Correction for light absorption in fluorescence studies of protein-ligand interactions

It is shown that absorption of the excitation light can lead to substantial systematic errors in fluorescence measurements of equilibrium constants for formation of protein-ligand complexes. The assumptions about the optical arrangement of the fluorescence spectrometer involved in the calculation of the correction of this absorption are discussed. A general semiempirical correction procedure which can be used for (calculated) absorbance values as high as 5 is described. The importance of choosing the excitation wavelength so as to minimize the necessity for these corrections is emphasized.     

Applicability of the induced-fit model to glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. Study of the binding of oxidized nicotinamide adenine dinucleotide and nicotinamide 8-bromoadenine dinucleotide

A new method of calculation, based on a direct fitting of the protein fluorescence intensity observed upon coenzyme binding (H.-P. Lutz, unpublished results), is used to study the negative cooperative behavior of glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. The calculation procedure simultaneously elaborates data obtained for four different protein concentrations, and it is able to compare different models by computing the minimal and critical sum of squares. Using this approach, it is shown that the induced-fit model [Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5,365] and the dimer of dimer model [Malhotra, O. P., & Bernhard, S. A. (1968) J. Biol. Chem. 243, 1243-1252] can both be applied for explaining the negative cooperativity observed upon coenzyme binding to sturgeon glyceraldehyde-3-phosphate dehydrogenase. In addition to the progressive modification of the binding affinity during ligand binding, different maximal fluorescence quenchings for the binding steps must be postulated; and furthermore, the binding capability decreases by decreasing the protein concentration. The fact that the induced-fit model can also be applied is rather in contradiction with the view generally accepted of a dimer of dimer structure of sturgeon glyceraldehyde-3-phosphate dehydrogenase. By use of the same approach, nicotinamide 8-bromoadenine dinucleotide is shown to bind to glyceraldehyde-3-phosphate dehydrogenase from sturgeon in a negative cooperative manner.     

Interaction of the dissociable glycerol-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase. Quantitative analysis by an extrinsic fluorescence probe

Cytoplasmic sn-glycerol-3-phosphate dehydrogenase, labelled covalently with fluorescein isothiocyanate, shows an enzyme-concentration-dependent fluorescence anisotropy. The anisotropy versus enzyme concentration curve is shifted towards higher concentrations when substrates are present. The comparison of the dissociation constants estimated from anisotropy measurements and derived from kinetic experiments suggests that the substrate-induced dissociation of the dimeric dehydrogenase is slow with respect to the enzymatic reaction catalyzed by either its monomeric or dimeric form. The fluorescence anisotropy of the fluorescent dye-labelled dehydrogenase increase with time upon addition of unlabelled fructose-1,6-bisphosphate aldolase approaching a limiting value. This fact indicates the binding of fructose-1,6-bisphosphate aldolase aldose aldolase to glycerolphosphate dehydrogenase. A model is proposed assuming simultaneous binding of tetrameric fructose-1,6-bisphosphate aldolase to monomeric and dimeric glycerolphosphate dehydrogenase with 1:1 stoichiometry. The dissociation constants, as parameters fitted to the experimental curves, were estimated as 0.2 microM and 1 microM for aldolase-dimeric-glycerolphosphate-dehydrogenase and aldolase-monomeric-glycerolphosphate-dehydrogenase complexes respectively.     

Fluorescence response and sensitivity determination for ATC 3000 flow cytometer

The fluorescence emitted by labeled particles after interaction with exciting light is conditioned by laser beam geometry and by the mode of fluorescence collection and filtration. A laser elliptic focusing mode is described, and the fluorescence characteristics of the sample cell flow are calculated. Fluorescence collection and detection through optical filters were analyzed, and efficiency was calculated for the ATC 3000 flow cytometer (Odam-Bruker, Wissembourg, France). A mathematical model is proposed for calculation of the fluorescence signal and its fluctuations. The background noise for the ATC 3000 was quantified experimentally using fluorescent microspheres of a known number of bound equivalent fluorescein isothiocyanate (FITC) molecules. These experimental measurements were found to fit the theoretical predictions, thus validating the proposed model.     

