Automated bioluminescent assays for NADH, glucose 6-phosphate, primary bile acids, and ATP

An automated flow system for the bioluminescent assay of various metabolites have been developed. The enzymes used in the assays have been coimmobilized onto Sepharose and packed into small flow cells. Assays for NADH, glucose 6-phosphate, and primary bile acids utilize the bacterial NADH:FMN oxidoreductase/luciferase and either glucose-6-phosphate dehydrogenase or 7 alpha-hydroxysteroid dehydrogenase. ATP assays were performed using immobilized firefly luciferase. In general, the lower limit of detection of the metabolites was at the picomole level, and light intensity was proportional to the substrate concentration from several picomoles to several hundred picomoles. The reproducibility was good with coefficient of variations in the range of 2-5%. The carryover was less than 5% and 30 samples per hour could be assayed. The flow cells were reusable for up to 700 consecutive assays. The major factor limiting their continued use was bacterial contamination of the Sepharose. The results obtained for serum primary bile acids using the bioluminescent assay wer in good agreement with independent measurements on the same samples using gas-liquid chromatography. The immobilized firefly luciferase system was successfully used to measure high levels of bacteria in urine specimens.     

Continuous monitoring of reactions that produce NADH and NADPH using immobilized luciferase and oxidoreductases from Beneckea harveyi.

Highly purified NADH and NADPH:FMN oxidoreductase and luciferase isolated from Beneckea harveyi have been immobilized to arylamine glass beads which were cemented to glass rods. The immobilized enzyme rods are stable, reuseable, and specific for either NADH or NADPH. These rods have been used to monitor reactions producing NADH or NADPH. Picomole levels of malate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase, and hexokinase have been assayed using these rods. Glucose determination has been carried out using soluble hexokinase and glucose-6-phosphate dehydrogenase and the immobilized luciferase-oxidoreductase enzymes. Determination of ethanol concentrations as low as 0.0004% has been achieved with an immobilized alcohol dehydrogenase-NADH:FMN oxidoreductase-luciferase rod.     

Glycogen phosphorylase from human leukocytes. Isolation and kinetic properties

Homogeneous glycogen phosphorylase from human leukocytes has been obtained. A one-step bioluminescent procedure for the enzyme activity assay has been developed. This method is based on a continuous recording of the product of the glycogen phosphorylase-catalyzed reaction using a coimmobilized multienzyme system (phosphoglucomutase, glucose-6-phosphate dehydrogenase, NADH:FMN oxidoreductase and bacterial luciferase). The method sensitivity is 10 times as high compared to earlier described methods. The Km values for glycogen (0.2 mg/ml) and phosphate (3.9 mM) at pH 7.9 were determined. AMP was shown to be the enzyme effector.     

Photokinetic microanalysis of NADP+, using bacterial luciferase

Summary Bioluminescence photokinetic assay of NADP⁺ is described, using the glucose-6-phosphate dehydrogenase reaction for conversion to its reduced form and subsequent measurement of this with luciferase extracts of Vibria fisherii. The analyses were applied to the determination of the activity of minute amounts of glutathione reductase using NADP⁺ as measurable product and for nucleotide assay in cell samples of 0.5–10 g dry weight. The sensitivity was sufficient for determining 0.5 picomoles NADP⁺.Previously, FMN, NADH, NAD⁺ and NADH have been analysed with the bacterial luciferase system. Its applicability has now been extended by the assay of NADP⁺.     

Bioluminescent immunosorbent for rapid immunoassays

We describe a bioluminescent immunoassay procedure which does not require a separation step to remove excess free label. A luminescent immunosorbent constituted of bacterial luciferase, FMN oxidoreductase, and an antibody coimmobilized on Sepharose is used to determine specifically the label enzyme (glucose-6-phosphate dehydrogenase, coupled to an antigen) bound by a specific antibody. The immunosorbent confines the bioluminescent reaction in a small volume, and the bound label produces NADH, which is directly used by the nearby luciferase FMN oxidoreductase enzyme system. On the contrary NADH produced by dehydrogenases in solution is directly oxidized without emitting light. Dehydrogenases contained in the biological sample do not interfere with the assay, which can be performed directly on 25 microliter of serum. In this paper we describe the general procedure and we analyze the different parameters that must be optimized.     

Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.     

Purification and molecular and enzymic properties of mitochondrial NADH dehydrogenase.

