Real-time detection and quantification of adenosine triphosphate sulfurylase activity by a bioluminometric approach.

A real-time, sensitive, and simple assay for detection and quantification of adenosine triphosphate sulfurylase (ATP:sulfate adenylytransferase, EC 2.7.7.4) activity has been developed. The method is based on detection of ATP generated in the ATP sulfurylase reaction between APS and PPi by the firefly luciferase system. For the Saccharomyces cerevisiae ATP sulfurylase, the concentrations of APS and PPi at the half-maximal rate were found to be about 0.5 and 7 microM, respectively. The assay is sensitive and yields linear response between 0.1 microU and 50 mU. The method can be used for monitoring and quantification of recombinant ATP sulfurylase activity in Escherichia coli lysate, as well as for detection of the activity during different purification procedures.     

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status

Abstract. Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum , a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 pM 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 pM 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 pM 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S.(Ps.)ochraceum (24.8 pM 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum , a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S.(N.) exiguum (10.9 pM 0.6mU) than in Simulium (Notolepria) gonzalezi (5.9pM1.9mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O. volvulus.     

Real-time bioluminometric method for detection of nucleoside diphosphate kinase activity.

A real-time, simple and sensitive method for detection of nucleoside diphosphate (NDP) kinase activity has been developed. The assay is based on detection of ATP, generated in the NDP kinase reaction between a nucleoside triphosphate and adenosine diphosphate (ADP), by the firefly luciferase system. In the presence of 0.3 mM dGTP, the Km for ADP was found to be approximately 30 microM for the NDP kinase from Baker's yeast. In the presence of 250 microM ADP, the Km for dATP alpha S, dTTP alpha S, dGTP, dTTP, dCTP and GTP was found to be approximately 0.01, 0.03, 0.05, 0.25, 0.75 and 0.2 mM, respectively. The assay is sensitive and yields linear responses between 0.05-50 mU. The detection limit was found to be 0.05 mU of NDP kinase. The method was used to detect NDP kinase contamination in commercial enzyme preparations.     

The p-coumaryl CoA ligase of potato tubers

The demonstration of activity of p-coumaryl CoA ligase in extracts of aged potato disks proved difficult owing to the presence of extremely high levels of apyrase which caused rapid hydrolysis of ATP, a co-factor for ligase activity. This problem was largely overcome by including an inhibitor of apyrase, sodium fluoride in the ligase assay and by initiation of the reaction with ATP. A method for the separation of apyrase and p-coumaryl CoA ligase by chromatography on DEAE-cellulose is described. p-Coumaryl CoA ligase was not detectable in freshly prepared disks of potato tubers. However on ageing in the light a large increase in the activity of this enzyme occurs, The enzyme of aged potato disks shows high activity with p-coumaric, ferulic, caffeic and with m- and p-methoxycinnamic acids. However the affinity of the enzyme for the methoxy derivatives is much lower than for cinnamic acids bearing free hydroxyl groups.     

Microanalysis for MDR1 ATPase by high-performance liquid chromatography with a titanium dioxide column

MDR1 is clinically important because it is involved in multidrug resistance of cancer cells and affects the pharmacokinetics of various drugs. Because MDR1 harnesses adenosine 5'-triphosphate (ATP) hydrolysis for transporting drugs, examining the effect on ATPase activity is imperative for understanding the interactions between drugs and MDR1. However, conventional assay systems for ATPase activity are not sensitive enough for screening drugs using purified MDR1. Here we report a novel method to measure ATPase activity of MDR1 using high-performance liquid chromatography equipped with a titanium dioxide column. The amount of adenosine 5'-diphosphate (ADP) produced by the ATPase reaction was determined within 2 min with a titanium dioxide column (4.6 mm ID x 100 mm). The relationship between ADP amount and chromatogram peak area was linear from 5 pmol to 10 nmol. This method made it possible to reduce the amount of purified MDR1 required for a reaction to 0.5 ng, about 1/20th of the conventional colorimetric inorganic phosphate detection assay. This method is sensitive enough to detect any subtle changes in ATPase activity of MDR1 induced by drugs and can be applied to measure ATPase activity of any protein.     

