Antarctic permafrost degassing in Taylor Valley by extensive soil gas investigation.

Ongoing studies conducted in northern polar regions reveal that permafrost stability plays a key role in the modern carbon cycle as it potentially stores considerable quantities of greenhouse gases. Rapid and recent warming of the Arctic permafrost is resulting in significant greenhouse gas emissions, both from physical and microbial processes. The potential impact of greenhouse gas release from the Antarctic region has not, to date, been investigated. In Antarctica, the McMurdo Dry Valleys comprise 10 % of the ice-free soil surface areas in Antarctica and like the northern polar regions are also warming albeit at a slower rate. The work presented herein examines a comprehensive sample suite of soil gas (e.g., CO2, CH4 and He) concentrations and CO2 flux measurements conducted in Taylor Valley during austral summer 2019/2020. Analytical results reveal the presence of significant concentrations of CO2, CH4 and He (up to 3.44 vol%, 18,447 ppmv and 6.49 ppmv, respectively) at the base of the active layer. When compared with the few previously obtained measurements, we observe increased CO2 flux rates (estimated CO2 emissions in the study area of 21.6 km2 ≈ 15 tons day-1). We suggest that the gas source is connected with the deep brines migrating from inland (potentially from beneath the Antarctic Ice Sheet) towards the coast beneath the permafrost layer. These data provide a baseline for future investigations aimed at monitoring the changing rate of greenhouse gas emissions from Antarctic permafrost, and the potential origin of gases, as the southern polar region warms.

Erythrocyte viscoelastic recovery after liver transplantation in a cirrhotic patient affected by spur cell anaemia.

In physiological conditions, red blood cells (RBCs) are capable of dramatic deformations when passing through the microvasculature. This extreme deformability is closely related to the RBC biconcave shape, to the fluidic nature of the haemoglobin and the cell membrane structure, primarily consisting of a phospholipid bilayer with an underlying two-dimensional spectrin network. In many pathological and inflammatory conditions, the shape and the extreme deformability of erythrocytes appear to be significantly altered. These findings have stimulated intense research towards the search and validation of novel erythrocyte-based mechanical biomarkers, useful for disease diagnosis and therapy monitoring. In this study, we investigated with Atomic Force Microscopy (AFM) the mechanical properties of erythrocytes obtained from a 68 years old cirrhotic man diagnosed with spur cell anaemia and cold agglutinated disease, before and after liver transplantation. Mechanical changes are compared with ultrastructural alterations as studied by scanning electron microscopy and discussed according to confocal fluorescence microscopy results, showing possible alterations induced by the cirrhotic environment at the level of the RBCs cytoskeletal organisation and lipidic composition. Taken together, the results here presented show that liver transplantation not only contributes to restoring the proper RBC morphology, but it also induces recovery of the physiological viscous behaviour of cells, further stressing the relevance of viscous and dissipative forces in determining the RBC biomechanical response.

Evaluation of cytokines concentration and percentage of survival of rabies virus-infected mice submitted to anti-rabies Vero-cell propagated vaccine and P. acnes.

Previously, survival of rabies infection was shown to correlate with low IL-6 serum concentration in mice subjected to post-exposure treatment with the Fuenzalida Palacios rabies vaccine in conjunction with the immunomodulator Propionibacterium acnes, previously Corynebacterium parvum. Considering the substitution of the Fuenzalida Palacios rabies vaccine by the Vero cell raised anti-rabies vaccine in almost all countries, the objective of this work was to evaluate the survival and cytokine serum concentration of rabies virus-infected mice treated with P. acnes in conjunction with or the anti-rabies-VERO vaccine. For this, Swiss mice were experimentally infected with street rabies virus and subjected to vaccine and/or P. acnes following infection. Animals were killed at different times and serum was collected to evaluate cytokines. The greatest survival was observed in animals given one or two does of P. acnes in the absence of vaccination. Animals given anti-rabies VERO vaccine alone or with three doses of P. acnes had the second highest survival rate. The group that had the highest percentage of mortality also had the highest IL-6 concentration on the 10th day, a time correlating with clinical symptoms of the animals. The results reinforce the inefficacy of anti-rabies vaccine in only one dose as a post-exposure treatment irrespective of the type of vaccine used, the immunomodulation activity of P. acnes in rabies post-exposure treatment and suggest a role for IL-6 in rabies virus pathogenesis.

