Mutating the Conserved Q-loop Glutamine 1291 Selectively Disrupts Adenylate Kinase-dependent Channel Gating of the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and Reduces Channel Function in Primary Human Airway Epithelia

The ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) and two other non-membrane-bound ABC proteins, Rad50 and a structural maintenance of chromosome (SMC) protein, exhibit adenylate kinase activity in the presence of physiologic concentrations of ATP and AMP or ADP (ATP + AMP ⇆ 2 ADP). The crystal structure of the nucleotide-binding domain of an SMC protein in complex with the adenylate kinase bisubstrate inhibitor P¹,P⁵-di(adenosine-5′) pentaphosphate (Ap₅A) suggests that AMP binds to the conserved Q-loop glutamine during the adenylate kinase reaction. Therefore, we hypothesized that mutating the corresponding residue in CFTR, Gln-1291, selectively disrupts adenylate kinase-dependent channel gating at physiologic nucleotide concentrations. We found that substituting Gln-1291 with bulky side-chain amino acids abolished the effects of Ap₅A, AMP, and adenosine 5′-monophosphoramidate on CFTR channel function. 8-Azidoadenosine 5′-monophosphate photolabeling of the AMP-binding site and adenylate kinase activity were disrupted in Q1291F CFTR. The Gln-1291 mutations did not alter the potency of ATP at stimulating current or ATP-dependent gating when ATP was the only nucleotide present. However, when physiologic concentrations of ADP and AMP were added, adenylate kinase-deficient Q1291F channels opened significantly less than wild type. Consistent with this result, we found that Q1291F CFTR displayed significantly reduced Cl⁻ channel function in well differentiated primary human airway epithelia. These results indicate that a highly conserved residue of an ABC transporter plays an important role in adenylate kinase-dependent CFTR gating. Furthermore, the results suggest that adenylate kinase activity is important for normal CFTR channel function in airway epithelia.     

Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity

Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5′-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5′-monophosphate (AMP), CFTR Cl⁻ channel function is coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the ³²P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.     

Role of CFTR’s intrinsic adenylate kinase activity in gating of the Cl− channel

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl^?channel in the ATP-binding cassette (ABC) transporter protein family. CFTR features the modular design characteristic of ABC transporters, which includes two membrane-spanning domains forming the channel pore, and two ABC nucleotide-binding domains that interact with ATP and contain the enzymatic activity coupled to normal gating. Like other ABC transporters CFTR is an ATPase (ATP + H₂O → ADP + P_i). Recent work has shown that CFTR also possesses intrinsic adenylate kinase activity (ATP + AMP ? ADP + ADP). This finding raises important questions: How does AMP influence CFTR gating? Why does ADP inhibit CFTR current? Which enzymatic activity gates CFTR in vivo? Are there implications for other ABC transporters? This minireview attempts to shed light on these questions by summarizing recent advances in our understanding of the role of the CFTR adenylate kinase activity for channel gating.     

Stable ATP binding mediated by a partial NBD dimer of the CFTR chloride channel

Cystic fibrosis transmembrane conductance regulator (CFTR), a member of the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, is an ATP-gated chloride channel. Like other ABC proteins, CFTR encompasses two nucleotide binding domains (NBDs), NBD1 and NBD2, each accommodating an ATP binding site. It is generally accepted that CFTR’s opening–closing cycles, each completed within 1 s, are driven by rapid ATP binding and hydrolysis events in NBD2. Here, by recording CFTR currents in real time with a ligand exchange protocol, we demonstrated that during many of these gating cycles, NBD1 is constantly occupied by a stably bound ATP or 8-N₃-ATP molecule for tens of seconds. We provided evidence that this tightly bound ATP or 8-N₃-ATP also interacts with residues in the signature sequence of NBD2, a telltale sign for an event occurring at the NBD1–NBD2 interface. The open state of CFTR has been shown to represent a two-ATP–bound NBD dimer. Our results indicate that upon ATP hydrolysis in NBD2, the channel closes into a “partial NBD dimer” state where the NBD interface remains partially closed, preventing ATP dissociation from NBD1 but allowing the release of hydrolytic products and binding of the next ATP to occur in NBD2. Opening and closing of CFTR can then be coupled to the formation and “partial” separation of the NBD dimer. The tightly bound ATP molecule in NBD1 can occasionally dissociate from the partial dimer state, resulting in a nucleotide-free monomeric state of NBDs. Our data, together with other structural/functional studies of CFTR’s NBDs, suggest that this process is poorly reversible, implying that the channel in the partial dimer state or monomeric state enters the open state through different pathways. We therefore proposed a gating model for CFTR with two distinct cycles. The structural and functional significance of our results to other ABC proteins is discussed.     

