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1.
This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC(50) values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low K(m) value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases. 相似文献
2.
A Transcreener kinase fluorescence polarization (FP) assay has been developed for the serine/threonine kinase protein kinase A (PKA). The PKA Transcreener kinase assay is an homogenous, competitive antibody-based FP assay that uses Far Red Alexa Fluor 633-labeled adenosine 5' disphosphate (ADP) tracer and mouse monoclonal anti-ADP antibody. The Transcreener PKA assay was validated with both known PKA inhibitors and library compounds. The Transcreener PKA assay is resistant to low-wavelength (or common) fluorescent interference from small-molecule library compounds and generates IC50 results comparable with current radioactive filter-binding assay. 相似文献
3.
Benicchi T Iozzi S Svahn A Axelsson H Mori E Bernocco S Cappelli F Caramelli C Fanti P Genesio E Maccari L Markova N Micco I Porcari V Schultz J Fecke W 《Journal of biomolecular screening》2012,17(7):933-945
The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction. 相似文献
4.
Sugita H Dan S Kong D Tomida A Yamori T 《Biochemical and biophysical research communications》2008,377(3):941-945
PIK3CA, coding a catalytic subunit of PI3K p110α, is frequently mutated in cancer. In previous studies, p110α with hotspot mutations such as E545K and H1047R were shown to be gain-of-function mutations. However, quantitative evaluation of these mutants was not well established. Recently, a new method for measuring PI3K activity using homogeneous time-resolved fluorescence (HTRF) has been developed. Using this method, we constructed a quantitative evaluation system for PI3K activity. Serial dilutions of standard PIP3 were subjected to the PI3K-HTRF assay in order to establish a regression line for calibration. The recombinant FLAG-tagged p110α proteins were engineered together with a regulatory subunit p85α in human embryonic kidney 293T cells. Anti-FLAG-Ig immunoprecipitates were then subjected to the assay, which enabled us to quantitatively evaluate the activities of hotspot mutants of p110α. We believe this method will also be applicable to the evaluation of p110α having uncharacterized mutations found in cancer. 相似文献
5.
L Missiaen F Wuytack Y Kanmura H Van Belle J Wynants J Minten R Casteels 《Biochimica et biophysica acta》1989,990(1):40-44
The specific activity of the Mg2+-ATPase and the (Ca2+ + Mg2+)-ATPase has been measured in a microsomal fraction from pig antral smooth muscle with the phosphate-release assay and the NADH-coupled enzyme assay, and the release of inorganic phosphate as a function of time is compared with the concomitant production of ADP. Both assays are found to overestimate the true Mg2+-ATPase activity. The adenylate kinase inhibitor P1,P5-di(adenosine-5'-)pentaphosphate (Ap5A) reduces the specific activity of the Mg2+-ATPase measured in the NADH-coupled enzyme assay to about half of its original value; however, it does not affect the specific activity of the Mg2+-ATPase in the Pi-release assay. The considerable overestimation of the Mg2+-ATPase activity in the NADH-coupled enzyme assay results from a combined action of an ATP pyrophosphatase (ATP in equilibrium AMP + PPi) and adenylate kinase activity contaminating the microsomes. The adenylate kinase activity in the microsomes catalyses the conversion of AMP formed by the ATP pyrophosphatase together with ATP into two ADP's. Also the phosphate-release assay is prone to an overestimation artefact because an inorganic pyrophosphatase will degrade the pyrophosphate and thus lead to additional Pi-production. Measurements of AMP and NAD+ production by HPLC confirmed our proposed reaction scheme. The same (Ca2+ + Mg2+)-ATPase activity is found in both assays, because the (Ca2+ + Mg2+)-ATPase activity is calculated from the difference in ATPase activity in the presence and absence of Ca2+, so that as a consequence the interfering activities are automatically subtracted. 相似文献
6.
Park YW Cummings RT Wu L Zheng S Cameron PM Woods A Zaller DM Marcy AI Hermes JD 《Analytical biochemistry》1999,269(1):94-104
Two homogeneous proximity assays for tyrosine kinases, scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence (HTRF), have been developed and compared. In both formats, the kinase assay was performed using biotinylated peptide substrate, ATP ([33P]ATP in the case of SPA), and tyrosine kinase in a 96-well assay format. After the kinase reaction was stopped, streptavidin-coated SPA beads or europium cryptate-labeled anti-phosphotyrosine antibody and streptavidin-labeled allophycocyanin were added as detection reagents for SPA or HTRF assays, respectively. Since the assay signal was detected only when the energy donor (radioactivity for SPA, Eu for HTRF) and the energy acceptor molecules (SPA beads for SPA, allophycocyanin for HTRF) were in close proximity, both assays required no wash or liquid transfer steps. This homogeneous ("mix-and-measure") nature allows these assays to be much simpler, more robust, and easier to automate than traditional protein kinase assays, such as a filter binding assay or ELISA. Both assays have been miniaturized to a 384-well format to reduce the assay volume, thereby saving the valuable screening samples as well as assay reagents, and automated using automated pipetting stations to increase the assay throughput. Several advantages and disadvantages for each assay are described. 相似文献
7.
