Resonance Raman spectra of copper(II)-substituted liver alcohol dehydrogenase: a type 1 copper analogue

Liver alcohol dehydrogenase (LADH) with copper in place of the catalytic zinc has recently been proposed to contain a type 1 site analogous to that in "blue" copper proteins. Resonance Raman spectra for the copper-substituted enzyme, Cu(II) X LADH, and its binary complexes with reduced nicotinamide adenine dinucleotide (NADH) and pyrazole support this viewpoint. These spectra have two dominant features: a sharp peak at approximately 415 cm-1, which is believed to be associated with vibration of the single histidine ligand, and a broader, asymmetric band at approximately 350 cm-1, whose components are assigned predominantly to vibrational modes of the two cysteinate ligands. The high frequency of these transitions, which is reminiscent of the blue copper proteins, is ascribed to the tetrahedral nature of the metal site that produces unusually short Cu-S bonds and coupled vibrational modes. Solvent exchange with H218O reveals no contribution to the resonance Raman spectrum of the water molecule, which is a metal ligand in free Cu(II) X LADH; however, the spectrum of the binary complex with pyrazole has several new peaks attributable, in part, to pyrazole ligation. The strong similarity among the vibrational spectra demonstrates that the Cu(II) environment in alcohol dehydrogenase maintains its near-tetrahedral geometry in the various enzyme derivatives. The resonance Raman spectrum of Ni(II) X LADH is close to that of Cu(II) X LADH and suggests a similar tetrahedral site. The Raman spectra presented here together with available optical and EPR data indicate that Cu(II) X LADH belongs to the type 1 copper classification and, thus, can provide new insights into this unusual coordination geometry.     

Spectroscopic investigation of binary and ternary coenzyme complexes of yeast alcohol dehydrogenase.

Corrected fluorescence properties of yeast alcohol dehydrogenase and its coenzyme complexes have been investigated as a function of temperature. Dissociation constants have been obtained for binary and ternary complexes of NAD and NADH by following the enhancement of NADH fluorescence or the quenching of the protein fluorescence. It is found that the presence of pyrazole increases the affinity of NAD to the enzyme approximately 100-fold. The formation of the ternary enzyme - NAD - pyrazole complex is accompanied by a large change in the ultraviolet absorption properties, with a new band in the 290-nm region. Significant optical changes also accompany the formation of the ternary enzyme-NADH-acetamide complex. The possible origin for the quenching of the protein fluorescence upon coenzyme binding is discussed, and it is suggested that a coenzyme-induced conformational change can cause it. Thermodynamic parameters associated with NAD and NADH binding have been evaluated on the basis of the change of the dissociation constants with temperature. Optical and thermodynamic properties of binary and ternary complexes of yeast alcohol dehydrogenase are compared with the analogous properties of horse liver alcohol dehydrogenase.     

Copper ion redox state is critical for its effects on ion transport pathways and methaemoglobin formation in trout erythrocytes.

We have studied the mechanism of copper uptake by the cells, its oxidative action and effects on ion transport systems using rainbow trout erythrocytes. Cupric ions enter trout erythrocytes as negatively charged complexes with chloride and hydroxyl anions via the band 3-mediated Cl-/HCO3- exchanger. Replacement of Cl- by gluconate, and complexation of cupric ions with histidine abolish rapid Cu2+ uptake. Within the cell cupric ions interact with haemoglobin, causing methaemoglobin formation by direct electron transfer from heme Fe2+ to Cu2+, and consecutive proton release. Ascorbate-mediated reduction of cupric ions to cuprous decreases copper-induced metHb formation and proton release. Moreover, cuprous ions stimulate Na+H+ exchange and residual Na+ transport causing net Na+ accumulation in the cells. The effect requires copper binding to an externally facing thiol group. Copper-induced Na+ accumulation is accompanied by K+ loss occurring mainly via K+-Cl- cotransporter. Taurine efflux is also stimulated by copper exposure. However, net loss of osmolytes is not as pronounced as Na+ uptake and modest swelling of the cells occurs after 5 min of copper exposure. Taken together the results indicate that copper toxicity, including copper transport into the cells and its interactions with ion transport processes, depend on the valency and complex formation of copper ions.     

The role of an essential histidine residue of yeast alcohol dehydrogenase.

1. Inactivation of yeast alcohol dehydrogenase for diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. 2. Enzyme modified by diethyl pyrocarbonate can form the binary enzyme - NADH complex with the same maximum NADH-binding capacity as that of native enzyme. Modified enzyme cannot form normal ternary complexes of the type enzyme - NADH - acetamide and enzyme - NAD+ - pyrazole, which are characteristic of native enzyme. 3. The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5--9. The histidine residue involved has approximately the same pKa as free histidine, but is 10-fold more reactive than free histidine.     

