The effects of Mg2+ on certain steps in the mechanisms of the dehydrogenase and esterase reactions catalysed by sheep liver aldehyde dehydrogenase. Support for the view that dehydrogenase and esterase activities occur at the same site on the enzyme.

Stopped-flow experiments in spectrophotometric and fluorescence modes reveal different aspects of the aldehyde dehydrogenase mechanism. Spectrophotometric experiments show a rapid burst of NADH production whose course is not affected by Mg2+. The slower burst seen in the fluorescence mode is markedly accelerated by Mg2+. It is argued that the fluorescence burst accompanies acyl-enzyme hydrolysis and, therefore, that Mg2+ increases the rate of this process. Experiments on the hydrolysis of p-nitrophenyl propionate indicate that acyl-enzyme hydrolysis is indeed accelerated by Mg2+ and a combination of Mg2+ and NADH. Vmax. values for p-nitrophenyl propionate hydrolysis in the presence of NADH and NADH and Mg2+ agree closely with the specific rates of acyl hydrolysis from the E . NADH . acyl and E . NADH . acyl . Mg2+ complexes seen in the dehydrogenase reaction with propionaldehyde. These observations support the view that esterase and dehydrogenase activities occur at the same site on the enzyme. Other evidence is presented to support this conclusion.     

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.     

Effects of Mg2+, Ca2+ and Mn2+ on sheep liver cytoplasmic aldehyde dehydrogenase.

Sheep liver cytoplasmic aldehyde dehydrogenase is strongly inhibited by Mg2+, Ca2+ and Mn2+. The inhibition is only partial, however, with 8-15% of activity remaining at high concentrations of these agents. In 50 mM-Tris/Hcl, pH 7.5, the concentrations giving half-maximal effect were: Mg2+, 6.5 micrometers; Ca2+, 15.2 micrometers; Mn2+, 1.5 micrometer. The esterase activity of the enzyme is not affected by such low metal ion concentrations, but appears to be activated by high concentrations. Fluorescence-titration and stopped-flow experiments provide evidence for interaction of Mg2+ with NADH complexes of the enzyme. As no evidence for the presence of increased concentrations of functioning active centres was obtained in the presence of Mg2+, it is concluded that effects of Mg2+ (and presumably Ca2+ and Mn2+ also) are brought about by trapping increased concentrations of NADH in a Mg2+-containing complex. This complex must liberate products more slowly than any of the complexes involved in the non-inhibited mechanism.     

Aldehyde dehydrogenase from rat intestinal mucosa: purification and characterization of an isozyme with high affinity for gamma-aminobutyraldehyde.

In rat adrenal gland and gastric mucosa putrescine is efficiently oxidized to GABA via gamma-aminobutyraldehyde (ABAL) by action of diamine oxidase and aldehyde dehydrogenase. Having turned our attention on the rat intestinal mucosa, where putrescine uptake and diamine oxidase are active, we have purified and characterized an aldehyde dehydrogenase optimally active on gamma-aminobutyraldehyde. A dimer with a subunit molecular weight of 52,000, the native enzyme binds ABAL and NAD+ with high affinity: at pH 7.4, Km values are equal to 18 and 14 microM, respectively. Affinity for betaine aldehyde is much lower (Km = 285 microM), but the efficiency is equally good, thanks to a high value of V. Unaffected by disulfiram and Mg2+, the enzyme is activated by high NAD+ concentrations (Vnn = 1.6 x Vn) and is competitively inhibited by NADH. According to the best fitting model, the dimeric enzyme only binds one NADH and the mixed complex enzyme-NAD(+)-NADH is inactive. The increase of activity promoted by NAD+ can therefore be ascribed to an allosteric effect, rather than to the activation of a second reaction center. Highly stable at pH 6.8 in the presence of dithiothreitol and high phosphate concentrations, ABALDH is inactivated by ion-exchange resins and by cationic buffers. Our results show that the enzyme can be effectively involved in the metabolism of biogenic amines and, with a K(m) for ABAL lower than 20 microM, in the synthesis of GABA.     