Mitochondrial NADH dehydrogenase has been purified to homogeneity by resolution of Complex I from beef heart mitochondria with the chaotrope NaClO₄ and precipitation of the enzyme with ammonium sulfate. The enzyme is water-soluble, has a molecular weight of 69,000 ± 1000 as determined by gel filtration on Sephadex G-100 and agarose 1.5 M. It is an iron-sulfur flavoprotein, with the ratio of flavin (FMN) to nonheme iron to labile sulfide being 1:5–6:5–6. The FMN content suggests a minimum molecular weight of 74,000 ± 3000 for the enzyme. NADH dehydrogenase is composed of three subunits with apparent M_r values, as determined by acrylamide gel electrophoresis as well as by gel filtration on agarose 5 M both in the presence of sodium dodecyl sulfate, of about 51,000, 24,000, and 9–10,000. Coomassie blue stain intensities of the subunits on acrylamide gels suggest that they are present in NADH dehydrogenase in equimolar amounts. However, summation of the apparent M_r values of the dodecyl sulfate-treated subunits appears to overestimate the molecular weight of the native enzyme. The amino acid compositions of NADH dehydrogenase and of each of the isolated and purified subunits have been determined. NADH dehydrogenase catalyzes the oxidation of NADH and NADPH by quinones, ferric compounds, and NAD (3-acetylpyridine adenine dinucleotide was used). All the activities of NADH dehydrogenase are greatly stimulated by addition of guanidine (up to 150 mm), alkylguanidines, arginine, and arginine methyl ester to the assay medium. Phosphoarginine had no effect. These results pointed to the importance of the positively charged guanido group, which appears to interact with and neutralize the negative charges on NAD(P)H and thereby allow for better enzyme-substrate interaction. In the absence of guanidine, NADPH is essentially unoxidized by the enzyme at pH values above 6.0. However, both NADPH dehydrogenase and NADPH → NAD transhydrogenase activities increase dramatically as the assay pH is lowered below pH = 6. Since the pK of the 2′-phosphate of NADPH is 6.1, it appears that the above pH effect is related to protonation of the 2′-phosphate, thus rendering NADPH a closer electronic analog of NADH, which is the primary substrate of the enzyme.     

Bioluminescent enzyme immunoassay for progesterone using monoclonal antibodies and glucose-6-phosphate dehydrogenase labels

Bacterial luciferase, NAD(P): FMN oxidoreductase and anti-mouse immunoglobulin were co-immobilized on Sepharose 4B. This reagent together with a progesterone glucose-6-phosphate dehydrogenase conjugate and various anti-progesterone monoclonal antibodies was used to develop a non-separation bioluminescent immunoassay for progesterone. This monoclonal antibody based assay was sensitive and reliable and using the tracer progesterone-11-acetate-glucose-6-phosphate dehydrogenase, the majority of the monoclonal antibodies give a better sensitivity with this enzymatic tracer than that obtained with an iodinated tracer. In a second assay design progesterone-glutathione was co-immobilized with bacterial luciferase and NAD(P): FMN oxidoreductase on Sepharose 4B and three monoclonal antibodies were labelled with glucose-6-phosphate dehydrogenase. With aqueous progester-one standards, this assay gave comparable sensitivity to the bioluminescent enzyme immunoassay using the second antibody immunoadsorbant and to an RIA but was unsuitable for plasma samples.     

Mannitol production in fungi during glucose catabolism.

The levels of phosphofructokinase (EC 2.7.1.11) and mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) have been determined in a number of Mucor and Penicillium species. Mannitol-1-phosphate dehydrogenase was found in only one species of mucor, Mucor rouxii, and this with a specific activity much lower than that found in Penicillium species. All of the fungi tested in the Ascomycetes class exhibited mannitol-1-phosphate dehydrogenase activity. Interference from both mannitol-1-phosphate dehydrogenase and NADH oxidase (EC 1.6.99.5) caused some difficulty initially in detecting phosphofructokinase in Penicillium species; the Penicillium phosphofructokinase is very unstable. Penicillium notatum accumulates mannitol intracellularly; detection of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase (EC 3.1.3.22) activity in cell-free extracts indicates that the mannitol is formed from glucose via fructose-6-phosphate and mannitol-1-phosphate; no direct reduction of fructose to mannitol could be detected. The mannitol-1-phosphate dehydrogenase was specific for mannitol-1-phosphate and fructose-6-phosphate; NADP+(H) could not replace NAD+(H). The phosphatase (EC3.1.3.22) exhibited a distinct preference for mannitol-1-phosphate as substrate; all other substrates tested exhibited less than 25% of the activity observed with mannitol-1-phosphate.     

Studies of the control of luminescence in Beneckea harveyi: properties of the NADH and NADPH:FMN oxidoreductases.