Simultaneous measurement of oxidative phosphorylation and adenylate kinase in plant mitochondria

An assay system capable of simultaneously measuring ATP, ADP, and AMP concentrations was used for the measurement of oxidative phosphorylation and adenylate kinase (5′-ATP:5′-AMP phosphotransferase) activities in mitochondria which were isolated from etiolated corn, soybean, or cucumber seedlings. Data obtained by this system was correlated with colorimetric P_i uptake and spectrophotometric NADH oxidation measurements. Adenylate kinase was active in both phosphorylating and nonphosphorylating mitochondria. Studies using NaCN, 2,4-dinitrophenol, atractyloside, and 2′-AMP as inhibitors indicated that exogenously supplied [¹⁴C]AMP was converted to [¹⁴C]ADP either by NADH-linked phosphorylation or by translocation and transphosphorylation from intramitochondrial nucleotides.     

An intrinsic adenylate kinase activity regulates gating of the ABC transporter CFTR

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP binding cassette (ABC) transporter family. Like other ABC transporters, it can hydrolyze ATP. Yet while ATP hydrolysis influences channel gating, it has long seemed puzzling that CFTR would require this reaction because anions flow passively through CFTR. Moreover, no other ion channel is known to require the large energy of ATP hydrolysis to gate. We found that CFTR also has adenylate kinase activity (ATP + AMP <=> ADP + ADP) that regulates gating. When functioning as an adenylate kinase, CFTR showed positive cooperativity for ATP suggesting its two nucleotide binding domains may dimerize. Thus, channel activity could be regulated by two different enzymatic reactions, ATPase and adenylate kinase, that share a common ATP binding site in the second nucleotide binding domain. At physiologic nucleotide concentrations, adenylate kinase activity, rather than ATPase activity may control gating, and therefore involve little energy consumption.     

Structural Basis for Adenylate Kinase Activity in ABC ATPases

ATP-binding cassette (ABC) enzymes are involved in diverse biological processes ranging from transmembrane transport to chromosome cohesion and DNA repair. They typically use ATP hydrolysis to conduct energy-dependent biological reactions. However, the cystic fibrosis transmembrane conductance regulator and the DNA repair protein Rad50 can also catalyze the adenylate kinase reaction (ATP + AMP ↔ 2ADP). To clarify and provide a mechanistic basis for the adenylate kinase activity of ABC enzymes, we report the crystal structure of the nucleotide-binding domain of the Pyrococcus furiosus structural maintenance of chromosome protein (pfSMC^nbd) in complex with the adenylate kinase inhibitor P¹,P⁵-di(adenosine-5′)pentaphosphate. We show that pfSMC^nbd possesses reverse adenylate kinase activity. Our results suggest that in adenylate kinase reactions, ATP binds to its canonical binding site while AMP binds to the Q-loop glutamine and a hydration water of the Mg²⁺ ion. Furthermore, mutational analysis indicates that adenylate kinase reaction occurs in the engaged pfSMC^nbd dimer and requires the Signature motif for phosphate transfer. Our results explain how ATP hydrolysis and adenylate kinase reactions can be catalyzed by the same functional motifs within the structural framework of ABC enzymes. Thus, adenylate kinase activity is likely to be a latent activity in many ABC enzymes.     

Nucleotide-binding domains of cystic fibrosis transmembrane conductance regulator, an ABC transporter, catalyze adenylate kinase activity but not ATP hydrolysis

