Stopped-flow Fluorimetry Research Articles

Calcium influx evoked by Ca 2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptor-mediated calcium entry

We have previously reported that a component of ADP-evoked Ca 2+ entry in human platelets appears to be promoted following the release of Ca 2+ from intracellular stores. Other agonists may employ a similar mechanism. Here we have further investigated the relationship between the state of filling of the Ca 2+ stores and plasma membrane Ca 2+ permeability in Fura-2-loaded human platelets. Ca 2+ influx was promoted following store depletion by inhibitors of the endoplasmic reticulum Ca 2+-ATPase, thapsigargin (TG) and 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBuBHQ). Divalent cation entry was confirmed by quenching of Fura-2 fluorescence with externally added Mn 2+. It has been suggested that cytochrome P-450 may couple Ca 2+ store depletion to an increased plasma membrane Ca 2+ permeability. In apparent agreement with this, Mn 2+ influx promoted by TG and tBuBHQ, or by preincubation of cells in Ca 2+-free medium, was inhibited by the imidazole antimycotics, econazole and miconazole, which inhibit cytochrome P-450 activity. Agonist-evoked Mn 2+ influx was only partially inhibited by these compounds at the same concentration (3 μM). Econazole (3 μM) reduced the Mn 2+ quench evoked by ADP by 38% of the control value and that evoked by vasopressin, platelet activating factor (PAF) and thrombin no more than 15% of control, 20 s after agonist addition. Stopped-flow fluorimetry indicated that econazole had no detectable effect on the early time course of agonist-evoked Mn 2+ entry or rises in [Ca 2+] i. These data confirm the existence of a Ca 2+ entry pathway in human platelets which is activated by depletion of the intracellular Ca 2+ stores. Further, the results support the suggestion that cytochrome P-450 may participate in such a pathway. However, any physiological role for the cytochrome or its products in agonist-evoked events appears to be in the long-term maintenance or restoration of store Ca 2+ content, rather than in promoting Ca 2+ influx in the initial stages of platelet Ca 2+ signal generation.

Resting and ADP‐evoked changes in cytosolic free sodium concentration in human platelets loaded with the indicator SBFI.

1. Cytosolic free Na+ concentration, [Na+]i, was investigated in human platelets loaded with the fluorescent indicator SBFI (sodium-binding benzofuran isophthalate). 2. SBFI fluorescence from platelet suspensions was measured at excitation wavelengths of 340 and 385 nm and the 340/385 nm fluorescence ratio was calibrated in terms of [Na+]i in situ. [Na+]i was set to known values by resuspending cells in media with various [Na+], in the presence of the Na(+)-K+ ionophore, gramicidin. 3. Basal free [Na+]i was 5.5 +/- 0.3 mM (n = 50). This is considerably lower than estimates of total platelet Na+, suggesting that much intracellular Na+ is sequestered or bound. 4. ADP (40 microM) evoked a rise in [Na+]i from 6.4 +/- 0.7 to 18.3 +/- 1.1 mM (n = 8). The ADP-evoked rise in [Na+]i was abolished when external Na+ was replaced with N-methyl-D-glucamine. This indicates that the rise in [Na+]i was due to Na+ entry. 5. In platelets loaded with the fluorescent pH indicator, BCECF, 40 microM-ADP was shown to evoke a fall in cytosolic pH (pHi) from 7.21 +/- 0.03 to 7.12 +/- 0.03 (n = 10). Three minutes after ADP addition pHi had only recovered to 7.15 +/- 0.03. The recovery was dependent on external Na+, suggesting it was mediated by Na(+)-H+ exchange. However, this would only account for an increase in [Na+]i of approximately 0.5 mM, indicating most of the ADP-evoked Na+ entry occurred by other mechanisms. 6. Stopped-flow fluorimetry showed that the ADP-evoked rise in [Na+]i commenced without measurable delay and peaked within 1 s. The initial kinetics were thus similar to those reported for ADP-evoked rises in [Ca2+]i. 7. Cell-attached patch-clamp recordings showed that ADP evoked single-channel inward currents when included in the pipette-filling solution. The currents were similar whether Ca2+ was present or absent from the pipette. The slope conductance was 11 pS in the presence of external Ca2+ and 10 pS in its absence. Current-voltage relationships were similar and the reversal potentials were close to 0 mV under both conditions. 8. SK & F 96,365 (20 microM), a blocker of receptor-mediated Ca2+ entry in several non-excitable cells, blocked the ADP-evoked rise in [Na+]i. This compound has been shown to only partly block the biphasic ADP-evoked rise in [Ca2+]i, being selective for the fast, receptor-operated phase of entry. 9. These data suggest that ADP rapidly activates a channel in that platelet plasma membrane which is permeable to Na+ and divalent cations.