Affinity labeling of an allosteric ADP site of glutamate dehydrogenase by 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate

Bovine liver glutamate dehydrogenase reacts covalently with the adenine nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)adenosine 5'-monophosphate (2-BDB-TAMP) with incorporation of about 1 mol of reagent/mol of enzyme subunit. The modified enzyme is not inactivated by this reaction as measured in the absence of allosteric effectors. Native glutamate dehydrogenase is activated by ADP and inhibited by high concentrations of NADH; both of these effects are irreversibly decreased upon reaction of the enzyme with 2-BDB-TAMP. The decrease in activation by ADP was used to determine the rate constant for reaction with 2-BDB-TAMP. The rate constant (kobs) for loss of ADP activation exhibits a nonlinear dependence on 2-BDB-TAMP concentration, suggesting a reversible binding of reagent (KR = 0.74 mM) prior to irreversible modification. At 1.2 mM 2-BDB-TAMP, kobs = 0.060 min-1 and is not affected by alpha-ketoglutarate or GTP, but is decreased to 0.020 min-1 by 5 mM NADH and to zero by 5 mM ADP. Incorporation after incubation with 1.2 mM 2-BDB-TAMP for 1 h at pH 7.1 is 1.02 mol/mol enzyme subunit in the absence but only 0.09 mol/subunit in the presence of ADP. The enzyme protected with 5 mM ADP behaves like native enzyme in its activation by ADP and in its inhibition by NADH. Native enzyme binds reversibly 2 mol of [14C]ADP/subunit, whereas modified enzyme binds only 1 mol of ADP/peptide chain. These results indicate that incorporation of 1 mol of 2-BDB-TAMP causes elimination of one of the ADP sites of the native enzyme. 2-BDB-TAMP acts as an affinity label of an ADP site of glutamate dehydrogenase and indirectly influences the NADH inhibitory site.     

Kinetic behaviour and properties of aldehyde dehydrogenase from rat testis mitochondria. Effect of Mg2+ ions.

1. Mitochondrial aldehyde dehydrogenase is purified to near homogeneity by hydroxylapatite-, affinity- and hydrophobic interaction-chromatography. 2. The enzyme is an oligomeric protein and its molecular weight, as determined by gel-filtration, is 117,000 +/- 5000. 3. Active only in the presence of exogenous sulfhydryl compounds and NAD(+)-dependent, aldehyde dehydrogenase works optimally with linear-chain aliphatic aldehydes and is practically inactive with benzaldehyde. The pH-optimum is at about pH 8.5. 4. Km-Values for aliphatic aldehydes (C2-C6) range between 0.17 and 0.32 microM. The Km for NAD+ increases from 16 microM with acetaldehyde to 71 microM with capronaldehyde. 5. Millimolar concentrations of Mg2+ promote high increases of both V and Km for NAD+. At the same time, saturation curves with C4-C6 aldehydes can be simulated with a substrate inhibition model. 6. Inhibition by NADH is competitive: with capronaldehyde, the inhibition constant for NADH is 52 microM in the absence of Mg2+ and 14 microM in the presence of 4 mM Mg2+; with acetaldehyde, the inhibition constant is about three times higher (36 and 159 microM, respectively).     

Equilibrium binding of coenzymes and substrates to nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase from bovine heart mitochondria.