The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter family. CFTR consists of two transmembrane domains, two nucleotide-binding domains (NBD1 and NBD2), and a regulatory domain. Previous biochemical reports suggest NBD1 is a site of stable nucleotide interaction with low ATPase activity, whereas NBD2 is the site of active ATP hydrolysis. It has also been reported that NBD2 additionally possessed adenylate kinase (AK) activity. Knowledge about the intrinsic biochemical activities of the NBDs is essential to understanding the Cl(-) ion gating mechanism. We find that purified mouse NBD1, human NBD1, and human NBD2 function as adenylate kinases but not as ATPases. AK activity is strictly dependent on the addition of the adenosine monophosphate (AMP) substrate. No liberation of [(33)P]phosphate is observed from the gamma-(33)P-labeled ATP substrate in the presence or absence of AMP. AK activity is intrinsic to both human NBDs, as the Walker A box lysine mutations abolish this activity. At low protein concentration, the NBDs display an initial slower nonlinear phase in AK activity, suggesting that the activity results from homodimerization. Interestingly, the G551D gating mutation has an exaggerated nonlinear phase compared with the wild type and may indicate this mutation affects the ability of NBD1 to dimerize. hNBD1 and hNBD2 mixing experiments resulted in an 8-57-fold synergistic enhancement in AK activity suggesting heterodimer formation, which supports a common theme in ABC transporter models. A CFTR gating mechanism model based on adenylate kinase activity is proposed.     

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding

Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain (NBD2) of mouse P-glycoprotein were investigated by using two recombinantly expressed soluble proteins of different lengths and photoactive ATP analogues, 8-azidoadenosine triphosphate (8N(3)-ATP) and 2',3',4'-O-(2,4,6-trinitrophenyl)-8-azidoadenosine triphosphate (TNP-8N(3)-ATP). The two proteins, Thr(1044)-Thr(1224) (NBD2(short)) and Lys(1025)-Ser(1276) (NBD2(long)), both incorporated the four consensus sequences of ABC (ATP-binding cassette) transporters, Walker A and B motifs, the Q-loop, and the ABC signature, while differing in N-terminal and C-terminal extensions. Radioactive photolabeling of both proteins was characterized by hyperbolic dependence on nucleotide concentration and high-affinity binding with K(0.5)(8N(3)-ATP) = 36-37 microM and K(0.5)(TNP-8N(3)-ATP) = 0.8-2.6 microM and was maximal at acidic pH. Photolabeling was strongly inhibited by TNP-ATP (K(D) = 0.1-5 microM) and ATP (K(D) = 0.5-2.7 mM). Since flavonoids display bifunctional interactions at the ATP-binding site and a vicinal steroid-interacting hydrophobic sequence [Conseil, G., Baubichon-Cortay, H., Dayan, G., Jault, J.-M., Barron, D., and Di Pietro, A. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9831-9836], a series of 30 flavonoids from different classes were investigated for structure-activity relationships toward binding to the ATP site, monitored by protection against photolabeling. The 3-OH and aromaticity of conjugated rings A and C appeared important, whereas opening of ring C abolished the binding in all but one case. It can be concluded that the benzopyrone portion of the flavonoids binds at the adenyl site and the phenyl ring B at the ribosyl site. The Walker A and B motifs, intervening sequences, and small segments on both sides are sufficient to constitute the ATP site.     

Potentiation of Disease-associated Cystic Fibrosis Transmembrane Conductance Regulator Mutants by Hydrolyzable ATP Analogs

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel belonging to the ATP-binding cassette transporter superfamily. CFTR is gated by ATP binding and hydrolysis at its two nucleotide-binding domains (NBDs), which dimerize in the presence of ATP to form two ATP-binding pockets (ABP1 and ABP2). Mutations reducing the activity of CFTR result in the genetic disease cystic fibrosis. Two of the most common mutations causing a severe phenotype are G551D and ΔF508. Previously we found that the ATP analog N⁶-(2-phenylethyl)-ATP (P-ATP) potentiates the activity of G551D by ∼7-fold. Here we show that 2′-deoxy-ATP (dATP), but not 3′-deoxy-ATP, increases the activity of G551D-CFTR by ∼8-fold. We custom synthesized N⁶-(2-phenylethyl)-2′-deoxy-ATP (P-dATP), an analog combining the chemical modifications in dATP and P-ATP. This new analog enhances G551D current by 36.2 ± 5.4-fold suggesting an independent but energetically additive action of these two different chemical modifications. We show that P-dATP binds to ABP1 to potentiate the activity of G551D, and mutations in both sides of ABP1 (W401G and S1347G) decrease its potentiation effect, suggesting that the action of P-dATP takes place at the interface of both NBDs. Interestingly, P-dATP completely rectified the gating abnormality of ΔF508-CFTR by increasing its activity by 19.5 ± 3.8-fold through binding to both ABPs. This result highlights the severity of the gating defect associated with ΔF508, the most prevalent disease-associated mutation. The new analog P-dATP can be not only an invaluable tool to study CFTR gating, but it can also serve as a proof-of-principle that, by combining elements that potentiate the channel activity independently, the increase in chloride transport necessary to reach a therapeutic target is attainable.     