The goal of this investigation was to develop an assay whereby we could measure changes in ATP, ADP, and phosphocreatine (PCr) during stimulation of the sarcoplasmic reticulum (SR) Ca2+ ATPase. After stopping the enzyme reaction, compounds were extracted by perchloric acid and separated by reversed-phase high-performance liquid chromatography (HPLC). Absorbance of ATP and ADP was monitored at 260 nm, and detection of PCr was done at 205 nm. Chromatograms show that peaks associated with each compound are clearly separated and easily detected. The SR Ca2+ ATPase assay was run for various time periods and using varying free [Ca2+]. The changes in ATP and ADP contents were linear with increasing time and varied as expected with increasing free [Ca2+]. The ATPase activities determined using changes in ATP and ADP were nearly identical to those determined using previously established assays. When PCr was added to the assay, we were able to confirm that the Ca2+ ATPase uses ATP that is synthesized locally from PCr via creatine kinase (CK). The results indicate that this is a valid and reliable method for examining SR Ca2+ ATPase activity and for investigating its interaction with CK. 相似文献
8.
9.
G.J. Kemp D.N. Manners J.F. Clark M.E. Bastin G.K. Radda 《Molecular and cellular biochemistry》1997,174(1-2):29-32
We describe a model of mitochondrial regulation in vivo which takes account of spatial diffusion of high-energy (ATP and phosphocreatine) and low-energy metabolites (ADP and creatine), their interconversion by creatine kinase (which is not assumed to be at equilibrium), and possible functional 'coupling' between the components of creatine kinase associated with the mitochondrial adenine nucleotide translocase and the myofibrillar ATPase. At high creatine kinase activity, the degree of functional coupling at either the mitochondrial or ATPase end has little effect on relationships between oxidative ATP synthesis rate and spatially-averaged metabolite concentrations. However, lowering the creatine kinase activity raises the mean steady state ADP and creatine concentrations, to a degree which depends on the degree of coupling. At high creatine kinase activity, the fraction of flow carried by ATP is small. Lowering the creatine kinase activity raises this fraction, especially when there is little functional coupling. All metabolites show small spatial gradients, more so at low cytosolic creatine kinase activity, and unless there is near-complete coupling, so does net creatine kinase flux. During workjump transitions, spatial-average responses exhibit near-exponential kinetics as expected, while concentration changes start at the ATPase end and propagate towards the mitochondrion, damped in time and space. (Mol Cell Biochem 174: 29–32, 1997) 相似文献
10.
Matthew L. Fowler Cheryl J. Ingram-Smith Kerry S. Smith 《Journal of visualized experiments : JoVE》2011,(58)
Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya6. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila7-14. An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD+ by the enzymes pyruvate kinase and lactate dehydrogenase21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine23. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP+ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase24.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi. 相似文献
11.
Christoph O. Randak Amanda R. Ver Heul Michael J. Welsh 《The Journal of biological chemistry》2012,287(43):36105-36110
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. 相似文献
12.
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. 相似文献
13.
Kashem MA Nelson RM Yingling JD Pullen SS Prokopowicz AS Jones JW Wolak JP Rogers GR Morelock MM Snow RJ Homon CA Jakes S 《Journal of biomolecular screening》2007,12(1):70-83
Numerous assay methods have been developed to identify small-molecule effectors of protein kinases, but no single method can be applied to all isolated kinases. The authors developed a set of 3 high-throughput screening (HTS)-compatible biochemical assays that can measure 3 mechanistically distinct properties of a kinase active site, with the goal that at least 1 of the 3 would be applicable to any kinase selected as a target for drug discovery efforts. Two assays measure catalytically active enzyme: A dissociation-enhanced lanthanide fluoroimmuno assay (DELFIA) uses an antibody to quantitate the generation of phosphorylated substrate; a second assay uses luciferase to measure the consumption of adenosine triphosphate (ATP) during either phosphoryl-transfer to a peptide substrate or to water (intrinsic ATPase activity). A third assay, which is not dependent on a catalytically active enzyme, measures the competition for binding to kinase between an inhibitor and a fluorescent ATP binding site probe. To evaluate the suitability of these assays for drug discovery, the authors compared their ability to identify inhibitors of a nonreceptor protein tyrosine kinase from the Tec family, interleukin-2-inducible T cell kinase (ITK). The 3 assays agreed on 57% of the combined confirmed hit set identified from screening a 10,208-compound library enriched with known kinase inhibitors and molecules that were structurally similar. Among the 3 assays, the one measuring intrinsic ATPase activity produced the largest number of unique hits, the fewest unique misses, and the most comprehensive hit set, missing only 2.7% of the confirmed inhibitors identified by the other 2 assays combined. Based on these data, all 3 assay formats are viable for screening and together provide greater options for assay design depending on the targeted kinase. 相似文献
14.