1. The stoicheiometries and affinities of ligand binding to isocitrate dehydrogenase were studied at pH 7.0, mainly by measuring changes in NADPH and protein fluorescence. 2. The affinity of the enzyme for NADPH is about 100-fold greater than it is for NADP+ in various buffer/salt solutions, and the affinities for both coenzymes are decreased by Mg2+, phosphate and increase in ionic strength. 3. The maximum binding capacity of the dimeric enzyme for NADPH, from coenzyme fluorescence and protein-fluorescence measurements, and also for NADP+, by ultrafiltration, is 2 mol/mol of enzyme. Protein-fluorescence titrations of the enzyme with NADP+ are apparently inconsistent with this conclusion, indicating that the increase in protein fluorescence caused by NADP+ binding is not proportional to fractional saturation of the binding sites. 4. Changes in protein fluorescence caused by changes in ionic strength and by the binding of substrates, Mg2+ or NADP+ (but not NADPH) are relatively slow, suggesting conformation changes. 5. In the presence of Mg2+, the enzyme binds isocitrate very strongly, and 2-oxoglutarate rather weakly. 6. Evidence is presented for the formation of an abortive complex of enzyme-Mg2+-isocitrate-NADPH in which isocitrate and NADPH are bound much more weakly than in their complexes with enzyme and Mg2+ alone. 7. The results are discussed in relation to the interpretation of the kinetic properties of the enzyme and its behaviour in the mitochondrion.     

Kinetics of conformational changes in Nereis sarcoplasmic calcium-binding protein upon binding of divalent ions

The sarcoplasmic calcium-binding protein (SCP) of the sandworm Nereis possesses three Ca2(+)-Mg2+ sites but no Ca2(+)-specific site. Binding of Mg2+, but not of Ca2+, displays a marked positive cooperativity. The apparent cooperativity of Ca2+ binding in the presence of Mg2+ results from the allostery in Mg2+ dissociation. Binding of the first Ca2+ or Mg2+ induces all the conformational change, monitored by Trp fluorescence. In displacement reactions the conformational changes occur in the step SCP.Mg3----SCP.Ca1Mg2. Stopped-flow experiments indicate that Trp fluorescence changes upon Ca2(+)-binding are instantaneous whereas Mg2(+)-binding involves a fast pre-equilibrium (Keq = 28 M-1), followed by two slow consecutive conformational changes with k1 = 13.5 s-1 and k2 = 0.21 s-1. The fluorescence change after dissociation of Ca2+ from SCP is monophasic with k = 0.02 s-1; that after Mg2+ dissociation is biphasic with k1 = 0.8 s-1 and k2 = 0.1 s-1. Trp life time measurements also indicate that Ca2(+)- and Mg2(+)-induced conformational changes are completely different. Displacement of bound Ca2+ by Mg2+ can be described by two consecutive reactions in which the first (without fluorescence change) corresponds to the dissociation of the last Ca2+ (k1 = 2.4 s-1) and the second (k2 = 0.45 s-1) to the final conformational change observed upon direct Mg2+ binding. Displacement of bound Mg2+ by Ca2+ follows the kinetic scheme of simple competition; the conformational rate constant approaches asymptotically (up to the limit of 129 s-1) the dissociation rate of Mg2+ as the concentration of Ca2+ increases. In summary, after fast dissociation of Ca2+ or Mg2+, Nereis SCP slowly converts to the metal-free configuration, but in Ca2(+)-Mg2+ exchange reactions, the conformational changes are nearly as fast as the cation dissociation reactions.     

Partial reversal of the acetaldehyde and butyraldehyde oxidation reactions catalysed by aldehyde dehydrogenases from sheep liver.

In the presence of acetic anhydride or butyric anhydride, liver aldehyde dehydrogenases catalyse the oxidation of NADH at pH 7.0 and 25 degrees C. The maximum velocities and Michaelis constants for NADH at saturating anhydride concentrations are independent of which anhydride is used, the values being V'max. = 12 min-1 and Km for NADH = 9 micrometer for the mitochondrial enzyme and V'max = 25 min-1 and Km for NADH = 20 micrometer for the cytoplasmic enzyme. Substitution of [4A-2H]NADH for NADH resulted in 2-fold and 4-fold decreases in rate for the mitochondrial and cytoplasmic enzymes respectively.     