Adenosine 5'-sulphatophosphate kinase activity in spinach leaf tissue

1. An F⁻-insensitive 3′-nucleotidase was purified from spinach leaf tissue; the enzyme hydrolysed 3′-AMP, 3′-CMP and adenosine 3′-phosphate 5′-sulphatophosphate but not adenosine 5′-nucleotides nor PP_i. The pH optimum of the enzyme was 7.5; K_m (3′-AMP) was approx. 0.8mm and K_m (3′-CMP) was approx. 3.3mm. 3′-Nucleotidase activity was not associated with chloroplasts. Purified Mg²⁺-dependent pyrophosphatase, free from F⁻-insensitive 3′-nucleotidase, catalysed some hydrolysis of 3′-AMP; this activity was F⁻-sensitive. 2. Adenosine 5′-sulphatophosphate kinase activity was demonstrated in crude spinach extracts supplied with 3′-AMP by the synthesis of the sulphate ester of 2-naphthol in the presence of purified phenol sulphotransferase; purified ATP sulphurylase and pyrophosphatase were also added to synthesize adenosine 5′-sulphatophosphate. Adenosine 5′-sulphatophosphate kinase activity was associated with chloroplasts and was released by sonication. 3. Isolated chloroplasts synthesized adenosine 3′-phosphate 5′-sulphatophosphate from sulphate and ATP in the presence of a 3′-nucleotide; the formation of adenosine 5′-sulphatophosphate was negligible. In the absence of a 3′-nucleotide the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate was negligible, but the formation of adenosine 5′-sulphatophosphate was readily detected. Some properties of the synthesis of adenosine 3′-phosphate 5′-sulphatophosphate by isolated chloroplasts are described. 4. Adenosine 3′-phosphate 5′-sulphatophosphate, synthesized by isolated chloroplasts, was characterized by specific enzyme methods, electrophoresis and i.r. spectrophotometry. 5. Isolated chloroplasts catalysed the incorporation of sulphur from sulphate into cystine/cysteine; the incorporation was enhanced by 3′-AMP and l-serine. It was concluded that adenosine 3′-phosphate 5′-sulphatophosphate is an intermediate in the incorporation of sulphur from sulphate into cystine/cysteine.     

Photoaffinity labeling of ribulose-bisphosphate carboxylase/oxygenase with 8-azidoadenosine 5′-triphosphate

Photoaffinity labeling with [³²P] 8-azidoadenosine 5-triphosphate (8-N₃ATP) was used to identify putative binding sites on tobacco (Nicotiana tabacum L. and N. rustica L.) leaf ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase, EC 4.1.1.39). Incorporation of ³²P was observed in polypeptides corresponding to both RuBPCase subunits when desalted leaf and chloroplast extracts, and purified RuBPCase were irradiated with ultraviolet light in the presence of [³²P] 8-N₃ATP. ³²P-labeling was dependent upon ultraviolet irradiation and occurred with [³²P] 8-N₃ATP labeled in the -position, indicating covalent incorporation of the photoprobe. Both [³²P] 8-N₃ATP and [³²P] 8-N₃GTP were incorporated to a similar extent into the 53-kilodalton (kDa) large subunit (LSu), but incorporation of [³²P] 8-N₃GTP into the 14-kDa small subunit (SSu) of RuBPCase was <5% of that measured with [³²P] 8-N₃ATP. Distinct binding sites for 8-N₃ATP on the two subunits were indicated by different apparent K _D values, 3 and 18 M for the SSu and LSu, respectively, and differences in the response of photoaffinity labeling to Mg²⁺, anions and enzyme activation. Active-site-directed compounds, including the non-gaseous substrate ribulose 1,5-bisphosphate, the reaction intermediate analog 2-carboxyarabinitol-1,5-bisphosphate and several phosphorylated effectors afforded protection to the LSu site against photoincorporation but provided almost no protection to the SSu. These results indicate that 8-N₃ATP binds to the active-site region of the LSu and a distinct site on the SSu of RuBPCase. Experiments conducted with intact pea (Pisum sativum L.) and tobacco chloroplasts showed that the SSu was not photolabeled with [³²P] 8-N₃ATP in organello or in undesalted chloroplast lysates but was photolabeled when lysates were ultrafiltered or desalted. These results indicate that 8-N₃ATP binds to a site on the SSu that has physiological significance.Abbreviations kDa kilodalton - LSu large subunit - 8-N₃ATP 8-azidoadenosine 5-triphosphate - RuBP ribulose-1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase - SSu small subunit Kentucky Agricultural Experiment Station Journal Article No. 89-3-150The authors acknowledge the technical assistance of J.C. Anderson. This work was supported in part by National Institute of Health grant GM 35766 to B.E.H.     

