Chemical cross-linking of cyclic AMP-dependent protein kinase and its dissimilar subunits

Previous kinetic studies have demonstrated that the activation of cyclic AMP-dependent protein kinase by cyclic AMP involves the formation of a ternary complex of cyclic AMP, the regulatory subunit (R) and the catalytic subunit (C). It is suggested that only this ternary complex breaks down to liberate the enzymically active catalytic subunit. We have performed cross-linking experiments with the holoenzyme and its dissimilar subunits in the presence of MgATP and various concentrations of cyclic AMP. Results from these cross-linking studies indicate that regulatory subunits exist as dimers in the native form. Moreover, dissociation of the holoenzyme or the reconstituted enzyme is promoted by cyclic AMP, and the effect of MgATP is to stabilize the enzyme in the tetrameric form. The success in cross-linking the regulatory and the catalytic subunits of protein kinase with the lysine-specific bifunctional cross-linking reagent dimethyl suberimidate may be attributed to the presence of a large number of lysine residues in the enzyme.     

Dissociation of the ribulosebisphosphate-carboxylase large-subunit binding protein into dissimilar subunits

The ribulosebisphosphate-carboxylase large-subunit binding protein from Pisum sativum chloroplasts is an oligomer of two types of subunit with the composition alpha 6 beta 6. These two subunits are immunologically distinct, show different partial protease digestion patterns and have different amino-terminal sequences. Leaves of Hordeum vulgare also contain an oligomeric binding protein composed of equal amounts of two types of subunit. Treatment of either P. sativum stromal extracts or purified binding protein with ATP and Mg2+ ions causes the dissociation of the oligomeric form of the binding protein to the monomeric subunits. This effect is highly specific for ATP since CTP, UTP, GTP, ADP, AMP, cyclic AMP, NADPH and pyrophosphate do not cause dissociation.     

Purification and regulatory properties of phosphofructokinase from Trypanosoma (Trypanozoon) brucei brucei.

Phosphofructokinase (EC 2.7.1.11) from Trypanosoma (Trypanozoon) brucei brucei was purified to homogeneity by using a three-step procedure that may be performed within 1 day. Proteolysis, which removes a fragment of Mr approx. 2000, may occur during the purification, but this can be prevented by including antipain, an inhibitor of cysteine proteinases, in the buffers during the purification. The subunits of the enzyme appear to be identical in size, with an Mr of 49 000. The Mr of the native enzyme was estimated to be approx. 220 000, suggesting a tetrameric structure. Kinetic studies showed the activity to depend hyperbolically on the concentration of ATP but sigmoidally on the concentration of fructose 6-phosphate. Although cyclic AMP, AMP and ADP stimulated the enzyme activity at low concentrations of fructose 6-phosphate, the last two nucleotides were inhibitory at high concentrations of this substrate. Phosphoenolpyruvate behaved as an allosteric inhibitor of the phosphofructokinase. Citrate, fructose 1,6-bisphosphate, fructose 2,6-bisphosphate and Pi did not influence significantly the activity of the enzyme.     

The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.

Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.     

Subunit exchange between membranous and soluble forms of bovine dopamine beta-hydroxylase

Dopamine beta-hydroxylase is present in the bovine adrenal medulla in two forms: soluble and membrane-bound. In a previous study, it was shown that the tetrameric, soluble form of the enzyme undergoes dissociation into two identical dimeric subunits and that this subunit dissociation is dependent on pH and ADP binding (Dhawan, S., Hensley, P., Osborne, J. C., Jr., and Fleming, P. J. (1986) J. Biol. Chem. 261, 7680-7684). Here we report the effect of pH and ADP on the dissociation of the membranous form of dopamine beta-hydroxylase into two nonidentical subunits. Negative stain electron microscopy of purified membranous hydroxylase showed largely tetrameric species together with occasional dimeric species. The tetrameric images of membranous hydroxylase were similar to, but clearly different from, previously published negative stain images of soluble hydroxylase (Duong, L. T., Fleming, P. J., and Ornberg, R. L. (1985) J. Biol. Chem. 260, 2393-2398). Quantitative binding of ADP to the membranous hydroxylase revealed the existence of two binding sites per dimeric subunit. ADP binding and low pH both promote dissociation of a hydrophilic, catalytically active subunit from the membranous enzyme reconstituted onto phospholipid vesicles. Kinetic analyses of reconstituted membranous hydroxylase activity were consistent with the existence of tetrameric and dimeric catalytic species in equilibrium. All of the hydrophilic subunits of the purified soluble hydroxylase bind to the hydrophobic subunits of the reconstituted membranous hydroxylase. We propose that, in the chromaffin granules, the soluble hydroxylase subunits are in equilibrium association with the membrane-bound hydroxylase subunits and that the hydrophilic subunits of both soluble and membranous hydroxylase are identical.     