Rapid kinetics of agonist-evoked changes in cytosolic free Ca2+ concentration in fura-2-loaded human neutrophils

The initial kinetics of agonist-evoked rises in the cytosolic Ca2+ concentration [Ca2+]i were investigated in fura-2-loaded human neutrophils by stopped-flow fluorimetry. The rises in [Ca2+]i evoked by chemotactic peptide (fMet-Leu-Phe), platelet-activating factor and ADP all lagged behind agonist addition by 1-1.3 s. Lag times were not significantly different in the presence and in the absence of external Ca2+. Stimulation of the cells in the presence of extracellular Mn2+ resulted in a quench of fluorescence with a similar lag time to [Ca2+]i rise. The delay in onset of the rise in [Ca2+]i evoked by fMet-Leu-Phe was dependent on concentration, becoming longer at lower concentrations of agonist. These results indicate that both the agonist-evoked discharge of the intracellular Ca2+ stores and the generation of bivalent-cation influx lag behind agonist-receptor binding in neutrophils. Both pathways thus appear to be mediated by indirect mechanisms, rather than by a directly coupled process such as a receptor-operated channel. The temporal coincidence of the onset of store discharge with the commencement of bivalent-cation influx suggests that the two events may be causally linked.

Kinetics of calcium binding to fluo-3 determined by stopped-flow fluorescence

The kinetics of Ca 2+ dissociation from fluo-3 was measured using stopped flow fluorimetry. Analysis of dissociation revealed, in contrast to other commonly used fluorescent Ca 2+ indicators, a biexponential behaviour with two distinct dissociation rates of 550 s −1 and 200 s −1 at physiological pH and room temperature. The dissociation rate constant of the fast phase increases to 700 s −1 at physiological temperature, whereas that of the slow phase does not change markedly. While the rate constants do not depend on pH between 6.6 and 7.8, the dissociation turns out to be monoexponential at pH 5.86. The association rate of Ca 2+ to fluo-3 could not be measured within the mixing dead time and is estimated to be above 10 9M −1 s −1. Since the rate constants of fluo-3 are larger than those of other fluorescent Ca 2+ indicators, fluo-3 is well suited for investigations of Ca 2+ oscillations in biological systems.

The kinetics of changes in intracellular calcium concentration in fura-2-loaded human platelets.

We have investigated the sub-second kinetics of changes in cytosolic free calcium, [Ca2+]i, in fura-2-loaded human platelets by stopped-flow fluorimetry. Thrombin, vasopressin, platelet-activating factor, and the thromboxane A2 analogue U46619 all evoked a rise in [Ca2+]i which was delayed in onset by 200-400 ms in the presence of 1 mM external Ca2+. The responses to these agonists in media containing 1 mM EGTA or 1 mM Ni2+, to prevent Ca2+ influx, were delayed by an additional 60-100 ms. These results indicate that agonist-evoked Ca2+ influx precedes the release of Ca2+ from internal stores. The delays in onset of both responses are sufficient for one or more biochemical steps to lie between ligand-receptor binding and Ca2+ flux generation. ADP responses in media containing EGTA or Ni2+ were similar to those evoked by other agonists, but the response in the presence of external Ca2+ was markedly shorter, occurring without measurable delay at optimal ligand concentration. Analysis of this response showed some delay in ADP-evoked influx at lower concentrations, but this delay was markedly less than that observed with thrombin at doses giving the same elevation in [Ca2+]i. These results suggest that ADP evokes influx using a different transduction system, more closely coupled to the Ca2+ entry system than that used by other agonists. Differences between thrombin- and ADP-evoked influx were further demonstrated by the inhibitory actions of cAMP, which reduced and substantially increased the delay in onset of thrombin-evoked influx but did not measurably delay the influx evoked by an optimal concentration of ADP.

Comparison of rates of cation release and of conformational change in dog kidney Na, K‐ATPase.