Photophysical properties of porphyrin planar aggregates in liposomes.

This paper describes studies of some photophysical properties of non-covalent planar aggregates of hematoporphyrin and protoporphyrin. This porphyrin species has been recently discovered and can be generated in lipid bilayers such as liposomes and inner mitochondrial membranes. The relative weight of this species in different media, as compared to porphyrin monomers and stacked aggregates, has been deduced by fluorescence decay studies. In contrast with what is observed for stacked aggregates, promotion of planar suprastructures can occur both in aqueous and lipid environments. The spectroscopic properties are very similar to those of the corresponding monomers, in particular as regards the shape of the absorption and emission spectra. The fluorescence decay times are generally higher than those of the monomers, and depend on the medium in which the planar aggregates are formed. The photooxidation properties of porphyrin planar aggregates, as revealed by oxygen consumption and histidine photodegradation upon irradiation at 365 nm, were compared to those of the monomers. The extent of the photooxidation process is nearly 20-30% higher in planar aggregates than in the monomers. In contrast, it is well known that cofacial aggregates are photochemically inert and only monomeric species of porphyrin are efficient photosensitizers. The biological relevance of these findings is discussed.

Alkaline denaturation and partial refolding of pepsin investigated with DAPI as an extrinsic probe.

The binding parameters of DAPI to porcine stomach pepsin have been described in the previous article in this issue (A. Mazzini et al.). Here we exploit the differences in the spectroscopic (fluorescence and circular dichroism) properties of DAPI bound to either native or alkali denatured pepsin. We follow the kinetics of pepsin denaturation around neutrality (pH range 6.8-7.4), at several phosphate buffer ionic strengths (range 0.02-0.25). The dependence of the apparent dissociation rate constant on pH clearly shows that the rate limiting step follows the dissociation of about three acidic protein residues. The accelerating effect by ionic strength we observed can be accounted for by a simple treatment based on both transition state theory and Debye-Hueckel's limiting law. Furthermore, when a solution of pepsin, rapidly denatured at pH 7, is reacidified to a pH between 4.5 and 5.5, a substantial recovery of protein secondary structure, with no enzymatic activity, is observed, judging by the far UV circular dichroism of the protein. This process of partial refolding can easily be followed using DAPI as an extrinsic reporter group, able to monitor the kinetics of formation and decay of a highly fluorescent intermediate. This process becomes faster at a lower pH, at least in the limited range investigated (pH 4.5-5.5), in which the refolded protein does not aggregate, but, in contrast to unfolding, is almost independent in ionic strength.

Interaction of DAPI with pepsin as a function of pH and ionic strength.

The fluorescent probe 4',6-diamidino-2-phenylindole (DAPI), extensively used with nucleic acids, has also recently been used with membranes and proteins (Favilla et al., Biophys. Chem., 46 (1993) 217-226 and Mazzini et al., Biophys. Chem. 52 (1994) 145-156, respectively). The spectroscopic changes of DAPI observed, namely a considerable enhancement of fluorescence, induced circular dichroism (CD) and absorbance spectral shifts, have been exploited to study the binding of this dye to both native and alkali denatured pepsin. Fluorescence and CD titrations show that nearly two molecules of DAPI bind to either native or alkali denatured pepsin with pH and ionic strength dependent Kd values, whereas absorbance titrations evidentiate an interaction characterized by a lower affinity and a larger number of binding sites. Therefore two kinds of interaction are proposed: a specific one, involving both hydrophobic and electrostatic forces; and a non-specific one, involving surface protein negative charges only. Finally, the behaviour of DAPI as a competitive inhibitor and the remarkable effect of pepstatin A, a specific inhibitor of pepsin, on both the CD and fluorescence spectra of DAPI in the presence of pepsin, show the involvement of the protein active site in the interaction.