Highly purified NADH and NADPH:FMN oxidoreductases from Beneckea harveyi have been characterized with regard to kinetic parameters, association with luciferase, activity with artificial electron acceptors, and the effects of inhibitors. The NADH:FMN oxidoreductase exhibits single displacement kinetics while the NADPH:FMN oxidoreductase exhibits double displacement or ping-pong kinetics. This is consistent with the formation of a reduced enzyme as an intermediate in the reaction of catalyzed by the NADPH:FMN oxidoreductase. Coupling of either of the oxidoreductases to the luciferase reaction decreases the apparent Kms for NADH, NADPH, and FMN, supporting the suggestion of a complex between the oxidoreductases and luciferase. The soluble oxidoreductases are more efficient in producing light with luciferase than is a NADH dehydrogenase preparation obtained from the membranes of these bacteria. The soluble enzymes use either FMN or FAD as substrates for the oxidation of reduced pyridine nucleotides while the membrane NADH dehydrogenase is much more active with artificial electron acceptors such as ferricyanide and methylene blue. FMN and FAD are very poor acceptors. The evidence indicates that neither of the soluble oxidoreductases is derived from the membranes. Both enzymes are constitutive and do not depend on the synthesis of luciferase.     

Evidence for the in vivo regulation of glucose 6-phosphate dehydrogenase activity in Hydrogenomonas eutropha H 16 from measurements of the intracellular concentrations of metabolic intermediates

The inhibition of fructose utilization by whole cells of Hydrogenomonas eutropha H 16, following the addition of hydrogen to the gas phase, has been explained as an inhibition of glucose 6-phosphate dehydrogenase (Blackkolb and Schlegel, 1968a, b). The intracellular concentrations of glucose 6-phosphate, 6-phosphogluconate, three inhibitors of the enzyme (NADH, ATP and phosphoenolpyruvate) and some related metabolites were measured in cells incubated in the presence and absence of hydrogen. Inhibition of glucose 6-phosphate dehydrogenase was confirmed by an increase in the glucose 6-phosphate pool and a decrease in the 6-phosphogluconate concentration. The regulatory control is apparently due to a threefold increase in the NADH concentration while the concentrations of the other two inhibitors fell slightly. When the measured intracellular concentrations of intermediates were used in the in vitro assay of glucose 6-phosphate dehydrogenase activity, an almost total inhibition of the dehydrogenase was observed, therefore further regulatory factors must be considered.     

Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii

Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.     

Transfer of pro-R hydrogen from NADH to dihydroxyacetonephosphate by sn-glycerol-1-phosphate dehydrogenase from the archaeon Methanothermobacter thermautotrophicus

sn-Glycerol-1-phosphate dehydrogenase is responsible for the formation of the sn-glycerol-1-phosphate backbone of archaeal lipids. [4-3H]NADH that had 3H at the R side was produced from [4-3H]NAD and glucose with glucose dehydrogenase (a pro-S type enzyme). The 3H of this [4-3H]NADH was transferred to dihydroxyacetonephosphate during the sn-glycerol-1-phosphate dehydrogenase reaction. On the contrary, in a similar reaction using alcohol dehydrogenase (a pro-R type enzyme), 3H was not incorporated into glycerophosphate. These results confirmed a prediction of the tertiary structure of sn-glycerol-1-phosphate dehydrogenase by homology modeling.     

A sensitive bioluminescent immunoassay for dinitrophenol and trinitrotoluene

A bioluminescent immunoassay for measuring dinitrophenol and trinitrotoluene (TNT) has been developed. The DNP and TNT were covalently linked to firefly luciferase, resulting in a conjugate containing 1 mol of DNP or trinitrophenyl (TNP) per mole of luciferase. The conjugate retained 90% of its original catalytic activity. When the conjugate was incubated with immobillzed anti-TNT or anti-DNP and varying concentrations of free TNT or DNP-leucine, the amount of conjugate bound was inversely proportional to the concentration of the free compound. Using this procedure it is possible to detect 2.5 pmol of DNP-leucine and 1.0 pmol of TNT. If the TNP or DNP is linked to glucose-6-phosphate dehydrogenase instead of luciferase, much lower quantities of antigen can be detected. This is due to the fact that this enzyme has a large turnover number so that amplification is possible. The NADH produced is measured using immobilized bacterial NADH:FMN oxidoreductase and luciferase. With this procedure, 10 amol (10^?17 mol) of antigen can be measured. These procedures should be suitable for measuring any antigen.     

The activation of glucose dehydrogenase by p-chloromercuribenzoate

Summary P-Chloromercuribenzoate alters various reactions of rat liver glucose (hexose phosphate) dehydrogenase differently. The reagent has little effect on the glucose: NAD or the glucose: NADP oxidoreductases, doubles the rates of oxidations of galactose-6-phosphate and glucose-6-phosphate by NADP and greatly stimulates the oxidations of glucose-6-phosphate and galactose-6-phosphate by NAD. The reagent appears to react with a sulfhydryl group of the enzyme since activation is reversed and prevented by mercaptoethanol. The direct reaction of the reagent with the enzyme is indicated by its lower thermal stability in the presence of the p-chloromercuribenzoate. The size of the enzyme appears to be the same when determined by sucrose gradient centrifugation in the presence or absence of p-chloromercuribenzoate. In microsomes, the oxidation of NADH or NADPH hampers measurements of glucose dehydrogenase. Since p-chloromercuribenzoate inhibits microsomal oxidation of reduced nicontinamide nucleotides, it is possible to assay for glucose dehydrogenase accurately in the presence of the mercurial in microsomes and microsomal extracts and thus measure the effectiveness of a detergent in extracting the enzyme from microsomes.Abbreviation pcMB p-chloromercuribenzoic acid     