Activation of adenylate cyclase in human platelet membranes by guanosine 5''-[beta gamma-imido]triphosphate is inhibited by cyclic-AMP-dependent phosphorylation. Slow activation occurs in the absence of ATP.

Incubation of platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate causes a slow increase in GS (stimulatory GTP-binding protein) activation of adenylate cyclase. Mg2+ is necessary for this slow activation. This process is inhibited in the presence of ATP, and inhibition is greater if cyclic AMP is also included in the incubation. Adenosine 5'-[beta gamma-imido]triphosphate instead of ATP in the incubation facilitates the slow activation in the presence of cyclic AMP, and incubation of membranes with cyclic-AMP-dependent protein kinase inhibitor decreased inhibition of the slow activation of adenylate cyclase by ATP and cyclic AMP. A protein of 45 kDa in platelet membranes is phosphorylated in a cyclic-AMP-dependent manner. The transition from a reversibly activated form of GS to an irreversibly activated form is substantially slower in the presence of ATP and cyclic AMP. We propose that guanosine 5'-[beta gamma-imido]triphosphate-activated GS may exist in phosphorylated or non-phosphorylated forms, and that the non-phosphorylated form is the more active of the two species. The non-phosphorylated form of GS may correspond to the irreversibly activated state.     

Characterization of two adenosine 3':5'-phosphate-dependent protein kinase species from Chinese hamster ovary cells.

Chinese hamster ovary cells exhibit several characteristic morphological and physiological responses upon treatment with agents which increase the intracellular level of adenosine 3':5'-phosphate (cyclic AMP). To better understand the mechanism of these cyclic AMP-mediated responses, we separated two cyclic AMP-dependent protein kinases (ATP:protein phosphotransferase, EC 2.7.1.37) (protein kinase I and protein kinase II) from the cytosol of Chinese hamster ovary cells by DEAE-cellulose chromatography and studied their properties. Protein kinase I is eluted at a lower salt concentration than protein kinase II and is stimulable to 10 times its basal catalytic activity, while protein kinase II is stimulable only 2-fold. Both kinases are completely dissociated by cyclic AMP and inhibited by specific cyclic AMP-dependent protein kinase inhibitor. They have similar Km values for magnesium (approximately 1 mM), cyclic AMP (approximately 60 nM), and ATP (approximately 0.1 mM), and the dissociation constant (Kdis) for cyclic AMP (approximately 13 nM) is the same for both enzymes. However, they appear to have different substrate preferences and cyclic AMP-binding properties in that cyclic AMP bound to protein kinase II exchanges readily with free cyclic AMP, while that bound to protein kinase I is not exchangeable. The native enzymes have different sedimentation coefficients (6.4 S for protein kinase I and 4.8 S for protein kinase II), whereas those of the activated enzymes are the same (2.9--3.0 S). It appears that the two cyclic AMP-dependent protein kinases which differ from each other in their regulatory subunits may play different roles in the mediation of cyclic AMP action in Chinese hamster ovary cells.     

Biosynthesis of bacterial glycogen. The nature of the binding of substrates and effectors to ADP-glucose synthase.