The action of progesterone and diethylstilboestrol on the dehydrogenase and esterase activities of a purified aldehyde dehydrogenase from rabbit liver.

A steroid-sensitive aldehyde dehydrogenase (EC 1.2.1.3) was purified from rabbit liver and is homogeneous by the criterion of electrophoresis in polyacrylamide gels with or without sodium dodecyl sulphate. The enzyme is tetrameric, of subunit mo.wt. 48 300, and contains no tightly bound zinc. The fluorescence of the protein is decreased in the presence of progesterone, which is inhibitory to the reactions catalysed by the enzyme. When NADH is bound to the enzyme, the fluorescence of the coenzyme is augmented to an extent independent of the presence of steroids or acetaldehyde. The purified enzyme catalyses the oxidation of acetaldehyde and glucuronolactone, and the hydrolysis of 4-nitrophenyl acetate. Each of these reactions is inhibited by progesterone in such a manner as to suggest the formation of a catalytically active enzyme-hormone complex. Diethylstilboestrol inhibits the hydrolysis of esters by this enzyme, but stimulates the oxidation of aldehydes, except at low aldehyde concentrations; the ligand is then inhibitory. NADH inhibits the hydrolysis of 4-nitrophenyl acetate by the enzyme in a partially competitive fashion.     

Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.     

Mg2+ -dependent inactivation / H+ -dependent activation equilibrium of chloroplast F1-ATPase

The catalytic part of chloroplast thylakoid ATPase, the chloroplast coupling factor CF1, is reversibly inactivated during incubation in the presence of Mg2+. The inactivation has two phases. Its fast phase occurs at basic pH of the incubation medium (k = 6 min-1), while the slow phase ( k = 0.1-0.2 min-1) depends on pH only slightly throughout the studied range (5.5-9.0). As followed from changes in the inactivation effect of magnesium ions, Mg2+ affinity for the enzyme decreases dramatically with decreasing medium pH. The pH-dependence of Mg2+ dissociation apparent constant suggests that the binding/dissociation equilibrium is determined by protonation/deprotonation of specific acid-base groups of the enzyme. The analysis of pH-dependence plots gives the equilibrium constant of magnesium dissociation (3-9 M) and the dissociation constant of the protonated groups pK 5.8-6.7). Sodium azide is known to stabilize the inactive CF1-MgADP complex; when added to the incubation medium it diminishes the Mg2+ dissociation constant and has no effect on the dissociation constant of the acid-base groups. At lower pH, Mg2+-inactivated CF1-ATPase reactivates. Octyl glucoside accelerates the reactivation, while Triton-100 affects it only slightly. The reactivation rate of membrane-bound CF1 (thylakoid ATPase) inactivated by preincubation with Mg2+ in the presence of gramicidin is a few times higher than that of isolated CF1. These results suggest that the reactivation of isolated and membrane-bound CF1-ATPase is determined by protonation of a limited number of acid-base groups buried in the enzyme molecule.     

Purification and properties of sheep-liver aldehyde dehydrogenases.

Sheep liver cytoplasmic aldehyde dehydrogenase was purified to homogeneity to give a sample with a specific activity of 380 nmol NADH min(-1) mg(-1). An amino acid analysis of the enzyme gave results similar to those reported for aldehyde dehydrogenases from other sources. The isoelectric point was at pH 5.25 and the enzyme contained no significant amounts of metal ions. On the binding of NADH to the enzyme there is a shift in absorption maximum of NADH to 344 nm, and a 5.6-fold enhancement of nucleotide fluorescence. The protein fluorescence (lambdaexcit = 290 nm, lambdaemisson = 340 nm) is quenched on the binding of NAD+ and NADH. The enhancement of nucleotide fluorescence on the binding of NADH has been utilised to determine the dissociation constant for the enzyme . NADH complex (Kd = 1.2 +/- 0.2 muM). A Hill plot of the data gave a straight line with a slope of 1.0 +/- 0.3 indicating the absence of co-operative effects. Ellman's reagent reacted only slowly with the enzyme but in the presence of sodium dodecylsulphate complete reaction occurred within a few minutes to an extent corresponding to 36 thiol groups/enzyme. Molecular weights were determined for both cytoplasmic and mitochondrial aldehyde dehydrogenases and were 212 000 +/- 8 000 and 205 000 respectively. Each enzyme consisted of four subunits with molecular weight of 53 000 +/- 2 000. Properties of the cytoplasmic and mitochondrial aldehyde dehydrogenases from sheep liver were compared with other mammalian liver aldehyde dehydrogenases.     