A model for the explanation of the thermally induced increase of the overall fluorescence in tryptophan-X peptides.

In the range of temperature 10-35 degrees C, Trp-X dipeptides show an unusual increase of fluorescence intensity in solution at pH 7. This effect has been recently studied by means of steady-state fluorescence. Although a model involving the deprotonation at the ground state of the zwitterion was proposed, the activation energy for that process could not rule out the involvement of excited state. In order to understand the mechanism of the thermal-induced increase of fluorescence, we present here time-resolved fluorescence experiments on Trp-X and X-Trp dipeptides at different pH and excitation wave-length. The fluorescence lifetimes (tau i) decrease in accord to thermal quenching, with activation energies (Ei) ranging from 4.0 to 6.4 kcal/mol. Under those circumstances where the anomaly was detected the preexponential factors of the longer-lived component increased as well as their fractional fluorescence. This component can be assigned to the anion species. Because of its larger (three- to fourfold) fluorescence quantum yield, compared to that of the corresponding zwitterion, the large increase of the concentration of the anion leads to an increase of the overall emission despite the thermal quenching. Also the decay-associated spectra well account for the red shift of the emission fluorescence spectrum, which accompanies the anomaly. Our model well fits the experimental data using a simple equation which combines Van't Hoff and Arrhenius equations; it also explains the presence of the anomalous thermal quenching exclusively in Trp-X dipeptides excited above 290 nm and at pH around neutrality.

An oxygenation-sensitive dye binding to Carcinus maenas hemocyanin.

The interaction of 4',6-diamidino-2-phenylindole (DAPI) with Carcinus maenas hemocyanin has been investigated by steady state fluorescence, dynamic fluorescence and circular dichroism measurements. The dye binds to apohemocyanin (without copper) as well as to oxygenated hemocyanin and to deoxygenated hemocyanin with very similar affinities (kd approximately equal to 1 microM ) and number of binding sites (one per subunit). In contrast, the fluorescence quantum yield enhancement of DAPI bound to oxygenated hemocyanin is nearly 60% lower than that observed for deoxygenated and apo forms. The decrease of fluorescence of the dye bound to deoxygenated hemocyanin is a sigmoidal function of the oxygen partial pressure, specular to that observed by following the absorbance of the copper-oxygen charge transfer band at 340 nm. This result provides preliminary evidence that DAPI may be used as a functional probe to monitor the cooperative binding of oxygen to the protein. The higher fluorescence quantum yield of DAPI bound to either apohemocyanin or deoxygenated protein is characterized by a single fluorescence decay with lifetime of about 3 ns, while with the oxygenated protein two components of about 1 ns and 3.0 ns are observed. This result is interpreted assuming the existence of two rotamers of DAPI in solution (Szabo et al. Photochem. Photobiol. 44 (1986) 143-150) both able to interact with oxygenated hemocyanin but only one to deoxygenated and apo forms. We conclude that the different fluorescence behaviour of the dye induced by the presence of oxygen bound to the protein is probably due to a structural change of hemocyanin in cooperative interaction with oxygen. Furthermore, the interaction is confirmed by the induced negative ellipticity of DAPI bound to apohemocyanin and deoxy-hemocyanin and by the increase of fluorescence anisotropy of DAPI bound to all forms of protein investigated.

The interaction of DAPI with phospholipid vesicles and micelles.

The interactions of the dye 4',6-diamidino-2-phenylindole (DAPI) with phospholipids ordered in single bilayer vesicles of dioleylphosphatidylserine (DOPS) or dimyristoylphosphatidylcholine (DMPC) or micelles of monomyristoylphosphatidylcholine (MPC) have been investigated. Somewhat unexpectedly, the binding of this dye to such ordered structures is not affected by the ionic strength of the external medium, which suggests an embedding of DAPI into the hydrocarbon phase. The fluorescence enhancement of DAPI bound can be accomodated within a model previously proposed for the behaviour of DAPI bound to proteins (Mazzini et al., Biophys. Chem. 42 (1992) 101). From both static and dynamic anisotropy measurements, bound DAPI results severely restricted in its rotational freedom but insensitive to the temperature dependent phase transition of the saturated DMPC vesicles. The considerable tightness and specificity of the interactions between DAPI and ordered phospholipids are also deduced from preliminary fluorescence quenching studies (reduced accessibility of iodide ions towards DAPI bound and quenching effects by the chaotrop Nonidet P-40).