Kinetic studies with ADP-glucose synthase show that 1,6-hexanediol bisphosphate (1,6-hexanediol-P2) is an effective activator that causes the enzyme to have a higher apparent affinity for ATP- and ADP-glucose than when fructose-1,6-P2 is the activator. Furthermore, in the presence of 1,6-hexanediol-P2, substrate saturation curves are hyperbolic shaped rather than sigmoidal shaped. CrATP behaves like a nonreactive analogue of ATP. Kinetic studies show that it is competitive with ATP. CrATP is not a competitive inhibitor of ADP-glucose. However, the combined addition of CrATP and glucose-1-P inhibits the enzyme competitively when ADP-glucose is the substrate. In binding experiments, CrATP, ATP, and fructose-P2 appear to bind to only half of the expected sites in the tetrameric enzyme, while ADP-glucose, the activators, pyridoxal-P and 1,6-hexanediol-P2, and the inhibitor, AMP, bind to four sites/tetrameric enzyme. Fructose-P2 inhibits 1,6-hexanediol-P2 binding, suggesting competition for the same sites. Glucose-1-P does not bind to the enzyme unless MgCl2 and CrATP are present and binds to four sites/tetrameric enzyme. Alternatively, CrATP in the presence of glucose-1-P binds to four sites/tetrameric enzyme. Thus, there are binding sites for the substrates, activators, and inhibitor located on each subunit and the binding sites can interact homotropically and heterotropically. ATP and fructose-P2 binding is synergistic showing heterotropic cooperativity. ATP and fructose-P2 must also be present together to effectively inhibit AMP binding. A mechanism is proposed which explains some of the kinetic and binding properties in terms of an asymmetry in the distribution of the conformational states of the four identical subunits.     

Stopped flow kinetic studies of adenosine triphosphate phosphoribosyl transferase, the first enzyme in the histidine biosynthesis of Escherichia coli

1. Stopped flow kinetic studies of ATP phosphoribosyl transferase (EC 2.4.2.17) from Escherichia coli showed that high protein concentration and elevated temperature do not give a drastic reduction of the transferase activity when measured before inhibitory amounts of PRibATP (product) has accumulated. 2. A small and slow increase in activity follows a reduction of the protein concentration showing a slow dissociation of the enzyme from an inactive to an active species. 3. By lowering the concentration of the inhibitor histidine, a fairly slow increase in activity is observed indicating a dissociation of the enzyme. 4. AMP and histidine together give a strong inhibition of the activity, while AMP alone stimulates the enzyme activity.     

The pH-dependent subunit dissociation and catalytic activity of bovine dopamine beta-hydroxylase

The soluble form of dopamine beta-hydroxylase from bovine adrenal medulla has previously been shown to exist as a tetrameric species of Mr = 290,000 composed of two disulfide-linked dimers. Here we report that this enzyme can also undergo a reversible tetramerdimer dissociation which is dependent on pH. Gel permeation chromatography of dopamine beta-hydroxylase at pH 5.0 demonstrates a Stokes radius of 5.8 nm. When the pH is shifted to 5.7, the Stokes radius changes to 6.9 nm. Sedimentation equilibrium analysis of the purified enzyme demonstrates that this change in molecular size is due to a change in molecular weight. At low protein concentration, the estimated Mr of the enzyme is 145,000 at pH 5.0 and at high protein concentration approaches 290,000 at pH 5.7. This change in Mr is consistent with the existence of a tetramer-dimer dissociation and a change in the equilibrium constant from 1.8 X 10(-6) M to 1.16 X 10(-9) M when the pH is increased from 5.0 to 5.7. This pH-dependent subunit dissociation is correlated with pH-dependent changes in enzyme activity. Purified bovine-soluble dopamine beta-hydroxylase activity is a hyperbolic function of tyramine concentration at pH 5.0. However, the hydroxylase activity displays non-hyperbolic kinetics at pH 6.0. The kinetic data obtained at pH 6.0 can be accounted for by fitting to a model containing two nonidentical catalytic forms of enzyme generated by the pH-dependent partial dissociation of tetrameric enzyme to dimeric subunits. The two catalytic forms have apparently identical maximal velocities; however, they differ in their Michaelis constants for the substrate; the dimeric form having a low Km and the tetrameric form having a high Km. Since the pH inside bovine adrenal medullary chromaffin granules is approximately 5.5, we conclude that the subunits of dopamine beta-hydroxylase are in dynamic dissociation in a physiologically important pH range.     

Variations in Ca(2+)-mediated activation of Ca(2+)-ATPase and its associated inhibitor in erythrocyte membrane.

1. Two distinct patterns of Ca(2+)-mediated activation of Ca(2+)-ATPase were identified in calmodulin-depleted membranes. 2. In membranes showing no activation (type A), preincubation with micromolar concentration of cyclic AMP and ATP made possible stimulation of the enzyme while in membranes already exhibiting activation (type B), preincubation with cyclic AMP and ATP abolished the activation. 3. ATPase stimulation in type A membranes was suppressible by leupeptin. 4. Triton extractable inhibitor isolated from type A membranes was as active as that derived from type B membranes only after preincubating the membranes with cyclic AMP and ATP. 5. The inhibitor could be inactivated by alkaline phosphatase.     