A cAMP receptor from mouse liver cytosol whose binding capacity is enhanced by Mg++-ATP.

A receptor with a dissociation constant of 2·10^?6M for cyclic 3′,5′-AMP (cAMP) has been found in mouse liver cytosol. This cAMP binding activity can be differentiated from the cAMP-dependent protein kinase holoenzymes and the free regulatory subunits also found in the cytosol. Mg⁺⁺-ATP increases the number of binding sites for cAMP several fold. This increased capacity for cAMP binding persists after Sephadex G-25 filtration, and incubation for 14 hours in the presence of 5 mM EDTA. Among several adenosine- and guanosine-derivatives tested, only AMP, ADP and ATP compete efficiently with [³H] cAMP for the cAMP binding site.     

A mutation in CFTR modifies the effects of the adenylate kinase inhibitor Ap5A on channel gating

Mutations in the gene that encodes the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis. The CFTR anion channel is controlled by ATP binding and enzymatic activity at the two nucleotide-binding domains. CFTR exhibits two types of enzymatic activity: 1), ATPase activity in the presence of ATP and 2), adenylate kinase activity in the presence of ATP plus physiologic concentrations of AMP or ADP. Previous work showed that P¹,P⁵-di(adenosine-5′)pentaphosphate (Ap₅A), a specific adenylate kinases inhibitor, inhibited wild-type CFTR. In this study, we report that Ap₅A increased activity of CFTR with an L1254A mutation. This mutation increased the EC50 for ATP by >10-fold and reduced channel activity by prolonging the closed state. Ap₅A did not elicit current on its own nor did it alter ATP EC50 or maximal current. However, it changed the relationship between ATP concentration and current. At submaximal ATP concentrations, Ap₅A stimulated current by stabilizing the channel open state. Whereas previous work indicated that adenylate kinase activity regulated channel opening, our data suggest that Ap₅A binding may also influence channel closing. These results also suggest that a better understanding of the adenylate kinase activity of CFTR may be of value in developing new therapeutic strategies for cystic fibrosis.     

Cyclic AMP-binding proteins in human blood platelets detected by photoaffinity labelling

Cyclic AMP inhibits platelet aggregation induced by physiological agents. 8 Azido [³²P]cyclic AMP (N₃ cyclic AMP) has been utilized as a photoaffinity probe to define the cyclic AMP-binding proteins present in unperturbed human platelets and their subcellular fractions. Specificity of cyclic AMP binding was determined by contrasting binding in the presence and absence of excess unlabelled cyclic AMP, cyclic GMP and 5′-AMP. Binding was unaffected by 5′-AMP and obliterated by cyclic AMP. Four major species of binding proteins, 49 000, 42 000, 39 000, 37 000, were obtained in all platelet fractions (crude homeogenate, cytosol, membranes and granules). Two-dimensional gel electrophoresis of platelet cytosol resolved the major molecular weight species into 15 specific cyclic AMP binding proteins of four molecular weight classes differing by charge density. These studies suggest that platelets contain an array of specific cyclic AMP-binding proteins which may function in hemostatic regulation.     

Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound,cAMP-activated protein kinase

Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3′,5′-cyclic monophosphate (8-N₃ cAMP), and [γ³²P] ATP, the membranebinding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [³²P] 8-N₃ cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [γ³²P] ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg · ATP concentration is below approximately 10 μM. The catalytic subunit is not required for the Mg · ATP inhibition of 8-N₃ cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N₃ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N₃ cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied in other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg · ATP.     