1. It is now widely believed that the main rate-limiting step in the sodium-potassium pump (Na, K-ATPase) cycle is a conformational change between two forms of the dephosphoenzyme (E2 and E1) and that this change releases to the cell interior potassium ions occluded within the E2 form. 2. If this hypothesis is correct, and if occluded ions cannot be released directly from dephosphoenzyme in the E2 conformation, we should expect that, under any given conditions, the rate of release of the occluded ions would be identical with the rate of the conformational change. 3. Using the potassium congeners 86Rb, 137Cs and 204Tl, the rates of release of the occluded ions can be measured by a rapid ion-exchange technique. Using the fluorescence probes fluorescein isothiocyanate (FITC), eosin or 5-iodoacetamido-fluorescein (5-IAF), the rates of the conformational change can be measured by stopped-flow fluorimetry. 4. A comparison of the two rates in the absence of ATP showed that the rate of release of the occluded ions was usually somewhat faster than the rate of the fluorescence change. The discrepancy was probably caused by a very slow direct release of occluded ions from enzyme in the E2 form, but we cannot exclude the possibility that it is the result of systematic errors. In the presence of 5 microM-ATP, both rates were increased and there was no significant difference between them. 5. The results are compatible with the hypothesis that the same conformational change alters the fluorescence of the fluorescent probes and releases the occluded potassium congener ions.

Transient kinetics of heparin-catalyzed protease inactivation by antithrombin III. The reaction step limiting heparin turnover in thrombin neutralization.

The intrinsic protein fluorescence quenching which accompanies the heparin-accelerated inhibition of thrombin (T) by antithrombin III (AT) was resolved into a heparin-independent component associated with formation of the product T-AT complex and a component associated with an AT conformational change linked to heparin dissociation. To determine whether dissociation of heparin from the product T-AT complex limits the rate at which heparin can turn over catalytically, the kinetics of protein fluorescence quenching during the reaction of thrombin with AT X heparin complex (AT X H) were investigated by stopped-flow fluorimetry under pseudo-first order conditions ([AT X H]o much greater than [T]o). Both fluorescence components were quenched in a single exponential reaction with a hyperbolic dependence of the first order rate constant (kobs) on [AT X H]o. An indistinguishable hyperbolic dependence of kobs on [AT X H]o was measured by displacement of p-aminobenzamidine from the T active site, with both signals extrapolating to a limiting rate constant of 5 s-1. These results indicate that heparin dissociation occurs concomitant with T-AT complex formation at the limiting 5 s-1 rate constant. In reasonable agreement with this value, a kcat of 2.3 s-1 was determined for heparin turnover at catalytic concentrations. We conclude that formation of the T-AT complex is the primary rate-limiting step in heparin catalytic turnover and that this reaction is accompanied by a change in conformation of the AT component resulting in facile heparin dissociation.

Kinetic differences between thrombin-induced and ADP-induced calcium influx and release from internal stores in fura-2-loaded human platelets

The rapid (<1 sec) time course of [Ca 2+] i rises was measured in fura-2-loaded, aspirin treated human platelets by stopped flow fluorimetry. With thrombin, which is known to cause substantial, rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate, the mean delay before a detectible rise in [Ca 2+] i in medium containing 1 mM Ca 2+ o was 250 +/− 10 msec (S.E.M., n=11). With ADP, which is reported not to stimulate phosphatidylinositol-4,5-bisphosphate hydrolysis, the delay under the same conditions was only 20 +/− 10 msec (S.E.M., n=26). In the absence of external Ca 2+, with 1 mM EGTA, the measured delays were 300 +/− 20 msec for thrombin and 210 +/− 10 msec for ADP. Times to peak were also faster for ADP than thrombin. These results suggest that thrombin and ADP promote Ca 2+ influx in different ways. It is also possible that the process generating Ca 2+ influx differs from that which triggers release from internal stores.

Stopped-flow kinetics of the interaction of the fluorescent calcium indicator Quin 2 with calcium ions

The kinetics of the Quin 2-Ca 2+ interaction have been studied using stopped-flow fluorimetry. Mixing the Quin 2-Ca 2+ complex with a large excess of EGTA, EDTA or MgCl 2 resulted in first order dissociation kinetics. The observed dissociation rate increased slightly with increasing EGTA concentration yielding a limiting value of 83±4 s −1 for the dissociation rate constant (k −) at pH 7.2, 37°C, ± 3mM Mg 2+. The temperature dependence of the dissociation was weak (activation energy = 22±1 kJ/mol) and around neutral pH the pH dependence was negligible. The association reaction was too fast to be monitored directly. From this and the instrument dead-time, the second order rate constant k + was estimated to be ≥10 9 M −1s −1, in agreement with the calculation from k + = k − K . These data should be useful in evaluating the potential of Quin 2 to measure fast intracellular Ca 2+ transients.