Dismutation of superoxide radicals by ceruloplasmin--details of the mechanism

Like superoxide dismutase (SOD), human ceruloplasmin (Cp) scavenges superoxide anion radicals injected into the solution with the aid a high-voltage generator, hydrogen peroxide being the product of reaction. The O2-/H2O2 ratio is close to 2:1. The dismutase activity of Cp is about 1500 times lower than that of Cu, Zn-SOD isolated from human erythrocytes. The dismutation of O2- accomplished by SOD, "free" copper ions, native Cp or partly copper-depleted Cp, is inhibited with equal efficiency by cyanide. All the copper ions of the multicopper catalytic center of Cp are not essentially required for the dismutation of O2-, since the enzyme depleted of all type 2 Cu2+ and partly of type 1 Cu2+ lost none of its dismutase activity. Type 1 copper ions of Cp seem to play the leading role in the one-electron transfer occurring upon dismutation of O2-.     

Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

Carboxymethyl horse-liver alcohol dehydrogenase. Ligand-binding and kinetic properties of the cysteine-46-modified enzyme.

Horse-liver alcohol dehydrogenase was carboxymethylated with iodoacetate, which is known to selectively alkylate cysteine-46 in the polypeptide sequence. Carboxymethyl and native enzyme had the same electrophoretic mobility on starch or polyacrylamide gel, but some separation was achieved when isobutyramide and a low concentration of NADH were present (under these conditions NADH was bound by native enzyme but not by Carboxymethyl enzyme).The Carboxymethyl enzyme formed ternary complexes with NAD⁺ and pyrazole or decanoate. The fluorescence emission of NADH was enhanced 7- to 8-fold (at 410 nm), and a dissociation-constant of 1.7 μM was calculated at pH 7.4; but, in contrast to native enzyme, neither the affinity nor fluorescence were increased by amides (acetamide or isobutyramide).Carboxymethyl alcohol dehydrogenase possesses catalytic activity. Higher alcohols gave maximum velocities up to 7-fold higher than ethanol (reaching nearly 20% of the activity of native enzyme) while [²H]ethanol showed an isotope-rate effect of 3.3. Although the affinity for aldehydes was considerably increased, the maximum velocity of aldehyde-reduction was always at least 20% of that shown by native enzyme, and at pH 9.9 it was almost 2-fold greater than with native enzyme. The rate-limiting step in alcohol-oxidation is likely to be the interconversion of ternary complexes (possibly the hydride-transfer step), while in aldehyde-reduction it could still be the dissociation of the enzyme/NAD⁺ complex. This is also indicated by inhibition experiments with decanoate, pyrazole, and isobutyramide.These results suggest that a major effect of carboxymethylation is upon ternary complexes of enzyme and NADH, which become much more reluctant to form, either by combination of NADH and ligand with the modified enzyme, or by catalytic conversion of the enzyme/NAD ⁺/alcohol complex.     

The reactions of 1,10-phenanthroline with yeast alcohol dehydrogenase.

Freshly prepared samples of yeast alcohol dehydrogenase (EC 1.1.1.1) were inhibited by 1,10-phenanthroline at pH 7.0 and 0 degrees C in a two-stage process. The first step appeared to be slowly established, but was rendered reversible by removal of reagent or by addition of excess Zn2+ ions. The second step was irreversible and was associated with the dissociation of the tetrameric enzyme. The presence of saturating concentrations of NAD+ or NADH promoted and enhanced inhibition by the slowly established reversible process, but prevented dissociation of the enzyme. For the incubation mixtures containing NAD+, removal of the 1,10-phenanthroline resulted in virtually complete recovery of activity, whereas, for the incubation mixtures containing NADH, removal of the reagent gave only partial re-activation. The presence of NAD+ and pyrazole, or NADH and acetamide, in incubation mixtures with the enzyme gave rise to ternary complexes that gave protection against both forms of inactivation by 1,10-phenanthroline. The results support the view that at least some of the Zn2+ ions associated with yeast alcohol dehydrogenase have a catalytic, as opposed to a purely structural, role.     

The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.     