Salivary apyrase of Aedes aegypti: characterization and secretory fate

Salivary gland homogenates of female adult Aedes aegypti hydrolyzed ATP and ADP thereby defining an apyrase activity. Activity is divalent cation dependent with an optimum pH of 9.0. ATPase and ADPase activities could not be dissociated thus suggesting the presence of a true apyrase enzyme. Apyrase activity is low on the day of emergence but increases to 160 mU per pair of glands on the second day. The site at which mosquitoes probed into warm polyacrylamide gels retains apyrase activity, confirming the secretory fate of this activity.     

Effects of protein-modifying reagents on an isoenzyme of potato apyrase

Treatment of an isoenzyme of potato apyrase of high adenosine triphosphatase/adenosine diphosphatase (ATPase/ADPase) ratio with iodine, N-acetylimidazole or tetranitromethane inactivates the ATPase activity of this enzyme faster than its ADPase activity. There was protection by substrates with the two last-named substances. This and the appearance of nitrotyrosine suggests the participation of tyrosyl residues in both enzymic activities of potato apyrase. The participation of thiol groups is excluded by the insensitivity of apyrase to p-chloromercuribenzoate. Also, 2-hydroxy-5-nitrobenzyl bromide or carboxymethylation produce the same rate of inactivation of ATPase and ADPase activities. Substrates protect both activities from inactivation. Hydrogen peroxide and photo-oxidation inactivate ATPase activity faster than ADPase activity. There is no protection by substrates. Analysis of pH effects on V_max. and K_m suggest different pK values for the amino acid residues at the ATP and ADP sites.     

Method for real-time detection of inorganic pyrophosphatase activity

A sensitive and simple method for real-time detection of inorganic pyrophosphatase (PPase) (EC 3.6.1.1) activity has been developed. The method is based on PPase-induced activation of the firefly luciferase activity in the presence of inorganic pyrophosphate (PPi). PPi inhibits the luciferase activity, but in the presence of PPase the luciferase activity is restored and the luminescence output increases. The assay yields linear responses between 8 and 500 mU. The detection limit was found to be 8 mU PPase. The method was used to detect the hydrolytic activity of PPases from Saccharomyces cerevisiae, Escherichia coli, and Bacillus stearothermophilus. As substrate for the luciferase, adenosine 5'-phosphosulfate can replace ATP, which is an advantage for detection of PPase activity in crude extracts containing ATP-hydrolyzing activities. The method can be used for kinetic and inhibition studies as well as for detection of PPase activity during different purification procedures.     

Salivary gland apyrase determines probing time in anopheline mosquitoes

Salivary gland homogenates of adult female anopheline mosquitoes, of three different species, hydrolysed ATP and ADP, thereby demonstrating an apyrase activity. Total enzyme activity was greatest in the vector species A. freeborni (20.7 ± 2.4 mU/pair of glands) and least in the autogenous mosquito A. sp. nr. salbaii (3.0 ± 0.4 mU/pair of glands); another vector species, A. stephensi, produced intermediate levels of the enzyme (7.8 ± 0.7 mU/pair of glands). In all cases, the reaction was activated by divalent cations and maximal at pH 9.0 and in the presence of 2-mercaptoethanol. Apyrase activity in each salivary gland correlated with the degree of inhibition of ADP-induced platelet aggregation in vitro. Duration of probing correlated inversely with salivary apyrase content. We conclude that salivary apyrase largely determines a mosquito's ability to locate blood. Differential selective pressures for facility of blood location would have influenced the level of salivary apyrase in these mosquitoes.     

Substitution of His59 converts CD39 apyrase into an ADPase in a quaternary structure dependent manner

The two transmembrane domains of CD39 ecto-apyrase regulate the formation of fully active homotetramers. We show that mutations in apyrase conserved region 1 (ACR1) have two dramatically different sets of effects determined by whether they occur in intact tetramers or in disrupted tetramers or monomers. In intact tetramers, substitution of H59 in the rat brain CD39 ACR1 with G or S abolishes more than 90% of the ATPase activity but less than 50% of the ADPase activity, converting the enzyme into an ADPase with relative ADP:ATP hydrolysis rates of 6:1 or 8:1, respectively. In contrast, the same substitutions in tetramers lacking either transmembrane domain, in monomers lacking both transmembrane domains, or in detergent-solubilized full-length monomers have no effect on ATPase activity and increase ADPase activity approximately 2-fold, resulting in equal ATPase and ADPase activities. N61R substitution has a much smaller effect on the ADPase:ATPase ratio in both cases. While the data for truncated and monomeric constructs are consistent with the proposed role of ACR1 as the beta-phosphate binding domain by analogy with the actin/hsp70/hexokinase superfamily, the finding that H59 substitutions in full-length CD39 primarily diminish the ATP hydrolysis rate suggests that ACR1 may play a different role in intact tetramers. We propose that CD39 uses different ATPase and ADPase mechanisms in different quaternary structure contexts, and that H59 in ACR1 plays a central role specifically in ATP hydrolysis in intact tetramers.     