A reaction pathway for transimination of the pyridoxal 5'-phosphate in D-serine dehydratase by amino acids.

Transimination of the enzyme-linked cofactor by an amino acid is the first chemical transformation in the reactions catalyzed by pyridoxal 5'-phosphate-requiring enzymes. In this work, stopped flow fluorimetry was used to characterize the kinetics of transimination of the cofactor in D-serine dehydratase by several amino acids. The results of these studies indicate that transimination is a multistep process, the first step of which is probably formation of a noncovalent complex between the enzyme and the amino acid. D-Serine dehydratase was found to exhibit considerable specificity in the transimination reaction. Furthermore, the enzyme was shown to facilitate the transimination reaction with amino acids and inhibit transimination of the bound cofactor by amines lacking a carboxylate group. A reaction pathway was proposed for the transimination process which accounts for the specificity of the enzyme and indicates the changes in the conformation of the bound cofactor as dictated by the stereoelectronic requirements of the transimination reaction.

Binding of high affinity heparin to antithrombin III. Stopped flow kinetic studies of the binding interaction.

The kinetics of high affinity heparin binding to human antithrombin III has been studied by stopped flow fluorimetry, using the 40% antithrombin fluorescence enhancement resulting from this interaction. At mu 0.15, pH 7.4, and 25 degrees C, the observed pseudo-first order rate constant varies hyperbolically with heparin concentration with a limiting rate constant of 440 +/- 90 s-1, demonstrating that heparin binding is a two-step process involving a conformational change in antithrombin III. An identical dependence is produced when antithrombin is varied, consistent with a symmetrical mechanism in which heparin binding induces a conformational change in antithrombin rather than perturbing an equilibrium between two conformational states of the protein. The rate constant for dissociation of the antithrombin-heparin complex is 1.1-1.5 s-1 at mu 0.15, as determined from the ordinate intercept at low heparin concentrations or by dissociation of the antithrombin-heparin complex with iodide. Observation of a single pseudo-first order binding rates over a 400-fold heparin concentration range with no detectable lags is compatible with the initial binding step being in rapid equilibrium with a KD of 4.3 +/- 1.3 X 10(-5) M at mu 0.15. Variation in ionic strength primarily affects the KD for the initial binding step with little effect on the conformational change rate constants, implying that binding involves ionic interactions. Calculation of the overall dissociation equilibrium constant from these rate parameters agrees with the directly determined value of 7.2 +/- 1.9 X 10(-8) M at mu 0.15. A major function of the conformational change is, thus, to increase the affinity of heparin for antithrombin III greater than 300-fold. The implications of these findings for the mechanism of the heparin-catalyzed inhibition of coagulation proteases by antithrombin III are discussed.

The kinetics of effector binding to phosphofructokinase. The binding of Mg2+-1,N6-ethenoadenosine triphosphate to the catalytic site.

1. The binding of the fluorescent ATP analogue, Mg2+-1,N6-etheno-ATP, to the catalytic site of rabbit skeletal muscle phosphofructokinase has been studied by stopped-flow fluorimetry [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. Binding of Mg2+-1,N6-etheno-ATP to the catalytic site is consistent with a two-step mechanism of the type: (formula: see text); in which the diffusion-controlled binding of ligand, L, is accompanied by prior interconversion of enzyme from one form, E, to another, E. 3. The allosteric activators, phosphate and cyclic AMP, which promote an R-type conformation, appear to stabilize slightly different conformations, R and R' respectively. 4. The binding of Mg2+-1,N6-etheno-ATP to the catalytic site is strongly affected by its binding to the inhibitory site. The rate constant for the displacement of Mg2+-1,N6-ethenol-ATP from the catalytic site, k32, is 470 +/- 35 s-1 for the R' conformation, whereas it is 6.0 +/- 0.09 s-1 for the T conformation induced by binding of Mg2+-1,N6-ethenol-ATP to the inhibitory site.