NADH fluorescence lifetime analysis of the effect of magnesium ions on ALDH2

Aldehyde dehydrogenase 2 (ALDH2) catalyzes oxidation of toxic aldehydes to carboxylic acids. Physiologic levels of Mg(2+) ions influence ALDH2 activity in part by increasing NADH binding affinity. Traditional fluorescence measurements monitor the blue shift of the NADH fluorescence spectrum to study ALDH2-NADH interactions. By using time-resolved fluorescence spectroscopy, we have resolved the fluorescent lifetimes (τ) of free NADH (τ=0.4 ns) and bound NADH (τ=6.0 ns). We used this technique to investigate the effects of Mg(2+) on the ALDH2-NADH binding characteristics and enzyme catalysis. From the resolved free and bound NADH fluorescence signatures, the K(D) for NADH with ALDH2 ranged from 468 μM to 12 μM for Mg(2+) ion concentrations of 20 to 6000 μM, respectively. The rate constant for dissociation of the enzyme-NADH complex ranged from 0.4s(-1) (6000 μM Mg(2+)) to 8.3s(-1) (0 μM Mg(2+)) as determined by addition of excess NAD(+) to prevent re-association of NADH and resolving the real-time NADH fluorescence signal. The apparent NADH association/re-association rate constants were approximately 0.04 μM(-1)s(-1) over the entire Mg(2+) ion concentration range and demonstrate that Mg(2+) ions slow the release of NADH from the enzyme rather than promoting its re-association. We applied NADH fluorescence lifetime analysis to the study of NADH binding during enzyme catalysis. Our fluorescence lifetime analysis confirmed complex behavior of the enzyme activity as a function of Mg(2+) concentration. Importantly, we observed no pre-steady state burst of NADH formation. Furthermore, we observed distinct fluorescence signatures from multiple ALDH2-NADH complexes corresponding to free NADH, enzyme-bound NADH, and, potentially, an abortive NADH-enzyme-propanal complex (τ=11.2 ns).     

Phosphorylation from adenosine triphosphate of sodium- and potassium-activated adenosine triphosphatase. Comparison of enzyme-ligand complexes as precursors to the phosphoenzyme.

The relative effectiveness of the ligands Mg2+, Na+, and ATP in preparing sodium plus potassium ion transport adenosine triphosphatase for phosphorylation was studied by means of a rapid mixing apparatus. Addition of 2 mM MgC12, 120 mM NaC1, and 5 muM [gamma-32P]ATP simultaneously to the free enzyme gave an initial phosphorylation rate of about 0.3 mu mol-mg-1-min-1 at 25 degrees and pH7.4. Addition of Mg2+ to the enzyme beforehand, separately or in combination with Na+ or ATP, had little effect on the initial rate. Addition of Na+ only to the enzyme beforehand increased this rate 1.5- to 3-fold. Early addition of ATP 130 ms before Na+ plus Mg2+ increased the rate 6- to 7-fold. Early addition of Na+ plus ATP was most effective; it increased the rate about 10-fold. The data indicate that Na+ and ATP bind in a random order and that each ligand potentiates the effect of the other. The rate of dissociation of ATP from the enzyme was estimated by a chase of unlabeled ATP of variable duration. This rate was slowest in the presence of Mg2+ (k = 540 min-1), most rapid in the presence of Na+ (k = 2000 min-1), and intermediate (k = 1100 min-1) in the absence of metal ions. The effect of Na+ concentration on the rate of phosphorylation was estimated when Na+ with Mg2+ was added to the enzyme-ATP complex. The rate followed Michaelis-Menten kinetics with a maximum of 2.9 mu mol-mg-1 and a Km of 8 mM. The effect of Na+ concentration was also estimated on the increment in the rate of phosphorylation produced by the presence of Na+ with the enzyme-ATP complex beforehand. The increment followed the same kinetics with a maximum of 3.75 mu mol-mg-1-min-1 and a Km of 5.4 mM. In both cases estimation of the Hill coefficient failed to show cooperativity between binding sites for Na+. In contrast, the dependence of ouabain-sensitive ATPase activity on Na+ concentration in the absence of K+ indicated two sites for Na+ with apparent Km values of 0.16 and 8.1 mM, respectively.     

Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites

The zinc-deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13-ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1-anilinonaphthalene 8-sulphonic acid is bound with comparable affinity. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc-deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 2,2'-Bipyridine, which chelates the active-site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc-depleted enzyme. The stopped-flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc-depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His-67, bound to the zinc ion, or the zinc-liganding water molecule in the case of the native enzyme (pK close to 9), or the free His-67 residue in the case of the zinc-deficient enzyme (pK about 8).     

Oxidation of fatty alcohols to acids in the caecum of a gourami (Trichogaster cosby).

Oxidation of fatty alcohols to acids in gourami caeca was investigated by measuring the reduction of NAD+ and the formation of labeled hexadecanoic acid from [1(-14)C]hexadecanol. Virtually all dehydrogenase activity is in the microsomal fraction. Maximal activity is obtained with NAD+ as cofactor whereas with NADP+ 60% of that activity is obtained. The enzyme is rather specific for long chain alcohols and 2 NADH are formed for each molecule of hexadecanol oxidized to acid. It is stabilized by mercaptoethanol, and completely inhibited by p-chloromercuribenzoate. The activity is optimal at pH 9.5. At higher pH, small amounts of aldehyde are found. The first reaction in the sequence, fatty alcohol leads to aldehyde leads to acid seems to occur under the more physiological condition at a much slower rate than the second reaction so that free aldehyde is not detected. Addition of palmitic acid indicated an uncompetitive product inhibition. The oxidation of alcohol to acid is reversible only to a very minor extent even in the presence of NADPH, CoA, ATP and Mg2+. Location, activity and properties of the enzyme are in agreement with the earlier observation from dietary experiments that in the gourami fatty alcohols of wax esters are oxidized to acids in the course of absorption.     

Anthroylouabain: a specific fluorescent probe for the cardiac glycoside receptor of the Na-K ATPase.

Anthroylouabain (AO) was synthesized by reaction of anthracene-9-carboxylic chloride with ouabain. Nuclear magnetic resonance spectroscopy of AO suggests that the anthracene is esterfied to the rhamnose in the glycoside. AO inhibits Na-K ATPase from human red cells, eel electroplax and rabbit and dog kidney with a KI less than 1muM. AO bound to rabbit or dog kidney Na-K ATPase shows enhanced fluorescence and characteristic spectral shifts. AO binding requires Mg and is optimum in the presence of Mg + Pi or MgATP + Na; ouabain prevents AO binding and fluorescence enhancement if added before AO or reverses it if added after AO is bound. Na inhibits AO binding in the presence of Mg + Pi and K inhibits it in the presence of MgATP + Na. AO binding and dissociation rate constants measured by fluorescence agree qualitatively with reported measurements for ouabain, using other methods, although AO shows faster kinetics than ouabain. Dissociation constants obtained from kinetic measurements are 1.5 X 10(-7) and 1.8 X 10(-7) M for the MgATP + Na complex and Mg + Pi complex, respectively. KD from fluorescence titrations is 2.3 X 10(-7) M for the latter. The enzyme has 2-2.5 nmol of AO binding sites/mg of protein. No differences in the fluorescence parameters of the Mg + Pi or MgATP + Na complexes were observed, suggesting that the same enzyme conformation binds AO under both ligand conditions. Comparison of the AO fluorescence parameters in the enzyme with those of model systems suggests that the binding site is hydrophobic and/or viscous and shielded from H2O. The results indicate that AO is a specific fluorescent probe of the cardiac glycoside receptor of the Na-K ATPase. Possible applications are discussed.     