Elementary steps in the reaction of the pyruvate dehydrogenase complex from pig heart. Kinetics of thiamine diphosphate binding to the complex

In the progress curve of the reaction of the pyruvate dehydrogenase complex, a lag phase was observed when the concentration of thiamin diphosphate was lower than usual (about 0.2-1 mM) in the enzyme assay. The length of the lag phase was dependent on thiamin diphosphate concentration, ranging from 0.2 min to 2 min as the thiamin diphosphate concentration varied from 800 nM to 22 nM. The lag phase was also observed in the elementary steps catalyzed by the pyruvate dehydrogenase component. A Km value of 107 nM was found for thiamin diphosphate with respect to the steady-state reaction rate following the lag phase. The pre-steady-state kinetic data indicate that the resulting lag phase was the consequence of a slow holoenzyme formation from apoenzyme and thiamin diphosphate. The thiamin diphosphate can bind to the pyruvate dehydrogenase complex in the absence of pyruvate, but the presence of 2 mM pyruvate increases the rate constant of binding from 1.4 X 10(4) M-1 S-1 to 1.3 X 10(5) M-1 S-1 and decreases the rate constant of dissociation from 2.3 X 10(-2) S-1 to 4.1 X 10(-3) S-1. On the other hand, the effect of pyruvate on the thiamin diphosphate binding revealed the existence of a thiamin-diphosphate-independent pyruvate-binding site in the pyruvate dehydrogenase complex. Direct evidence was also obtained with fluorescence techniques for the existence of this binding site and the dissociation constant of pyruvate was found to be 0.38 mM. On the basis of these data we have proposed a random mechanism for the binding of pyruvate and thiamin diphosphate to the complex. Binding of substrates to the enzyme complex caused an increase in the fluorescence of the dansylaziridine-labelled pyruvate dehydrogenase complex, showing that binding of substrates to the complex is accompanied by structural changes.     

Fluorescence of Dyes in Solutions with High Absorbance. Inner Filter Effect Correction

Fluorescence is a proven tool in all fields of knowledge, including biology and medicine. A significant obstacle in its use is the nonlinearity of the dependence of the fluorescence intensity on fluorophore concentration that is caused by the so-called primary inner filter effect. The existing methods for correcting the fluorescence intensity are hard to implement in practice; thus, it is generally considered best to use dilute solutions. We showed that correction must be performed always. Furthermore, high-concentration solutions (high absorbance) are inherent condition in studying of the photophysical properties of fluorescent dyes and the functionally significant interactions of biological macromolecules. We proposed an easy to use method to correct the experimentally recorded total fluorescence intensity and showed that informative component of fluorescence intensity numerically equals to the product of the absorbance and the fluorescence quantum yield of the object. It is shown that if dye molecules do not interact with each other and there is no reabsorption (as for NATA) and spectrofluorimeter provides the proportionality of the detected fluorescence intensity to the part of the absorbed light (that is possible for spectrofluorimeter with horizontal slits) then the dependence of experimentally detected total fluorescence intensity of the dye on its absorbance coincides with the calculated dependence and the correction factor for eliminating the primary inner filter effect can be calculated on the basis of solution absorbance. It was experimentally shown for NATA fluorescence in the wide range of absorbance (at least up to 60). For ATTO-425, which fluorescence and absorption spectra overlap, the elimination of the primary and secondary filter effects and additional spectral analysis allow to conclude that the most probable reason of the deviation of experimentally detected fluorescence intensity dependence on solution absorbance from the calculated dependence is the dye molecules self-quenching, which accompanies resonance radiationless excitation energy transfer.     

A simple method for high temporal resolution calcium imaging with dual excitation dyes.

Calcium-sensitive dual excitation dyes, such as fura-2, are now widely used to measure the free calcium concentration ([Ca2+]) in living cells. Preferentially, [Ca2+] is calculated in a ratiometric manner, but if calcium images need to be acquired at high temporal resolution, a potential drawback of ratiometry is that it requires equally fast switching of the excitation light between two wavelengths. To circumvent continuous excitation switching, some investigators have devised methods for calculating [Ca2+] from single-wavelength measurements combined with the acquisition of a single ratiometric pair of fluorescence images at the start of the recording. These methods, however, are based on the assumption that the concentration of the dye does not change during the experiment, a condition that is often not fulfilled. We describe here a method of single-wavelength calcium imaging, in which the dye concentration is estimated from ratiometric fluorescence image pairs acquired at regular intervals during the recording period, that furthermore includes a correction for the changing dye concentration in the calculation of [Ca2+].     

