching mechanisms.

Collisional relaxation of vibrationally excited OCS in rare gas mixtures

Following excitation of the OCS bending overtone 2ν2, with a Q-switched CO2 laser, time-dependent fluorescence from the symmetric stretching fundamental ν1, has been observed for mixtures of OCS with 4He, Ne, Ar, Kr, and Xe. Analysis of the exponential rise and fall of fluorescence provides relaxation rates corresponding to the collisional coupling of the ν1 and ν2 manifolds and the overall vibration–translation/rotation relaxation for collisions of OCS with the various rare gases. For the process 2ν2→ν1, the experimentally derived probabilities per collision are OCS–Ne?1.7×10−4, OCS–Ar 1.2±0.4×10−4, OCS–Kr 0.8±0.2×10−4 and OCS–Xe 1.0±0.2×10−4. For the vibration–translation/rotation relaxation, the probabilities per collision are OCS–Ne 1.4±0.8×10−4, OCS–Ar 0.3±0.3×10−4, OCS–Kr 0.2±0.2 ×10−4, and OCS–Xe 0.1±0.1×10−4. Model calculated probabilities, using a spherically symmetric, short-range repulsive interaction potential, qualitatively agree with these experimental probabilities. If in the case of OCS–4He, only the path 2ν2→ν1 is assumed to couple the bending and stretching (ν1) modes, then the experimental probabilities are 6±2×10−4, for the process 2ν2→ν1 and 10 ± 3×10−4 for the vibration–translation/rotation process. This intermode vibration– vibration probability, however, is unexpectedly large when compared to both the model calculations and the experimental values for the other OCS–rare gas mixtures. A cyclic kinetic path allowing for both 2ν2 and ν2 to couple to ν1 in OCS–4He collisions is suggested by the model calculations and is also consistent with the OCS–4He experimental data. Assuming the complete kinetic cycle results in OCS–4He probabilities with the following ranges: 0–8×10−4 for the process 2ν2→ν1, 0–1×10−4 for the process ν2→ν1 and 5–13×10−4 for the vibration–translation/rotation process, depending on the relative contribution (which cannot be ascertained from these experiments) of the two competing steps filling the ν1 level. The cyclic path kinetics of OCS–4He system exemplifies the general phenomenon of cyclic mechanisms which may be important in promoting rapid vibration–vibration and vibration–translation/rotation energy transfer, particularly for complex or highly excited molecules.

Elementary steps of the (Na + + K +)-ATPase mechanism, studied with formycin nucleotides

1. 1. Formycin triphosphate (FTP), a fluorescent analogue of ATP, is a substrate for (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.3), with properties similar to those of ATP. 2. 2. FTP and formycin diphosphate (FDP) bind to the enzyme with high affinity and, on binding, the nucleotide fluorescence is enhanced 3–4-fold. It is therefore possible, with a stopped-flow fluorimeter, to measure the rates of binding and release of FTP and FDP under conditions in which turnover does not occur. 3. 3. When the enzyme · FTP complex is exposed to conditions permitting turnover ( Mg p2+, Na + ± K + ), changes in fluorescence occur which can be explained by supposing that they reflect the interconversion of states with or without bound nucleotides. A rapid fall in fluorescence, that we attribute to the rapid release of FDP from newly phosphorylated enzyme, is followed by a steady state in which low fluorescence suggests that little nucleotide is bound. Eventually, exhaustion of FTP allows rebinding of FDP to the enzyme, which is signalled by a rise in fluorescence. 4. 4. The estimated rate of FDP release from newly formed phosphoenzyme is unaffected by the presence of K + (0–2mM) or the concentration of FTP (1–20 μM). 5. 5. Experiments with [γ- 32P]FTP show that about 1 mol of 32P is incorporated per mol of enzyme. The rate of phosphorylation of the enzyme by [γ- 32P]FTP has been measured with a rapid-mixing-and-quenching apparatus. 6. 6. Kinetic data from the fluorescence and phosphorylation experiments show that the behaviour of the enzyme, at least at the low nucleotide concentrations employed, is consistent with the Albers-Post model, and is difficult to reconcile with models in which K + acts at or before the step in which FDP is released during turnover.