Steady-state kinetics of horse-liver alcohol dehydrogenase with a covalently bound coenzyme analogue

The steady-state kinetics of the enzyme modified by affinity labelling with NAD analogue, nicotinamide-N6-[N-(6-aminohexyl)carbamoylmethyl]-adenine dinucleotide, has been investigated using a recycling reaction with p-nitrosodimethylaniline and n-butanol as substrates and compared to the kinetics of native alcohol dehydrogenase. The modified enzyme obeys a ping-pong mechanism involving two inactive enzyme forms (enzyme-NAD and enzyme-NADH complexes in the 'open' conformations, the nicotinamide moieties of the coenzymes being out of the active center). The rate of p-nitrosodimethylaniline reduction in the reaction catalyzed by the modified enzyme is comparable to that observed in the presence of the native enzyme. On the other hand, the oxidation of butanol by the modified enzyme is essentially slower under our experimental conditions (pH 8.5). The measurements in the presence of specific alcohol dehydrogenase inhibitors competing with substrates and coenzymes (isobutyramide, pyrazole and AMP) revealed that the relative portion of the inactive 'open' form of the enzyme-NADH complex is negligible, whereas the 'open' form of the enzyme-NAD complex seems to represent a more significant portion (about 30%) under the conditions used.     

Active site specific cadmium(II)-substituted horse liver alcohol dehydrogenase: crystal structures of the free enzyme, its binary complex with NADH, and the ternary complex with NADH and bound p-bromobenzyl alcohol

Three crystal structures have been determined of active site specific substituted Cd(II) horse liver alcohol dehydrogenase and its complexes. Intensities were collected for the free, orthorhombic enzyme to 2.4-A resolution and for a triclinic binary complex with NADH to 2.7-A resolution. A ternary complex was crystallized from an equilibrium mixture of NAD+ and p-bromobenzyl alcohol. The microspectrophotometric analysis of these single crystals showed the protein-bound coenzyme to be largely NADH, which proves the complex to consist of CdII-LADH, NADH, and p-bromobenzyl alcohol. Intensity data for this abortive ternary complex were collected to 2.9-A resolution. The coordination geometry in the free Cd(II)-substituted enzyme is highly similar to that of the native enzyme. Cd(II) is bound to Cys-46, Cys-174, His-67, and a water molecule in a distorted tetrahedral geometry. Binding of coenzymes induces a conformational change similar to that in the native enzyme. The interactions between the coenzyme and the protein in the binary and ternary complexes are highly similar to those in the native ternary complexes. The substrate binds directly to the cadmium ion in a distorted tetrahedral geometry. No large, significant structural changes compared to the native ternary complex with coenzyme and p-bromobenzyl alcohol were found. The implications of these results for the use of active site specific Cd(II)-substituted horse liver alcohol dehydrogenase as a model system for the native enzyme are discussed.     

The role of metal in liver alcohol dehydrogenase catalysis. Spectral and kinetic studies with cobalt-substituted enzyme.

The kinetic and spectral properties of native and totally cobalt-substituted liver alcohol dehydrogenase have been compared. Based on titrimetric determinations of enzyme active site concentration, the turnover number at pH 7.0 for cobalt enzyme was the same as for native enzyme. At pH 10, however, the turnover number was slower for cobalt-substituted enzyme, 3.14 s-1 as compared with 4.05 s-1 for native enzyme. A comparison between native and totally cobalt-substituted enzyme showed a blue-shifted enzyme-NADH double difference spectrum and a splitting and red-shifted enzyme-NAD+-pyrazole double difference spectrum in the near-ultraviolet. The 655-nm peak of the cobalt-substituted enzyme was perturbed by the formation of enzyme-NADH binary complex, enzyme-NAD+-trifloroethanol ternary complex, and enzyme-NAD+ binary complex formation. At pH 7.0, the only observable step in the reaction sequence with a significantly different rate constant for cobalt enzyme was the catalytic hydrogen-transferring step. The rate constant for this step is 92 s-1 for totally cobalt-substituted enzyme as compared with 138 s-1 for native liver alcohol dehydrogenase. The results of this study indicate that zinc is involved in catalysis alcohol and NADH.     

Studies on the interactions of glycerol dehydrogenase from Bacillus stearothermophilus with Zn2+ ions and NADH

The interactions of the essential divalent cation, Zn2+, with the binary complex formed between glycerol dehydrogenase (glycerol:NAD+ 2-oxidoreductase, EC 1.1.1.6) and its coenzyme NADH have been examined by fluorescence spectroscopy. Both the metallo and non-metallo form of the enzyme bind the coenzyme NADH. The addition of Zn2+ ions to a solution of the binary complex formed between metal-depleted enzyme and NADH results in a rapid increase in fluorescence emission at 430 nm. This has been used to determine the on rate for Zn2+ to the enzyme/binary complex. A dissociation constant of 3.02 +/- 0.25.10(-9) M for the equilibrium between Zn2+ ions and the enzyme has been determined.     