Re-absorption of chlorophyll fluorescence in leaves revisited. A comparison of correction models.

The application of correction methods to account for re-absorption of chlorophyll fluorescence emission in leaves is subject to a number of controversies in the literature. These uncertainties lead to high discrepancies in the corrected spectral distribution of fluorescence and consequently in the interpretation of related physiological features of plants, according to the chosen method used in the process of correction. In this research, three correction methods, based on transmittance and/or reflectance measurements on leaves, were analysed comparatively. One method gave high values for the corrected fluorescence ratio between 685 nm and 737 nm (F685/F737 approximately 7 to 20 according to the different species of leaves). The two other methods were found to give similar results with corrected fluorescence ratios around a value of two (F685/F737 approximately 2). While the first method was developed in the light of empirical considerations, the latter two models are based upon defined physical approaches depicting interaction between light and matter. The theoretical basis of these methods, the validation methodologies used to support them and the similarity in the spectra corrected by light re-absorption for both models, all showed that they should be treated as confident and suitable approximations to solve the problem of light re-absorption in leaves.     

Analysis of fluorescence lifetime curves. A new method for correcting raw fluorescence lifetime curves for the parallel contribution of scattered light.

A method is described for estimating the fractional contribution of light scattered from the excitation lamp to the normalized raw fluorescence lifetime curve. The method depends on the ratio of the slope of the normalized light scatter spectrum to the slope of the normalized raw fluorescence spectrum in the vicinity of the intersection of the two spectra. The correction for scattered light is made prior to deconvolution, and hence, has the advantage of being independent of the method selected to calculate the true fluorescence life-time spectrum. It is simple and does not require a computer. Tested against curves synthesized from known additions of scattered light to fluorescence spectra exhibiting mono-, bi-, or triexponential decay, it yielded small absolute errors.     

Optical properties of the adaxial and abaxial faces of leaves. Chlorophyll fluorescence, absorption and scattering coefficients.

Emission fluorescence spectra were obtained for the adaxial and abaxial faces of dicotyledonous (Ficus benjamina L., Ficus elastica, Gardenia jasminoides and Hedera helix) and monocotyledonous leaves (Gladiolus spp. and Dracaena cincta bicolor). After correction by light-re-absorption processes, using a previously published physical model, the adaxial faces of dicotyledons showed a fluorescence ratio Fred/Ffar-red rather lower than the respective values for the abaxial faces. Monocotyledons and shade-adapted-plants showed similar values for the corrected fluorescence ratio for both faces. Even when differences in experimental fluorescence emission from adaxial and abaxial leaves in dicotyledons are mostly due to light re-absorption processes, the residual dissimilarity found after application of the correction model would point to the fact that fluorescence re-absorption is not the only responsible for the observed disparity. It was concluded that light re-absorption processes does not account entirely for the differences in the experimental emission spectra between adaxial and abaxial leaves. Differences that remains still present after correction might be interpreted in terms of a different photosystem ratio (PSII/PSI). Experiments at low temperature sustained this hypothesis. In dicotyledons, light reflectance for adaxial leaves was found to be lower than for the abaxial ones. It was mainly due to an increase in the scattering coefficient for the lower leaf-side. The absorption coefficient values were slightly higher for the upper leaf-side. During senescence of Ficus benjamina leaves, the scattering coefficient increased for both the upper and lower leaf-sides. With senescence time the absorption coefficient spectra broadened while the corrected fluorescence ratio (Fred/Ffar-red) decreased for both faces. The results pointed to a preferential destruction of photosystem II relative to photosystem I during senescence.     

Photosystem II Photosynthetic Unit Sizes from Fluorescence Induction in Leaves : CORRELATION TO PHOTOSYNTHETIC CAPACITY

The use of fluorescence induction measurements in leaves infiltrated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea has been evaluated as a routine method for estimation of the concentration of the reaction centers of photosystem II relative to total chlorophyll in a wide variety of plant species. The procedure is based on a simple theory that takes into account the attenuation of light in passing through the leaf and the linear dependence of the fluorescence induction time from different parts of the leaf on the inverse of the local light intensity. A formula to calculate the reaction center concentration of photosystem II was obtained. The effect of the light attenuation is accounted for by a correction factor which could become practically insignificant by an optimal choice of the excitation and emission wavelengths and the geometry of the photodetector with respect to the sample. Estimation of quantum yields for primary photochemistry and influence of light scattering were considered. The results demonstrate the effect of the above factors under various circumstances and are in agreement, to a first approximation, with the theory.     