The kinetics of effector binding to phosphofructokinase. The influence of effectors on the allosteric conformational transition.

1. The extent of the allosteric transition from the R into the T conformation of rabbit skeletal muscle phosphofructokinase induced by Mg2+-1,N6-etheno-ATP was determined by stopped-flow fluorimetry from the amplitude of the slow phase of the Mg2+-1,N6-etheno-ATP fluorescence enhancement [Roberts & Kellet (1979) Biochem. J. 183, 349--360]. 2. The amplitude of the slow phase was decreased by low concentrations of the activators cyclic AMP and fructose 1,6-bisphosphate, but increased in a complex manner by the inhibitor citrate. 3. Mg2+-1,N6-etheno-ATP and Mg2+-ATP are unable to induce the T conformation to a detectable extent in the presence of saturating cyclic AMP, but can do so readily in the presence of saturating fructose 1,6-bisphosphate. 4. The conformational transitions induced in enzyme alone by different ligands were observed by changes in intrinsic protein fluorescence. In general, an R-type conformation has diminished protein fluorescence compared with a T-type conformation. 5. Mg2+-ATP exerts a complex effect on protein fluorescence; both the enhancement at low concentrations and the quenching at high concentrations of Mg2+-ATP result from the binding of Mg2+-ATP to the inhibitory site and the ensuing allosteric transition. Enhancement reflects the extent of the allosteric transition and involves both tyrosine and tryptophan, probably in the region of the active site; quenching reflects occupation of the inhibitory site and involves tyrosine at the inhibitory site. 6. The mechanism of the allosteric transition from the R into the T conformation induced by Mg2+-1,N6-etheno-ATP at low concentrations occurs predominantly by a 'prior-isomerization' pathway; at higher concentrations a limited contribution from a 'substrate-guided' pathway occurs. 7. The allosteric behaviour of phosphofructokinase with respect to Mg2+-ATP and Mg2+-1,N6-ethenol-ATP binding may be accounted for in terms of the simple, concerted model.

Transient kinetics of a Ca2+-induced fluorescence change from membrane-associated and solubilized sarcoplasmic reticulum Ca2+-ATPase.

The kinetics and extent of the fluorescence change induced by Ca2+ interaction with the Ca2+-ATPase from sarcoplasmic reticulum have been compared by stopped flow fluorimetry for three preparations: sarcoplasmic reticulum; purified ATPase in membrane vesicles; and solubilized, delipidated ATPase. The kinetics of Ca2+ release and binding for both purified preparations could be described by a single exponential as has been observed for sarcoplasmic reticulum. The rate and extent of the fluorescence change for the solubilized and membrane-associated preparations are shown to be quite similar to those of the sarcoplasmic reticulum. From these results, it is concluded that all of the Ca2+-induced fluoescence change in sarcoplasmic reticulum originates from the Ca2+-ATPase. In addition, since the change in fluorescence is probably result of a conformational change in the ATPase during the Ca2+ pumping cycle, the results provide additional evidence that monomeric Ca2+-ATPase may be capable of Ca2+ transport since the delipidated preparation is monomeric under the conditions used for these experiments. Finally, it is concluded that phospholipid bilayer is not essential for this conformational change.

The kinetics of effector binding to phosphofructokinase. The allosteric conformational transition induced by 1,N6-ethenoadenosine triphosphate

1. The fluorescent ATP analogue 1,N6-etheno-ATP is a good substrate and an efficient allosteric inhibitor of rabbit skeletal-muscle phosphofructokinase. 2. Fluorescence energy transfer occurs between bound 1,N6-etheno-ATP and phosphofructokinase. 1,N6-Etheno-ATP fluorescence is enhanced, intrinsic protein fluorescence is quenched, and the excitation spectrum of 1,N6-etheno-ATP fluorescence is characteristic of protein absorption. 3. The binding reaction of 1,N6-etheno-ATP observed by stopped-flow fluorimetry is biphasic. The fast phase results from binding to the catalytic site alone. The slow phase results from the allosteric transition of the R conformation into the T conformation induced by the binding of 1,N6-etheno-ATP to the regulatory site. 4. The fluorescence signal that allows the transition of the R conformation into the T conformation to be observed does not arise from 1,N6-etheno-ATP bound to the regulatory site. It arises instead from 1,N6-etheno-ATP bound to the catalytic site as a consequence of changes at the catalytic site caused by the transition of the R conformation into the T conformation. 5. In the presence of excess of Mg2+, the affinity of 1,N6-etheno-ATP for the regulatory site is very much greater in the T state than in the R state.