Fluorescence studies on the conformation of litorin in solution and in the presence of model membranes.

The conformation of the nonapeptide hormone litorin has been studied in buffer and in the presence of lipids, using static and dynamic fluorescence. The results obtained show that, in buffer, the hormone probably exists in a collection of flexible conformers, slowly interconverting between them. The marked changes observed in fluorescence spectra and lifetimes upon addition of dimyristoylphosphatidylserine vesicles clearly show that the peptide interacts with lipids assuming lipid specific conformations. Interestingly, no significative spectroscopic changes are produced by exposure to dimirystoylphosphatidylcholine vesicles both in the gel and liquid-christalline phases, suggesting a requirement for negatively charged lipids during the process of hormone-membrane interaction.

The interaction of 4', 6-diamidino-2-phenylindole (DAPI) with pepsin.

We recently found that the fluorescent dye DAPI, well-known for its use with nucleic acids, is also able to interact with proteins as well as ordered phospholipids assemblies. The interaction of DAPI with pepsin under different conditions of pH and ionic strength was studied with fluorescence and circular dichroism techniques. From a comparison of the results obtained, the interaction appears to be rather tight and specific, dependent on both electrostatic and hydrophobic forces, and able to probe the tridimensional conformation of the protein.

The binding of 4',6-diamidino-2-phenylindole to bovine serum albumin.

The binding of 4',6-diamidino-2-phenylindole (DAPI) to bovine serum albumin (BSA) has been investigated between pH 6 and 8, in 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations and the results analyzed according to a procedure previously reported (R. Favilla and A. Mazzini, Biochim. Biophys. Acta 788 (1984) 48). The dye binds to the protein with a blue shift of about 4 nm in its fluorescence emission maximum, but with an enhancement factor of 10 of its fluorescence quantum yield. The dissociation constant decreases from 100 microM to 54 microM as the pH is increased from 6 to 8, with a constant number of nearly three equivalent binding sites. The complete displacement of DAPI bound to BSA by Ca2+ suggests a possible specificity of this substantially electrostatic interaction. The fluorescence decay of DAPI bound to the protein shows a double exponential kinetics, with a tau 1 = 0.97 ns and tau 2 = 2.78 ns. These results, compared with those obtained for DAPI alone, tau 1 = 0.16 ns and tau 2 = 2.8 ns, are rationalized in terms of two different rotamers of DAPI. Both rotamers are able to bind to the protein, but only one of them undergoes an intramolecular proton transfer, from the 6-amidinium group to the indole aromatic ring, in the excited singlet state of DAPI alone. When DAPI interacts with BSA this transfer does not occur and consequently a large increase of fluorescence is observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential adaptive response to hyperosmolarity of 3T3 and transformed SV3T3 cells.

Both 3T3 and simian virus 40-transformed 3T3 (SV3T3) cells were used to investigate differences in population kinetics, protein synthesis, monovalent ion levels, and amino acid accumulations between normal and transformed cells exposed to hyperosmolarity at 0.5 Osm. Under similar culture conditions, SV3T3 cells were found to be more sensitive in their proliferative response than normal cells to the hyperosmolar treatment. In the normal 3T3 cells, the increase in transport of amino acids was less sustained and was associated with higher levels of accumulated amino acids. The equilibrium distribution of intracellular monovalent cations and the rate of protein synthesis also returned faster to baseline values in the normal cells than in the transformed cells. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis revealed the induction of a 69-kDa polypeptide in the 3T3 cells but not in the SV3T3 cells after exposure to hyperosmolarity. On electrofocusing and relative mass analysis, this polypeptide closely migrated with the 70-kDa heat shock protein (hsp) family, although it was unrelated immunologically to the inducible 72-kDa hsp.