Resolution of the light-dependent modulation system of pea chloroplasts

The mechanism of the light-dependent inactivation of glucose-6-phosphate dehydrogenase and the light-dependent activation of NADP⁺-malate dehydrogenase has been studied in partially purified extracts of pea (Pisum sativum) chloroplasts. Neither partially purified enzyme could be light modulated by washed thylakoids alone. However, a factor (mol. wt. 50 000) was present in the stroma which could, when added to purified enzyme and thylakoid membranes, reconstitute a light-dependent modulation of either glucose-6-phosphate dehydrogenase or NADP⁺-malate dehydrogenase. This factor, which we term protein-modulating factor, is distinct from ferredoxin-thioredoxin reductase and from thioredoxin, the factors involved in another scheme for light modulation. The scheme proposed here for light modulation involves electron transfer from Photosystem I to a membrane-bound light-effect mediator and then to the soluble protein modulating factor which modulates chloroplast enzyme activity, probably by reduction of a regulatory disulfide bond.     

Biooptical characteristics of PSII and PSI in 33 species (13 pigment groups) of marine phytoplankton,and the relevance for pulse‐amplitude‐modulated and fast‐repetition‐rate fluorometry1

We studied the variability of in vivo absorption coefficients and PSII‐scaled fluorescence excitation (fl‐ex) spectra of high light (HL) and low light (LL) acclimated cultures of 33 phytoplankton species that belonged to 13 different pigment groups (PGs) and 10 different phytoplankton classes. By scaling fl‐ex spectra to the corresponding absorption spectra by matching them in the 540–650 nm range, we obtained estimates for the fraction of total chl a that resided in PSII, the absorption of light by PSII, PSI, and photoprotective carotenoids. The in vivo red peak absorption maxima ranged from 673 to 679 nm, reflecting bonding of chl a to different pigment proteins. A simple approach is presented for quantifying intracellular self‐shading and evaluating the impact of photoacclimation on biooptical characteristics of the different PGs examined. In view of these results, parameters used in the calculation of oxygenic photosynthesis based on pulse‐amplitude‐modulated (PAM) and fast‐repetition‐rate (FRR) fluorometers are discussed, showing that the ratio between light available to PSII and total absorption, essential for the calculation of the oxygen release rate (using the PSII‐scaled fluorescence spectrum as a proxy) was dependent on species and photoacclimation state. Three subgroups of chromophytes exhibited 70%–80%, 60%–80%, and 50%–60% chl a in PSII‐LHCII; the two subgroups of chlorophytes, 70% or 80%; and cyanobacteria, only 12%. In contrast, the mean fraction for chromo‐ and chlorophytes of quanta absorbed by PSII was 73% in LL‐ and 55% in HL‐acclimated cells; thus, the corresponding ratios 0.55 and 0.73 might be used as correction factors adjusting for quanta absorbed by PSII for PAM and FRR measurements.     

The influence of the electric diffusion potential on delayed fluorescence light curves of chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea

The potassium salt-induced transient increase of delayed fluorescence yield was studied in pea chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea.A simple kinetic model is proposed to account for the actinic light intensity dependence of the delayed fluorescence enhancement by the transmembrane diffusion potential induced by sudden salt addition. The electric field dependence of the rate constants for the recombination of primary separated charges with and without subsequent electronic excitation of reaction center chlorophyll was obtained.From the value of enhancement of delayed fluorescence by salt concentration gradients at saturating actinic light intensity, it is concluded that the distance, normal to thylakoid membrane surface, between the primary acceptor and the donor of Photosystem II is smaller than the membrane thickness.     