Purification and control of bovine adrenal cortical cholesterol ester hydrolase and evidence for the activation of the enzyme by a phosphorylation.

A procedure for the purification of cholesterol ester hydrolase from bovine adrenal cortical 105000 x g supernatant is described. Preincubation of a crude enzyme extract with [gamma-32P]ATP followed by purification resulted in the isolation of a phosphorylated preparation of cholesterol ester hydrolase. The phosphorylated cholesterol ester hydrolase appeared to be composed of 4 subunits, each having a molecular weight of 41000 +/- 280, only one of which may be phosphorylated. Preincubation of the crude enzyme preparation with [alpha-32P]ATP followed by purification did not produce a phosphorylated preparation of cholesterol ester hydrolase. Cyclic-AMP-dependent protein kinase, cyclic AMP, ATP and magnesium ions were required for activation of purified cholesterol ester hydrolase in vitro and the time course of activation closely paralleled the time course of phosphorylation of the enzyme. The addition of ATP, cyclic AMP and magnesium ions to the bovine adrenal cortical 105000 x g supernatant produced a 2.5-fold stimulation in cholesterol ester hydrolase activity. This stimulation was abolished if protein kinase inhibitor was added prior to the addition of ATP cyclic AMP and magensium ions. The addition of magnesium ions or calcium ions to a crude preparation of cholesterol ester hydrolase was found to inhibit activity; however the same additions made to a purified preparation of cholesterol ester hydrolase were not inhibitory. The decrease in cholesterol ester hydrolase activity on incubation with magnesium ion was accompanied by a loss of 32P radioactivity from the protein. Preincubation of a crude preparation of cholesterol ester hydrolase with alkaline phosphatase resulted in a deactivation of cholesterol ester hydrolase. It is suggested that bovine adrenal cortex cholesterol ester hydrolase is activated by a phosphorylation catalysed by a cyclic-AMP-dependent protein kinase. Deactivation of cholesterol ester hydrolase is accomplished by dephosphorylation catalysed by a phosphoprotein phosphatase, dependent on magnesium or calcium ions.     

Heart phosphofructokinase. Allosteric kinetics following affinity labeling modification of the enzyme by 8-[m-(m-fluorosulfonylbenzamido) benzylthio] adenine.

An adenine analog 8-[m-(m-fluorosulfonylbenzamido)benzylthio]adenine (FSB-adenine) reacts covalently with sheep heart phosphofructokinase. Under conditions optimal for allosteric kinetics the modified enzyme is less sensitive to inhibition by ATP and insensitive to activation by AMP, cyclic AMP, and ADP. The concentration of fructose-6-P necessary for half-maximal activity is markedly decreased, while the cooperativity to the same substrate is not changed under the same conditions. The modified enzyme is more stable at pH 6.5 when compared with the native enzyme. Changes in the allosteric kinetics of the enzyme are proportional to the extent of modification reaching maximal effect when 3.2 mol of the reagent were bound/mol of tetrameric enzyme. Affinity labeling of the enzyme by the adenine derivative does not affect significantly the catalytic site. This is evidenced by the demonstration that under assay conditions optimal for Michaelian kinetics neither the Km for ATP nor for fructose-6-P is significantly changed following chemical modification. Maximal activity of the modified enzyme was 60% of the native enzyme. ADP gives the best protection, while AMP gives less protection against modification by the reagent. ATP slows the rate of the reaction and causes a slight decrease in maximum binding of the reagent to the enzyme. Modification of the enzyme caused a marked reduction of AMP and ADP binding. The evidence indicates that the modified site is a nucleotide mono- and diphosphate activation site.     

Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.     

Regulation of skeletal-muscle AMP deaminase. Evidence for a highly pH-dependent inhibition by ATP of the homogeneous derivative of the rabbit enzyme yielded by limited proteolysis.