Control of protein synthesis by extracellular Na+ in cultured fibroblasts.

In chick embryo fibroblasts (CEFs), a partial substitution of extracellular Na+ with other cations or carbohydrates decreased the intracellular Na+ content without altering the K+ level. Concomitantly, a significant decrease in the serum-dependent rate of protein synthesis occurred. This phenomenon appeared to be quickly reversible upon reconstitution of the correct extracellular Na+ concentration in the culture medium. The presence of a transcriptional inhibitor such as actinomycin D during the treatment did not inhibit the reversibility of the phenomenon. The presence in the culture medium of K+ in such excess as to dissipate the membrane potential did not alter the observed relationship between the protein synthesis rate and the internal Na+ content. Analysis of the amino acid pool indicated that the observed inhibition of the rate of protein synthesis in CEFs incubated in low Na+ medium was not caused by an unbalanced availability of intracellular amino acids. In addition, intracellular pH, as estimated by the measurement of the equilibrium distribution of benzoic acid, did not show any significant alteration in cells incubated in the presence of bicarbonate buffer and in low extracellular Na+. Moreover, the relationship between the rate of protein synthesis and the internal Na+ content was still observed in CEFs cultured in bicarbonate-containing media, but at lower or higher than physiological pH. Analysis by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of the proteins synthesized by CEFs cultured at a reduced extracellular Na+ concentration showed that specific alterations of gene expression occurred.

Fast kinetics analysis of the peroxidatic reaction catalysed by horse liver alcohol dehydrogenase.

The kinetics of the enzymatic step of the peroxidatic reaction between NAD and hydrogen peroxide, catalysed by horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1), has been investigated at pH 7 at high enzyme concentration. Under such conditions no burst phase has been observed, thus indicating that the rate-limiting step in the process, which converts NAD into Compound I, either precedes or coincides with the chemical step responsible for the observed spectroscopic change. Kinetic analysis of the data, performed according to a simplified reaction scheme suggests that the rate-limiting step is coincident with the spectroscopic (i.e., chemical) step itself. Furthermore, the absence of a proton burst phase indicates the proton release step does not precede the chemical step, in contrast with the case of ethanol oxidation. A kinetic effect of different premixing conditions on the reaction rate has been observed and attributed to the presence of NADH formed in the 'blank reaction' between NAD and residual ethanol tightly bound to alcohol dehydrogenase. A molecular mechanism for the enzymatic peroxidation step is finally proposed, exploiting the knowledge of the much better known reaction of ethanol oxidation. Inhibition of this reaction by NADH has been investigated with respect to H2O2 (noncompetitive, Ki about 10 microM) and to NAD (competitive, Ki about 0.7 microM). The effect of temperature on the steady-state reaction state (about 65 kJ/mol activation energy) has also been studied.

Hyperosmolarity-induced stress proteins in chick embryo fibroblasts.

The effects of a short exposure of chick embryo fibroblasts to a hyperosmolar medium on monovalent cation content, rate of protein synthesis, and polypeptide pattern expression were studied. The hyperosmolar shock gave an immediate and pronounced inhibition of the protein-synthesis rate temporally related to a marked alteration of the intracellular Na+ content. Following the return of the cells to an osmolar environment, the internal Na+ content quickly resumed its previous level, while the recovery of the protein-synthesis rate was more gradual. During the recovery period, there was enhanced expression of at least 12 proteins. The 4 major induced proteins exhibited apparent molecular weights of 96, 87, 70, and 48 kDa. A reduction in the synthesis of five protein bands including three large polypeptides of 220, 160, and 140 kDa was also observed. A comparison with the 3 major proteins induced by a 44 degrees C heat shock indicated an apparent similarity with only two of the hyperosmolarity-inducible polypeptides. Moreover, evidence has been also obtained of the close similarity between the 96 and 75 kDa glucose-regulated proteins and the 96 and 75 kDa proteins inducible by a hyperosmolar shock or by a continuous hyperosmolar treatment, respectively. The kinetics of the stress-proteins appearance indicated nonsimultaneous induction. The presence of actinomycin D during the exposure of the cells to the stress and the recovery period suggested that the expression of some hyperosmolarity-enhanced proteins is regulated at the transcriptional level.