Structures of the dI2dIII1 complex of proton-translocating transhydrogenase with bound, inactive analogues of NADH and NADPH reveal active site geometries

Transhydrogenase couples the redox reaction between NADH and NADP+ to proton translocation across a membrane. The enzyme comprises three components; dI binds NAD(H), dIII binds NADP(H), and dII spans the membrane. The 1,4,5,6-tetrahydro analogue of NADH (designated H2NADH) bound to isolated dI from Rhodospirillum rubrum transhydrogenase with similar affinity to the physiological nucleotide. Binding of either NADH or H2NADH led to closure of the dI mobile loop. The 1,4,5,6-tetrahydro analogue of NADPH (H2NADPH) bound very tightly to isolated R. rubrum dIII, but the rate constant for dissociation was greater than that for NADPH. The replacement of NADP+ on dIII either with H2NADPH or with NADPH caused a similar set of chemical shift alterations, signifying an equivalent conformational change. Despite similar binding properties to the natural nucleotides, neither H2NADH nor H2NADPH could serve as a hydride donor in transhydrogenation reactions. Mixtures of dI and dIII form dI2dIII1 complexes. The nucleotide charge distribution of complexes loaded either with H2NADH and NADP+ or with NAD+ and H2NADPH should more closely mimic the ground states for forward and reverse hydride transfer, respectively, than previously studied dead-end species. Crystal structures of such complexes at 2.6 and 2.3 A resolution are described. A transition state for hydride transfer between dihydronicotinamide and nicotinamide derivatives determined in ab initio quantum mechanical calculations resembles the organization of nucleotides in the transhydrogenase active site in the crystal structure. Molecular dynamics simulations of the enzyme indicate that the (dihydro)nicotinamide rings remain close to a ground state for hydride transfer throughout a 1.4 ns trajectory.     

Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of D-galactosamine.

The displacement of NADH from cytoplasmic aldehyde dehydrogenase (EC 1.2.1.3) from sheep liver was studied by using NAD+, 1,10-phenanthroline, ADP-ribose, deamino-NAD+ and pyridine-3-aldehyde-adenine dinucleotide as displacing agents, by following the decrease in fluorescence as a function of time. The data obtained could be fitted by assuming two first-order processes were occurring, a faster process with an apparent rate constant of 0.85 +/- 0.20 s-1 and a relative amplitude of 60 +/- 10% and a slower process with an apparent rate constant of 0.20 +/- 0.05 s-1 and a relative amplitude of 40 +/- 10% (except for pyridine-3-aldehyde-adenine dinucleotide, where the apparent rate constant for the slow process was 0.05 s-1). The displacement rates did not change significantly when the pH was varied from 6.0 to 9.0. Kinetic data are also reported for the dependence of the rate of binding of NADH to the enzyme on the total concentration of NADH. Detailed arguments are presented based on the isolation and purification procedures, the equilibrium coenzyme-binding studies and the kinetic data, which lead to the following model for the release of NADH from the enzyme: (formula: see article). The parameters that best fit the data are: k + 1 = 0.2 s-1; k - 1 = 0.05 s-1; k + 2 = 0.8 s-1 and k - 2 = 5 X 10(5)litre-mol-1-s-1. The slow phase of the NADH release is similar to the steady-state turnover number for substrates such as acetaldehyde and propionaldehyde and appears to contribute significantly to the limitation of the steady-state rate.