Covalent binding of an NAD+ analogue to horse liver alcohol dehydrogenase in a ternary complex with pyrazole

Examination of the model of the fixation site of the adenosine phosphate part of NAD+ on horse liver alcohol dehydrogenase led us to synthesize a NAD+ analogue N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]-NAD+ in order to alkylate the carboxylic acid group of Asp-273 and to convert the normally dissociable coenzyme into a permanently bound prosthetic group. This NAD+ analogue is coupled to the horse liver alcohol dehydrogenase in the ternary complex formed with pyrazole. In these conditions the degree of fixation varies between 0.4 and 0.58 coenzyme molecule/enzyme subunit molecule. The N6-[N-(8-amino-3,6-dioxaoctyl)carbamoylmethyl]NAD+ acts as a true prosthetic group which can be reduced and reoxidized by a coupled substrate reaction and the internal activity of this holoenzyme corresponds to the amount of analogue incorporated.     

pH-dependent conformational states of horse liver alcohol dehydrogenase.

The quenching of liver alcohol dehydrogenase protein fluorescence at alkaline pH indicates two conformational states of the enzyme with a pKa of 9.8+/-0.2, shifted to 10.6+/-0.2 in D2O. NAD+ and 2-p-toluidinonaphthalene-6-sulfonate, a fluorescent probe competitive with coenzyme, bind to the acid conformation of the enzyme. The pKa of the protein-fluorescence quenching curve is shifted toward 7.6 in the presence of NAD+, and the ternary complex formation with NAD+ and trifluoroethanol results in a pH-independent maximal quench. At pH (pD) 10.5, the rate constant for NAD+ binding was 2.6 times faster in D2O2 than in H2O due to the shift of the pKa. Based on these results, a scheme has been proposed in which the state of protonation of an enzyme functional group with a pKa of 9.8 controls the conformational state of the enzyme. NAD+ binds to the acid conformation and subsequently causes another conformational change resulting in the perturbation of the pKa to 7.6. Alcohol then binds to the unprotonated form of the functional group with a pKa of 7.6 in the binary enzyme-NAD+ complex and converts the enzyme to the alkaline conformation. Thus, at neutral pH liver alcohol dehydrogenase undergoes two conformational changes en route to the ternary complex in which hydride transfer occurs.     

Covalent fixation of NAD+ to dehydrogenases and properties of the modified enzymes

Starting from 6-chloropurine riboside and NAD+, different reactive analogues of NAD+ have been obtained by introducing diazoniumaryl or aromatic imidoester groups via flexible spacers into the nonfunctional adenine moiety of the coenzyme. The analogues react with different amino-acid residues of dehydrogenases and form stable amidine or azobridges, respectively. After the formation of a ternary complex by the coenzyme, the enzyme and a pseudosubstrate, the reactive spacer is anchored in the vicinity of the active site. Thus, the coenzyme remains covalently attached to the protein even after decomposition of the complex. On addition of substrates the covalently bound coenzyme is converted to the dihydro-form. In enzymatic tests the modified dehydrogenases show 80-90% of the specific activity of the native enzymes, but they need remarkably higher concentrations of free NAD+ to achieve these values. The dihydro-coenzymes can be reoxidized by oxidizing agents like phenazine methosulfate or by a second enzyme system. Various systems for coenzyme regeneration were investigated; the modified enzymes were lactate dehydrogenase from pig heart and alcohol dehydrogenase from horse liver; the auxiliary enzymes were alcohol dehydrogenase from yeast and liver, lactate dehydrogenase from pig heart, glutamate dehydrogenase and alanine dehydrogenase. Lactate dehydrogenase from heart muscle is inhibited by pyruvate. With alanine dehydrogenase as the auxiliary enzyme, the coenzyme is regenerated and the reaction product, pyruvate, is removed. This system succeeds to convert lactate quantitatively to L-alanine. The thermostability of the binary enzyme systems indicates an interaction of covalently bound coenzymes with both dehydrogenases; both binding sites seem to compete for the coenzyme. The comparison of dehydrogenases with different degrees of modifications shows that product formation mainly depends on the amount of incorporated coenzyme.     

Purification and preliminary characterization of alcohol dehydrogenase from Aspergillus nidulans.