Limited proteolysis of rabbit skeletal-muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) with trypsin results in conversion of the enzyme into a species which over the pH range 6.5-7.1 exhibits hyperbolic kinetics at low K+ concentration even in the absence of ADP, but shows a 20% decrease in activity at saturating substrate concentration. Analysis by sedimentation-equilibrium techniques reveals the proteolysed enzyme to be homogeneous and to have a molecular mass of 222,000 Da, indicative of a trimeric structure with a subunit molecular mass of 72,000 Da, in contrast with the tetrameric structure of the native enzyme, composed of four 79,000-Da subunits. These observations suggest a role of the 7,000-Da fragment which is removed by proteolysis in the maintenance of the three-dimensional structure of the subunit that causes the enzyme at low K+ concentration to show homotropic positive co-operativity. Study of the influence of pH, isolated from that of K+, on the kinetics of AMP deaminase reveals a highly pH-dependent inhibitory effect by ATP which is completely absent at acid pH values and abruptly manifests itself just above neutrality. This phenomenon may have significance in the metabolism of exercising muscle, in connection with the pH-dependent interaction of AMP deaminase with the thick filament.     

Visual arrestin activity may be regulated by self-association.

Visual arrestin is the protein responsible for rapid quenching of G-protein-coupled receptor signaling. Arrestin exists as a latent inhibitor which must be 'activated' upon contact with a phosphorylated receptor. X-ray crystal structures of visual arrestin exhibit a tetrameric arrangement wherein an asymmetric dimer with an extensive interface between conformationally different subunits is related to a second asymmetric dimer by a local two-fold rotation axis. To test the biological relevance of this molecular organization in solution, we carried out a sedimentation equilibrium analysis of arrestin at both crystallographic and physiological protein concentrations. While the tetrameric form can exist at the high concentrations used in crystallography experiments, we find that arrestin participates in a monomer/dimer equilibrium at concentrations more likely to be physiologically relevant. Solution interaction analysis of a proteolytically modified, constitutively active form of arrestin shows diminished dimerization. We propose that self-association of arrestin may provide a mechanism for regulation of arrestin activity by (i) ensuring an adequate supply for rapid quenching of the visual signal and (ii) limiting the availability of active monomeric species, thereby preventing inappropriate signal termination.     

Novel activity of eukaryotic translocase, eEF2: dissociation of the 80S ribosome into subunits with ATP but not with GTP

Ribosomes must dissociate into subunits in order to begin protein biosynthesis. The enzymes that catalyze this fundamental process in eukaryotes remained unknown. Here, we demonstrate that eukaryotic translocase, eEF2, which catalyzes peptide elongation in the presence of GTP, dissociates yeast 80S ribosomes into subunits in the presence of ATP but not GTP or other nucleoside triphosphates. Dissociation was detected by light scattering or ultracentrifugation after the split subunits were stabilized. ATP was hydrolyzed during the eEF2-dependent dissociation, while a non-hydrolyzable analog of ATP was inactive in ribosome splitting by eEF2. GTP inhibited not only ATP hydrolysis but also dissociation. Sordarin, a fungal eEF2 inhibitor, averted the splitting but stimulated ATP hydrolysis. Another elongation inhibitor, cycloheximide, also prevented eEF2/ATP-dependent splitting, while the inhibitory effect of fusidic acid on the splitting was nominal. Upon dissociation of the 80S ribosome, eEF2 was found on the subunits. We propose that the dissociation activity of eEF2/ATP plays a role in mobilizing 80S ribosomes for protein synthesis during the shift up of physiological conditions.     

Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells.

Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.     

Adenine nucleotides promote dissociation of pertussis toxin subunits

Pertussis toxin is composed of an enzymatically active A subunit and a binding component (B oligomer). Both the holotoxin and the isolated A subunit have previously been shown to exhibit NAD glycohydrolase activity although the A subunit is more active on a molar basis than the holotoxin. We have investigated the mechanism by which ATP stimulates the activity of this toxin. Since dissociation of pertussis toxin subunits would result in increased NAD glycohydrolase activity, the ability of ATP to promote release of the A subunit from the B oligomer was examined. In the presence of the zwitterionic detergent 3-(3-cholamidopropyldimethyl)-1-ammonio)-propanesulfonate, concentrations of ATP as low as 1 microM promoted subunit dissociation. The concentration of ATP required for release of the A subunit was similar to that required for stimulation of NAD glycohydrolase activity. Both ATP and ADP promoted subunit dissociation and stimulated NAD glycohydrolase activity. In contrast, AMP and adenosine did not alter NAD glycohydrolase activity or affect subunit structure. The ability of ATP to decrease the affinity of the A subunit for the B oligomer may play a role in nucleotide stimulation of pertussis toxin activity.