Miller JR Herberg JT Tomilo M McCroskey MC Feilmeier BJ 《Analytical biochemistry》2007,365(1):132-143
The rise in bacterial resistance to antibiotics demonstrates the medical need for new antibacterial agents. One approach to this problem is to identify new antibacterials that act through validated drug targets such as bacterial DNA gyrase. DNA gyrase uses the energy of ATP hydrolysis to introduce negative supercoils into plasmid and chromosomal DNA and is essential for DNA replication. Inhibition of the ATPase activity of DNA gyrase is the mechanism by which coumarin-class antibiotics such as novobiocin inhibit bacterial growth. Although ATPase inhibitors exhibit potent antibacterial activity against gram-positive pathogens, no gyrase ATPase activity from a gram-positive organism is described in the literature. To address this, we developed and optimized an enzyme-coupled phosphate assay and used this assay to characterize the ATPase kinetics of Streptococcus pneumoniae gyrase. The S. pneumoniae enzyme exhibits cooperativity with ATP and requires organic potassium salts. We also studied inhibition of the enzyme by novobiocin. Apparent inhibition constants for novobiocin increased linearly with ATP concentration, indicative of an ATP-competitive mechanism. Similar binding affinities were measured by isothermal titration calorimetry. These results reveal unique features of the S. pneumoniae DNA gyrase ATPase and demonstrate the utility of the assay for screening and kinetic characterization of ATPase inhibitors. 相似文献
15.
The assay of acetyl-CoA carboxylase (EC 6.4.1.2) does not follow ideal zero-order kinetics when assayed in a crude extract from wheat (Triticum aestivum L.) germ. Our results show that the lack of ideality is the consequence of contamination by ATPase and adenylate kinase. These enzyme activities generate significant amounts of ADP and AMP in the assay mixture, thus limiting the availability of ATP for the carboxylase reaction. Moreover, ADP and AMP are competitive inhibitors, with respect to ATP, of acetyl-CoA carboxylase. Similar relationships between adenylate nucleotides and acetyl-CoA carboxylase are found in isolated chloroplasts. There is no evidence that acetyl-CoA carboxylase activity in the extracts of the plant systems examined is altered by covalent modification, such as a phosphorylation-dephosphorylation cycle. A scheme is presented that illustrates the dependency of acetyl-CoA carboxylase and fatty acid synthesis on the energy demands of the chloroplasts in vivo. 相似文献
16.
Glycosyltransferases catalyze transfer of sugar moieties from activated donor molecules to specific acceptor molecules, forming glycosidic bonds. Identification of selective modulators of glycosyltransferases is important both to provide new tools for investigating pathophysiological roles of glycosylation reactions in cells and tissues, and as new leads in drug discovery. Here we describe a universal enzyme-coupled fluorescence assay for glycosyltransferases, based on quantification of nucleotides produced in the glycosyl transfer reaction. GDP, UDP, and CMP are phosphorylated with nucleotide kinase in the presence of excess ATP, generating ADP. Via coupled enzyme reactions involving ADP-hexokinase, glucose-6-phosphate dehydrogenase, and diaphorase, the ADP is utilized for conversion of resazurin to resorufin, which is determined by fluorescence measurement. The method was validated by comparison with an HPLC method, and employed to screen the LOPAC1280 library for inhibitors in a 384-well plate format. The assay performed well, with a Z′-factor of 0.80. We identified 12 hits for human galactosyltransferase B4GALT1 after elimination of false positives that inhibited the enzyme-coupled assay system. The assay components are all commercially available and the reagent cost is only 2 to 10 US cents per well. This method is suitable for low-cost, high-throughput assay of various glycosyltransferases and screening of glycosyltransferase modulators. 相似文献
17.