Aspergillus alcohol dehydrogenase is produced in response to growth in the presence of a wide variety of inducers, of which the most effective are short-chain alcohols and ketones, e.g. butan-2-one and propan-2-ol. The enzyme can be readily extracted from fresh or freeze-dried cells and purified to homogeneity on Blue Sepharose in a single step by using specific elution with NAD+ and pyrazole. The pure enzyme has Mr 290 000 by electrophoresis or gel filtration; it is a homopolymer with subunit Mr 37 500 by electrophoresis in sodium dodecyl sulphate; its amino acid composition corresponds to Mr 37 900, and the native enzyme contains one zinc atom per subunit. The enzyme is NAD-specific and has a wide substrate activity in the forward and reverse reactions; its activity profile is not identical with those of other alcohol dehydrogenases.     

NAD+-dependent oxidation of 20-hydroxyleukotriene B4 to 20-carboxyleukotriene B4 by rat liver cytosol

20-Hydroxyleukotriene B4 was converted by rat liver homogenates in the presence of NAD+ to a more polar product on reverse-phase high-performance liquid chromatography. The product was identified as 20-carboxyleukotriene B4 by straight-phase high performance liquid chromatography, ultraviolet spectrophotometry and gas chromatography-mass spectrometry. The oxidative activity of the homogenates was located in the cytosol with an optimal pH of 8.0. The activity was dependent on NAD+, and NADP+ could not substitute for NAD+. 1 mol of 20-carboxyleukotriene B4 was formed with the reduction of 2 mol of NAD+. The reaction was inhibited by pyrazole and 4-methylpyrazole, inhibitors of alcohol dehydrogenase, and by various alcohols, such as ethanol, 12-hydroxylaurate, and 20-hydroxyprostaglandin E1. Disulfiram, an inhibitor of aldehyde dehydrogenase, also inhibited the activity. These results suggest that two discrete steps catalyzed by different enzymes, alcohol dehydrogenase and aldehyde dehydrogenase, are involved in the oxidation of 20-hydroxyleukotriene B4 in rat liver cytosol. The enzyme system seems to be different from that of human neutrophils.     

The AdhS alleloenzyme of alcohol dehydrogenase from Drosophila melanogaster. Variation of kinetic parameters with pH.

The variation with pH of the kinetic parameters for the alcohol and acetaldehyde reactions were studied for the alleloenzyme AdhS from Drosophila melanogaster. The variation of Ki (KEO,I) with pH for two ethanol-competitive inhibitors, pyrazole and 2,2,2-trifluoroethanol, was also studied. Both alcohol oxidation and acetaldehyde reduction follow a compulsory ordered pathway, with coenzyme binding first. The rate-limiting step for ethanol oxidation is complex and involves at least hydride transfer and dissociation of the enzyme-NADH complex (ER). In contrast with this, the rate-limiting step for the back reaction, i.e. the reduction of acetaldehyde, is dissociation of the enzyme-NAD+ complex (EO). A rate-limiting ER dissociation appears in the oxidation of the secondary alcohol propan-2-ol, whereas for the back reaction, i.e. acetone reduction, hydride transfer in the ternary complexes is rate-limiting. There is one group in the free enzyme, with a pK of approx. 8.0, that regulates the kon velocity for NADH, whereas for NAD+ several groups seem to be involved. A group in the enzyme is drastically perturbed by the formation of the binary EO complex. Protonation of this group with a pK of 7.6 in the EO complex resulted in weakened alcohol and inhibitor binding, in addition to an increased dissociation rate of NAD+ from the binary EO complex. Neither the binding of acetaldehyde nor the dissociation rate of NADH from the binary ER complex varied within the pH region studied.     

The binding of Ca2+ ions to pig heart NAD+-isocitrate dehydrogenase and the 2-oxoglutarate dehydrogenase complex.

1. The binding of Ca2+ ions to purified pig heart NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase, freed of contaminating Ca2+ by parvalbumin/polyacrylamide chromatography, has been studied by flow dialysis and by the use of fura-2. 2. For the 2-oxoglutarate dehydrogenase complex, 3.5 mol of Ca2+-binding sites/mol of complex were apparent, with an apparent dissociation constant (Kd value) for Ca2+ of 2.0 microM. These values were little affected by Mg2+ ions, ADP or 2-oxoglutarate. 3. By contrast, binding of Ca2+ to NAD+-isocitrate dehydrogenase (Kd = 14 microM) required ADP, isocitrate and Mg2+ ions. The number of Ca2+-binding sites associated with NAD+-isocitrate dehydrogenase was then 0.9 mol/mol of tetrameric enzyme. 4. The 2-oxoglutarate dehydrogenase complex bound ADP (as ADP3-) to a group of tight-binding sites (Kd = 3.1 microM) with a stoichiometry, 3.3 mol/mol of complex, similar to that for the binding of Ca2+; a variable number of much weaker sites (Kd = 100 microM) for ADP3- was also apparent.