Rapid intramolecular coupling of active sites in the pyruvate dehydrogenase complex of Escherichia coli : Mechanism for rate enhancement in a multimeric structure

In the absence of CoA and presence of pyruvate, the lipoic acid residues covalently bound to the lipoate acetyltransferase core component (acetyl-CoA:dihydrolipoate S-acetyltransferase, EC 2.3.1.12) of the pyruvate dehydrogenase multienzyme complex of Escherichia coli become reductively acetylated. A study of a series of reassembled complexes varying only in their content of pyruvate decarboxylase [pyruvate:lipoate-oxidoreductase (decarboxylating and acceptor-acetylating) EC 1.2.4.1] showed that the initial direct reductive acetylation of lipoic acid residues can be followed by extensive intramolecular transacetylation reaction between lipoic acid residues on neighboring polypeptide chains of the lipoate acetyltransferase core [Bates, D. L., Danson, M. J., Hale, G., Hooper, E. A. & Perham, R. N. (1977) Nature (London) 268, 313-316]. Pulsed-quenched-flow measurements of the rates of the acetylation reactions in the various complexes now demonstrate that the intramolecular transacetylation reactions are not rate-determining in the normal reaction mechanism of the enzyme. There is therefore the potential for rapid multiple coupling of active sites in the lipoate acetyltransferase core. The rate constant for the overall complex reaction, measured by stopped-flow fluorimetry, is found to be approximately twice that for the reductive acetylation reaction measured by pulsed-quenched flow. This result could mean that CoA is an allosteric stimulator of the reductive acetylation part of the overall reaction or that there are two active sites on each chain of the lipoate acetyltransferase component working in parallel. A system of rapid functional connection of active sites in a multienzyme complex ensures that sequential reactions can be successfully coupled even under conditions of low substrate concentrations for the different steps. The substantial rate enhancement thus achieved offers a plausible explanation for the unusual complexity of the quaternary structure of the enzyme.

Agonist-mediated changes of the acetylcholine receptor in its membrane environment

The interaction of a cholinergic agonist, suberyldicholine, with the membrane-bound acetylcholine receptor from Torpedo marmorata was studied in vitro by a combination of kinetic and steady-state techniques. Exposure of the receptor-rich membranes to the agonist resulted in a time and concentration dependent, saturable and reversible series of changes in receptor properties. The apparent affinity of the receptor for the ligand increased about 50-fold in the course of this exposure, as measured indirectly by the inhibition of α-toxin association kinetics. The modifications induced by suberyldicholine in the receptor could be studied in a more direct manner by following the kinetics of the (intrinsic) protein fluorescence quenching. An initial step occurring in the time scale of milliseconds could be observed with stopped-flow fluorimetry, its characteristics being consistent with a binding process of the agonist to a “low affinity” form of the receptor ( K app ~ 1 μM) with on and off-rates of 9.8 × 10 6 m −1 s −1 and 10 s −1, respectively. This step was followed by a larger fluorescence change taking place in a time range of seconds (forward rate 1.2 to 1.5 s −1; backward rate 0.06 to 0.1 s −1). The final, “high-affinity” state of the complex, which differed in its fluorescence properties from both the free (initial) and liganded-forms of the receptor, is tentatively attributed to the “desensitised” conformation postulated from in vivo studies (Katz & Thesleff, 1957). The rate of formation of this complex elicited by suberyldicholine was higher than those of acetyl- or carbamoylcholine. The extent of the total fluorescence quenching was, on the other hand, approximately the same for the three agonists, suggesting that the final conformation does not depend on the nature of the ligand. The specific agonist-induced quenching probably operates via a “static” mechanism, since the lifetime of the excited state did not change in the presence of the ligand. The accessibility of the intrinsic fluorophores to acrylamide or to nitroxide spin labels, as sensed by quenching of fluorescence from the bulk and lipid phases, respectively, was also affected by suberyldicholine. Both fluorescence and toxin-inhibition studies have been analysed in terms of a reaction mechanism derived from in vivo observations, involving interconvertible conformational states of the acetylcholine receptor in its membrane environment, differing in affinity for the agonist, and in their intrinsic fluorescence properties.