The identification of intermediates in the reaction of pig heart lactate dehydrogenase with its substrates

Pig heart lactate dehydrogenase was studied in the direction of pyruvate and NADH formation by recording rapid changes in extinction, proton concentration, nucleotide fluorescence and protein fluorescence. Experiments measuring extinction changes show that there is a very rapid formation of NADH within the first millisecond and that the amplitude of this phase (phase 1) increases threefold over the pH range 6-8. A second transient rate (phase 2) can also be distinguished (whose rate is pH-dependent), followed by a steady-state rate (phase 3) of NADH production. The sum of the amplitudes of the first two phases corresponds to 1mol of NADH produced/mol of active sites of lactate dehydrogenase. Experiments that measured the liberation of protons by using Phenol Red as an indicator show that no proton release occurs during the initial very rapid formation of NADH (phase 1), but protons are released during subsequent phases of NADH production. Fluorescence experiments help to characterize these phases, and show that the very rapid phase 1 corresponds to the establishment of an equilibrium between E(NAD) (Lactate) right harpoon over left harpoon H(+)E(NADH) (Pyruvate). This equilibrium can be altered by changing lactate concentration or pH, and the H(+)E(NADH) (Pyruvate) species formed has very low nucleotide fluorescence and quenched protein fluorescence. Phase 2 corresponds to the dissociation of pyruvate and a proton from the complex with a rate constant of 1150s(-1). The observed rate constant is slower than this and is proportional to the position of the preceding equilibrium. The E(NADH) formed has high nucleotide fluorescence and quenched protein fluorescence. The reaction, which is rate-limiting during steady-state turnover, must then follow this step and be involved with dissociation of NADH from the enzyme or some conformational change immediately preceding dissociation. Several inhibitory complexes have also been studied including E(NAD+) (Oxamate) and E(NADH) (Oxamate') and the abortive ternary complex E(NADH) (Lactate). The rate of NADH dissociation from the enzyme was measured and found to be the same whether measured by ligand displacement or by relaxation experiments. These results are discussed in relation to the overall mechanism of lactate dehydrogenase turnover and the independence of the four binding sites in the active tetramer.     

Decay-associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase

A procedure is described for using nanosecond time resolved fluorescence decay data to obtain decay-associated fluorescence spectra. It is demonstrated that the individual fluorescence spectra of two or more components in a mixture can be extracted without prior knowledge of their spectral shapes or degree of overlap. The procedure is also of value for eliminating scattered light artifacts in the fluorescence spectra of turbid samples. The method was used to separate the overlapping emission spectra of the two tryptophan residues in horse liver alcohol dehydrogenase. Formation of a ternary complex between the enzyme, NAD+, and pyrazole leads to a decrease in the total tryptophan fluorescence. It is shown that the emission of both tryptophan residues decreases. The buried tryptophan (residue 314) undergoes dynamic quenching with no change in the spectral distribution. Under the same conditions, the fluorescence intensity of tryptophan (residue 15) decreases without a change in decay time but with a red shift of the emission spectrum. There is also a decrease in tryptophan fluorescence intensity when the free enzyme is acid denatured (succinate buffer, pH 4.1). The denatured enzyme retains sufficient structure to provide different microenvironments for different tryptophan residues as reflected by biexponential decay and spectrally shifted emission spectra (revealed by decay association). The value of this technique for studies of microheterogeneity in biological macromolecules is discussed.     

NADPH dehydrogenase-mediated respiratory electron transport in thylakoid membranes of the cyanobacterium Synechocystis sp. PCC 6803 is inactive in the light

Non-photochemical redox changes of the plastoquinone pools in darkness were investigated in the cyanobacterium Synechocystis sp. PCC 6803 by monitoring changes in Chl fluorescence yield during light-to-dark transitions. The inhibitors rotenone and mercury with or without 1 mM succinate fully suppressed the post-illumination increase in Chl fluorescence in both NADPH dehydrogenase-defective (M55) and deltaCtaI cells. The latter cells lack subunit I of cytochrome aa3-type cytochrome c oxidase. These results strongly suggest that NADPH dehydrogenase plays the major role in electron donation in the non-photo-chemical reduction of plastoquinone. The rising phase of post-illumination Chl fluorescence in both wild type pretreated with KCN, and deltaCtaI cells, was significantly slowed by low light illumination. We detected comparable photochemical levels of both photosystem (PS) II and PSI during steady state illumination in wild type and deltaCtaI cells. From these results, we suggest that respiratory electron flow involved in the non-photochemical redox change of plastoquinone is not likely to occur in the light.     