The effect of guanidinium chloride on the self-association of bovine liver glutamate dehydrogenase: a gel filtration study.

The associative behaviour of bovine liver glutamate dehydrogenase has been studied by gel chromatography at neutral pH in 1 M guanidinium chloride and 1 M sodium chloride. In guanidinium chloride both the elution volume and the elution profile of the enzyme are independent of protein concentration, whereas in sodium chloride they are strongly dependent on it. In NaCl the enzyme behaves as expected according to the well established random association model, whereas in guanidinium chloride it appears to have completely lost the self-associative property. Furthermore, since the elution volume of the enzyme in guanidinium chloride corresponds to that of an hexamer, trimer formation reported to occur in these conditions is not confirmed by this technique.

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique.

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.

Effect of l-carnitine on human neutrophil activity.

The effect of l-carnitine on human neutrophil oxidative metabolism was investigated, both on superoxide production and luminol amplified chemiluminescence (CL) in phorbol-myristate-acetate (PMA) stimulated cells. L-carnitine either preincubated 10 minutes with the cells, before PMA challenge, or added simultaneously to the stimulator, inhibited superoxide generation. When tested in an O2- -generating system, such as xanthine-xanthine oxidase, l-carnitine did not act as an O2- scavenger. On the PMA induced CL response the drug was ineffective as an inhibitor, if preincubated with the cell suspension before activation. When added together with PMA, l-carnitine significantly inhibited the CL. Taken together the results reveal that the drug might affect the interaction of PMA with its specific receptor in human neutrophils, that is protein kinase C.

The binding of 1,N6-etheno-NAD to bovine liver glutamate dehydrogenase.

The binding of 1,N6-etheno-NAD (epsilon NAD) to bovine liver glutamate dehydrogenase (L-glutamate:NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) saturated with glutarate has been investigated at pH 7.0, 0.05 M phosphate buffer at 20 degrees C, by fluorescence titrations. epsilon NAD binds to the protein in a simple fashion: one molecule of coenzyme per enzyme polypeptide chain in the range of enzyme concentrations investigated (from above 50 to a few micromoles of enzyme polypeptide chains/liter). The fluorescence enhancement factor, Q, of bound epsilon NAD relative to free epsilon NAD is independent of the saturation degree, as deduced from the constant value of the long fluorescence decay lifetime (about 21 ns), and is about 17, as deduced from Fmax/F0 ratio values obtained after extrapolation from double reciprocal plots of 1/delta F vs. 1/[glutamate dehydrogenase]. This value for the Q factor is also independent of enzyme concentration, as well as of the presence of either GTP or ADP. At low enzyme concentrations (below 20 mumol polypeptide chains/liter), the dissociation constant of epsilon NAD increases progressively from a plateau value of about 50 microM to about 100 microM at infinite dilution. This is interpreted as being due to a minor affinity of glutamate dehydrogenase hexamers, with respect to higher aggregation states of the enzyme, towards epsilon NAD. As expected, GTP and ADP change the affinity of glutamate dehydrogenase towards epsilon NAD in an opposite manner: GTP strongly increases it, whereas ADP strongly decreases it (Kappd around 6 microM with saturating GTP and around 300 microM with saturating ADP). Furthermore, in the case of GTP, both GTP and epsilon NAD bind to glutamate dehydrogenase with positive cooperativity, with a Hill coefficient of approx. 1.8 for both and a Kappd approximately equal to 30 microM for the binding of GTP to glutamate dehydrogenase saturated with epsilon NAD and glutarate. The value of the Q factor remains the same, even in the presence of the effectors (again from lifetime measurements), as well as the number of epsilon NAD binding sites per enzyme polypeptide chain. These results are interpreted in terms of independent active sites, in the case without effectors. With ADP the binding appears to be simple, but no careful investigation has been attempted at low enzyme concentrations because of the low saturation degree achievable, whereas with GTP the cooperativity can be explained as due to a shift towards hexamers from higher aggregation states.