A conformational change in phosphoglycerate dehydrogenase induced by a shift in pH

The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD + oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0. Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s −1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer L.I. (1977) J. Biol. Chem. 252, 1527–1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5

Transient kinetic studies on the allosteric transition of phosphoglycerate dehydrogenase.

Stopped flow spectrophotometry was used to investigate the kinetics of the transition of the phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD oxidoreductase, EC 1.1.1.95) reaction from the active to the inhibited rate upon the addition of the physiological inhibitor serine. The transition was characterized by a single first order rate constant (kobs,i) which was independent of enzyme concentration. At pH 8.5, kobs,i increased in a hyperbolic manner with serine concentration from 2 to 8 s-1. The increase in kobs,i occurred at serine concentrations where the steady state inhibition was virtually complete. These results indicate that serine inhibition is an allosteric process involving a conformational change in the enzyme. A model is presented in which serine at low concentrations binds exclusively to the inhibited state of the enzyme and shifts the equilibrium toward that state; at high serine concentrations, serine binds to the active state, facilitating its conversion to the inhibited state. An alternative model, which we favor, proposes two classes of inhibitor binding sites. The kinetics of the fluorescence quenching of enzyme-bound NADH by serine (Sugimoto, E., and Pizer, L.I. (1968) J. Biol. Chem. 243, 2090-2098), measured by stopped flow fluorimetry, was also characterized by a single first order rate constant (kobs,f.q.) which was independent of enzyme concentration. At pH 8.5, kobs,f.q. ranged from 0.4 s-1 at low serine concentrations to 1.1 s-1 at high serine concentrations. These results indicate that the fluorescence quenching induced by serine is a manifestation of a structural change in the enzyme. Enzyme and excess NADH were mixed with substrate and serine in the stopped flow instrument, and enzyme-bound NADH fluorescence was monitored by exciting through the protein at 285 nm. A rapid fluorescence quenching process, which occurred within the mixing time, was followed by a slower fluorescence enhancement process which terminated in a steady state level corresponding to the quenched fluorescence of the enzyme NADH serine complex. The rapid quenching was the result of substrate binding (Dubrow, R., and Pizer, L.I. (1977) J. Biol. Chem. 252, 1539-1551). The fluorescence enhancement was characterized by a single first order rate constant whose value for a given serine concentration corresponded with Kobs,j. This data shows that the quenched state of the enzyme-NADH-complex is the state which is directly responsible for the inhibition of enzyme activity. During catalysis the quenched state is achieved from a different initial conformation, and consequently at a different rate, than in the absence of substrate. kobs,j and kobs,f.q. were also measured using glycine, another inhibitor. The ultraviolet difference spectrum between enzyme and enzyme plus serine was determined and proposed to be the result of the same structural change which is responsible for the fluorescence quenching by serine.

Transient kinetics of (Na+ +K+)-ATPase studied with a fluorescent substrate.

WE describe experiments, with a purified (Na++K+)-ATPase, designed to investigate details of the enzymatic process that couples ATP hydrolysis to the active transport of Na+ and K+ ions. We have found that formycin triphosphate1–3 (FTP), an analogue of ATP previously used in studies of myosin ATPase4, is a substrate for the (Na++K+)-ATPase, and that the binding of FTP or of formycin diphosphate (FDP) to the enzyme is accompanied by a two- to threefold enhancement of nucleotide fluorescence. The change in strength of the fluorescent signal has allowed us to measure equilibrium binding of the nucleotides to the enzyme, and using stopped-flow fluorimetry we have been able to measure the rates of binding and release. FTP and FDP bind with a high affinity and may be displaced by excess ATP or by K+ ions. During turnover, at least with FTP concentrations up to 24 µM, the FDP is released (or its fluorescence is quenched) before the rate-limiting step of the overall reaction, both in the presence and absence of K+ ions. When turnover is prevented by the absence of magnesium, the enzyme is still able to change its conformation according to whether Na+ or K+ is the predominant alkali-metal ion5. Because the Na and K conformations have different nucleotide affinites, we have been able to measure the rates of the conformational changes by monitoring the decrease or increase in fluorescence associated with the net release or binding of nucleotide. Finally, the effects of alterations in FTP concentration on the time course of the fluorescence changes have provided a clue to the non-phosphorylating role of ATP.