Catabolism of adenine derivatives in leaves: study of the role of light on the in vivo activity of xanthine dehydrogenase

The in vivo activity of xanthine dehydrogenase (E.C. 1.2.1.37) was followed in leaf discs excised from illuminated or darkened plants. In cotyledons of Pharbitis nil, 24 hours of darkness enhanced the in vivo activity of xanthine dehydrogenase which increased between 2 to 5-fold depending on the concentration of hypoxanthine of the solution where cotyledon discs were incubated. The same effect occurred in leaves of several other species, in plants with both high and low ureide content. However, the effect of light was not observed in leaves of Zea mays, Pennisetum americanum and Atriplex spongiosa, whereas, it appeared very clearly in other C₄ plants such as Sorghum sudanense and Portulaca oleracea. This enzymic activity in chlorophyll-deficient tobacco leaves was the same both for illuminated and darkened plants. In addition, the in vivo activity of xanthine dehydrogenase in roots of Pharbitis nil was not dependent upon the light conditions applied to leaves. In cotyledons of Pharbitis nil, the level of the in vivo activity of xanthine dehydrogenase was influenced by the energy of light and the duration of illumination. The supply of carbohydrates to darkened cotyledons had the same effect as light on the in vivo activity of xanthine dehydrogenase. It is proposed that the effect of light on the in vivo activity of xanthine dehydrogenase in leaves is mainly due to the production of photosynthates which changes the osmotic state of leaf tissue and thus modifies the level of the in vivo activity of xanthine dehydrogenase.     

Fluorescence imaging to quantify the fluorescent microspheres in cardiac tissue

To quantify the fluorescent microsphere (FM) content in cardiac tissue, which is an indicative of blood flow, fluorescence imaging of both sides of the pig heart slice was employed. Despite the light scattering inside the tissue and contributions from multiple tissue layers to the total emission, it is shown that the fluorescence intensity at any pixel is proportional to the FM content and the fluorescence image may be transformed to the image of the FM concentration. A convenient standard for the emission‐FM concentration transformation is proposed. The approach has several advantages in comparison with the traditional “digestion & extraction” method such as: non‐destructiveness, high spatial resolution, high throughput, repeatability and simplicity of operation. (© 2011 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Dynamic interactions of enzymes involved in triosephosphate metabolism

A steady-state kinetic analysis of the coupled reactions catalysed by the three-enzyme system, aldolase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, was performed. The kinetic parameters of the progress curves of end-product formation calculated for noninteracting enzymes were compared with those measured in the two-enzyme and three-enzyme systems. Changes in the fluorescence anisotropy of labelled dehydrogenase upon addition of aldolase and/or isomerase were also measured. Glyceraldehyde-3-phosphate oxidation catalysed by glyceraldehyde-3-phosphate dehydrogenase in the presence of isomerase (which ensures rapid equilibration of the triosephosphates) follows single first-order kinetics. The rate constant depends simply on the concentration of the dehydrogenase, indicating no kinetically significant isomerase-dehydrogenase interaction. Fluorescence anisotropy measurements also fail to reveal complex formation between the two enzymes. The steady-state velocity of 3-phosphoglycerate formation from fructose 1, 6-bisphosphate in the reactions catalysed by aldolase and dehydrogenase is not increased twofold on addition of the isomerase, even though a 1:2 stoichiometry of fructose 1,6-bisphosphate/glyceraldehyde 3-phosphate is expected. In fact, by increasing the concentration of the isomerase, the steady-state velocity actually decreases. This effect of the isomerase may be a kinetic consequence of an aldolase-isomerase interaction, which results in a decrease of aldolase activity. Furthermore, the fluorescence anisotropy of labelled dehydrogenase, measured at different aldolase concentrations, is significantly lower when the sample contains isomerase. The decrease in the steady-state velocity of the consecutive reactions caused by the elevation of isomerase concentration could be negated by increasing the dehydrogenase concentrations in the three-enzyme system. All of these observations fit the assumption that the amount of aldolase-dehydrogenase complex is reduced due to competition of isomerase with dehydrogenase. The alternate binding of dehydrogenase and isomerase to aldolase may regulate the flux rate of glycolysis.