Binding and reaction studies with adenine nucleotides on purified coupling factor fromRhodospirillum rubrum

Equilibrium binding studies with ATPase isolated fromRhodospirillum rubum chromotophores have been carried out using gel filtration.Binding experiments with variable concentrations of [¹⁴C]ADP show a biphasic saturation curve. With a parameter fitting computer program the dissociation constants for two distinct binding sites are determined as 7×10^–6 and 9×10^–5 M, respectively. The enzymebound radioactivity is recovered as ADP (80–90%), and the rest is converted to AMP and ATP. In the free nucleotides a large amount of AMP (about 70%) is found in addition to ADP.Analogous binding experiments with [¹⁴C]ATP are monophasic. Most of the bound radioactivity can be identified as ADP showing a dissociation constant corresponding to the high affinity site. The pattern of the free nucleotides is the same as in the experiments with ADP.These results indicate three separate binding sites on the enzyme: a low and a high affinity site for ADP, and a site at which ATP hydrolysis takes place. The analysis of the nucleotides suggests for the ADP sites a phosphoryl group transfer to produce ATP and AMP. Various experiments exclude the contamination of the enzyme preparation with adenylate kinase.Part of this work has been published in theBook of Abstracts, 4th International Congress on Photosynthesis, Reading, U.K., 1977.     

Determination of the cellular location of cyclic nucleotide binding sites using 8-azidoadenosine-3', 5'-monophosphate, a photoaffinity probe.

A photoaffinity analog of cyclic AMP, 8-azidoadenosine-3′,5′-monophosphate (8-N₃cAMP), and a linear gradient sodium dodecyl sulfate-polyacrylamide gel system have been used to photolabel and separate possible cyclic AMP binding sites in whole cells of the sarcoma 37 line. At least four sites within the whole sarcoma 37 cell bind [³²P]8-N₃cAMP such that they are saturated at 1.0 μm concentration. These four binding moieties appear to have measurably different affinities for 8-N₃cAMP and the number of total sites labeled per cell differs with each binding moiety. Cell fractionation allows separation of some of the photolabeled sites and gives an indication as to the geographical location of these binding sites within the whole cell. Enrichment by cell fractionation allows additional photolabeled sites to be observed.     

A Complex Containing SNF1-Related Kinase (SnRK1) and Adenosine Kinase in Arabidopsis

SNF1-related kinase (SnRK1) in plants belongs to a conserved family that includes sucrose non-fermenting 1 kinase (SNF1) in yeast and AMP-activated protein kinase (AMPK) in animals. These kinases play important roles in the regulation of cellular energy homeostasis and in response to stresses that deplete ATP, they inhibit energy consuming anabolic pathways and promote catabolism. Energy stress is sensed by increased AMP:ATP ratios and in plants, 5′-AMP inhibits inactivation of phosphorylated SnRK1 by phosphatase. In previous studies, we showed that geminivirus pathogenicity proteins interact with both SnRK1 and adenosine kinase (ADK), which phosphorylates adenosine to generate 5′-AMP. This suggested a relationship between SnRK1 and ADK, which we investigate in the studies described here. We demonstrate that SnRK1 and ADK physically associate in the cytoplasm, and that SnRK1 stimulates ADK in vitro by an unknown, non-enzymatic mechanism. Further, altering SnRK1 or ADK activity in transgenic plants altered the activity of the other kinase, providing evidence for in vivo linkage but also revealing that in vivo regulation of these activities is complex. This study establishes the existence of SnRK1-ADK complexes that may play important roles in energy homeostasis and cellular responses to biotic and abiotic stress.     

A study of adenosine 3′–5′ cyclic monophosphate binding sites of human erythrocyte membranes using 8-azidoadenosine 3′–5′ cyclic monophosphate,a photoaffinity probe

An earlier report (1a) has shown the utility of 8-N₃cAMP (8-azidoadenosine-3′, 5′-cyclic monophosphate) as a photoaffinity probe for cAMP binding sites in human erythrocyte membranes. The increased resolution obtained using a linear-gradient SDS polyacrylamide gel system now shows that: (1) both cAMP and 8-N₃cAMP stimulate the phosphorylation by [γ-³²P]-ATP of the same red cell membrane proteins; (2) the protein of approximately 48,000 molecular weight whose phosphorylation by [γ-³²P]-ATP is stimulated by cAMP and 8-N₃cAMP migrates at a solwer rate than the protein in the same molecular weight range which is heavily photolabeled with [³²P]-8-N₃cAMP; (3) other cyclic nucleotide binding sites exist besides those initailly reported; (4) the variation in the ratio of incorporation of ³²P-8-N₃cAMP into the two highest affinity binding sites appears to be the result of a specific proteolysis of the larger protein.