Flow injection determination of glucose,bile acid and ATP using immobilized enzyme reactor and chemiluminescent assay of NAD(P)H

We have developed a chemiluminescent flow injection method for analysis of bile acid, glucose and ATP using the chemiluminescent assay of NADH using 1-methoxy-5-methylphenazinium methyl sulphate (1-MPMS)/isoluminol(IL)/microperoxidase (m-POD) system and immobilized enzyme reactors such as 3α-hydroxysteroid dehydrogenase, glucosedehydrogenase, hexokinase and glucose-6-phosphate dehydrogenase. The standard curves were obtained in the range of 5 ～ 100 pmol for bile acid, 0.5 ～ 5.0 nmol for glucose and 10^?7 ～ 10^?5 mol/L for ATP. The coefficient of variation for each assay was not more than 4.1% for bile acid, 2.3% for glucose and 5.3% for ATP, respectively.     

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

Summary A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N₂ fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H₂ to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein. Evidence is available indicating that nodule bacteroids contain flavoproteins analogous to Azotobacter, azotoflavin, and spinach ferredoxin-NADP reductase. It is concluded that physiologically important systems involved in transport of electrons from dehydrogenases to nitrogenase in bacteroids very likely will include relatively specific electron transport proteins such as bacteroid non-heme iron protein and a flavoprotein from bacteroids that is analogous to azotoflavin.     

The pentose phosphate pathway of glucose metabolism. Measurement of the non-oxidative reactions of the cycle

Methods for the quantitative determination of ribose 5-phosphate isomerase, ribulose 5-phosphate 3-epimerase, transketolase and transaldolase in tissue extracts are described. The determinations depend on the measurement of glyceraldehyde 3-phosphate by using the coupled system triose phosphate isomerase, α-glycero-phosphate dehydrogenase and NADH. By using additional purified enzymes transketolase, ribose 5-phosphate isomerase and ribulose 5-phosphate epimerase conditions could be arranged so that each enzyme in turn was made rate-limiting in the overall system. Transaldolase was measured with fructose 6-phosphate and erythrose 4-phosphate as substrates, and again glyceraldehyde 3-phosphate was measured by using the same coupled system. Measurements of the activities of the non-oxidative reactions of the pentose phosphate pathway were made in a variety of tissues and the values compared with those of the two oxidative steps catalysed by glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase.     

Enzyme channeling immunoassay: A new homogeneous enzyme immunoassay technique

A homogeneous enzyme immunoassay has been developed in which an antigen and glucose-6-phosphate dehydrogenase are coimmobilized on agarose beads. Binding of hexokinase-labeled antibody to the bead-bound antigen results in an accelerated conversion of glucose, ATP, and NAD⁺ to 6-phosphogluconolactone, ADP, and NADH. Critical parameters affecting assay response are discussed.     

Pathway and regulation of erythritol formation in Leuconostoc oenos.

It was recently observed that Leuconostoc oenos GM, a wine lactic acid bacterium, produced erythritol anaerobically from glucose but not from fructose or ribose and that this production was almost absent in the presence of O2. In this study, the pathway of formation of erythritol from glucose in L. oenos was shown to involve the isomerization of glucose 6-phosphate to fructose 6-phosphate by a phosphoglucose isomerase, the cleavage of fructose 6-phosphate by a phosphoketolase, the reduction of erythrose 4-phosphate by an erythritol 4-phosphate dehydrogenase and, finally, the hydrolysis of erythritol 4-phosphate to erythritol by a phosphatase. Fructose 6-phosphate phosphoketolase was copurified with xylulose 5-phosphate phosphoketolase, and the activity of the latter was competitively inhibited by fructose 6-phosphate, with a Ki of 26 mM, corresponding to the Km of fructose 6-phosphate phosphoketolase (22 mM). These results suggest that the two phosphoketolase activities are borne by a single enzyme. Extracts of L. oenos were also found to contain NAD(P)H oxidase, which must be largely responsible for the reoxidation of NADPH and NADH in cells incubated in the presence of O2. In cells incubated with glucose, the concentrations of glucose 6-phosphate and of fructose 6-phosphate were higher in the absence of O2 than in its presence, explaining the stimulation by anaerobiosis of erythritol production. The increase in the hexose 6-phosphate concentration is presumably the result of a functional inhibition of glucose 6-phosphate dehydrogenase because of a reduction in the availability of NADP.