摘要:【目的】腺苷酸激酶(adenylate kinase, ADK)和多聚磷酸盐激酶(polyphosphate kinase, PPK)偶联催化的ATP扩增反应结合生物发光检测法能够对微量微生物进行检测。但是PPK当中结合的内源性的ADP会产生背景干扰,影响测定。本文旨在融合表达ADK和PPK,并建立一种方便有效的内源性ADP的去除方法,降低背景,使之与传统生物发光法结合,实现高灵敏生物发光法检测微量ATP及微生物。【方法】PCR扩增得到PPK、ADK基因,插入表达载体pET28a (+)中构建重组表达质粒pET28a (+)-PPKADK,表达PPK-ADK融合蛋白。利用表面包裹聚胺醇(Polyurethane)的磁珠(magnetic beads),通过化学反应将腺苷酸双磷酸酶(apyrase)固定于磁珠表面,制备固相腺苷酸双磷酸酶(Beads-apyrase),用于除去与融合蛋白结合的内源性ADP,降低ATP扩增反应的背景,从而使之与生物发光反应相结合,测定微量外源ATP及细菌菌落数。【结果】表达的融合蛋白具有PPK和ADK的活性,利用Beads-apyrase可以方便而有效的去除内源性ADP,显著地降低反应背景,从而实现了利用ATP扩增反应与传统生物发光反应结合,测定了小于1 fmol的外源微量ATP,使生物发光法检测ATP及微生物的灵敏度提高至少100倍。【结论】利用Beads-apyrase能够方便、有效地降低PPK-ADK中的ADP背景,从而使PPK-ADK催化的ATP扩增反应能够与传统生物发光法相结合,极大地提高了生物发光法的灵敏度。 相似文献
18.
Mezna M Wong AC Ainger M Scott RW Hammonds T Olson MF 《Journal of biomolecular screening》2012,17(4):460-468
Kinases are attractive drug targets because of the central roles they play in signal transduction pathways and human diseases. Their well-formed adenosine triphosphate (ATP)-binding pockets make ideal targets for small-molecule inhibitors. For drug discovery purposes, many peptide-based kinase assays have been developed that measure substrate phosphorylation using fluorescence-based readouts. However, for some kinases these assays may not be appropriate. In the case of the LIM kinases (LIMK), an inability to phosphorylate peptide substrates resulted in previous high-throughput screens (HTS) using radioactive labeling of recombinant cofilin protein as the readout. We describe the development of an HTS-compatible assay that measures relative ATP levels using luciferase-generated luminescence as a function of LIMK activity. The assay was inexpensive to perform, and proof-of-principle screening of kinase inhibitors demonstrated that compound potency against LIMK could be determined; ultimately, the assay was used for successful prosecution of automated HTS. Following HTS, the secondary assay format was changed to obtain more accurate measures of potency and mechanism of action using more complex (and expensive) assays. The luciferase assay nonetheless provides an inexpensive and reliable primary assay for HTS that allowed for the identification of LIMK inhibitors to initiate discovery programs for the eventual treatment of human diseases. 相似文献
19.
Shapiro AB Livchak S Gao N Whiteaker J Thresher J Jahić H Huang J Gu RF 《Journal of biomolecular screening》2012,17(3):327-338
A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB. 相似文献
20.
Kinetics of interaction of adenosine diphosphate and adenosine triphosphate with adenosine triphosphatase of bovine heart submitochondrial particles. 总被引:2,自引:2,他引:0
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The short preincubation of submitochondrial particles with low concentrations of ADP in the presence of Mg2+ results in a complete loss of their ATPase and inosine triphosphatase activities. Other nucleoside diphosphates (IDP and GDP) do not affect the ATPase activity. The ADP-inhibited ATPase can be activated in a time-dependent manner by treatment of submitochondrial particles with the enzyme converting ADP into ATP (phosphoenolpyruvate plus pyruvate kinase). The activaton is a first-order reaction with rate constant 0.2 min-1 at 25 degrees C. The rate constant of activation is increased in the presence of ATP up to 2 min-1, and this increase shows saturation kinetics with Km value equal to that for ATPase reaction itself (10(-4) M at 25 degrees C at pH 8.0). The experimental results obtained are consistent with the model where two alternative pathways of ADP dissociation from the inhibitory site of ATPase exist; one is spontaneous dissociation and the second is ATP-dependent dissociation through the formation of the ternary complex between ADP, the enzyme and ATP. ADP-induced inactivation and ATP-dependent activation of ATPase activity of submitochondrial particles is accompanied by the same directed change of their ability to catalyse the ATP-dependent reverse electron transport from succinate to NAD+. The possible implication of the model suggested is discussed in terms of functional role of the inhibitory high-affinity binding site for ADP in the mitochondrial ATPase. 相似文献