Stimulation of the weak ATPase activity of human hsp90 by a client protein.

Heat shock protein 90 (Hsp90) is a molecular chaperone involved in the folding and assembly of a limited set of "client" proteins, many of which are involved in signal transduction pathways. In vivo, it is found in complex with additional proteins, including the chaperones Hsp70, Hsp40, Hip and Hop (Hsp-interacting and Hsp-organising proteins, respectively), as well as high molecular mass immunophilins, such as FKBP59, and the small acidic protein p23. The role of these proteins in Hsp90-mediated assembly processes is poorly understood. It is known that ATP binding and hydrolysis are essential for Hsp90 function in vivo and in vitro.Here we show, for the first time, that human Hsp90 has ATPase activity in vitro. The ATPase activity is characterised using a sensitive assay based on a chemically modified form of the phosphate-binding protein from Escherichia coli. Human Hsp90 is a very weak ATPase, its activity is significantly lower than that of the yeast homologue, and it has a half-life of ATP hydrolysis of eight minutes at 37 degrees C. Using a physiological substrate of Hsp90, the ligand-binding domain of the glucocorticoid receptor, we show that this "client" protein can stimulate the ATPase activity up to 200-fold. This effect is highly specific and unfolded or partially folded proteins, which are known to bind to Hsp90, do not affect the ATPase activity. In addition, the peroxisome proliferator-activated receptor, which is related in both sequence and structure to the glucocorticoid receptor but which does not bind Hsp90, has no observable effect on the ATPase activity.We establish the effect of the co-chaperones Hop, FKBP59 and p23 on the basal ATPase activity as well as the client protein-stimulated ATPase activity of human Hsp90. In contrast with the yeast system, human Hop has little effect on the basal rate of ATP hydrolysis but significantly inhibits the client-protein stimulated rate. Similarly, FKBP59 has little effect on the basal rate but stimulates the client-protein stimulated rate further. In contrast, p23 inhibits both the basal and stimulated rates of ATP hydrolysis.Our results show that the ATPase activity of human Hsp90 is highly regulated by both client protein and co-chaperone binding. We suggest that the rate of ATP hydrolysis is critical to the mode of action of Hsp90, consistent with results that have shown that both over and under-active ATPase mutants of yeast Hsp90 have impaired function in vivo. We suggest that the tight regulation of the ATPase activity of Hsp90 is important and allows the client protein to remain bound to Hsp90 for sufficient time for activation to occur.     

Apyrase from pea stems: Isolation, purification, characterization and identification of a NTPase from the cytoskeleton fraction of pea stem tissue

The cytoskeleton pellet from the first internode of dark-grown pea stems was disintegrated in a high salt buffer, ultracentrifuged to remove ribosomes and the post-ribosomal supernatant was applied to a heparin affinity column. Significant ATPase activity was present in the cytoskeleton fraction and this was eluted from the column at 0.6–0.7 M KOAc, in the same fractions as a 49-kDa protein (which we called B3). B3 was desalted and further purified by cation exchange column chromatography. Purified B3 catalyzed hydrolysis of ATP, CTP, GTP, TTP, UTP and ADP and thus appears to be an apyrase (ATP diphosphohydrolase, EC 3.6.1.5). Partial amino acid sequences of three major fragments were obtained by digestion of B3 by Staphylococcus aureus V8 protease (EC 3.4.21.19), and all these sequences were consistent with the previously reported amino acid sequences for pea nucleoside triphosphatase (NTPase, EC 3.6.1.15) (PIR S48859), which is thought to be an apyrase.     

Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.     

A microassay for ATPase

A newly developed microtechnique for quantitating activity of myosin ATPase (EC 3.6.1.32) is more sensitive and less time-consuming than existing spectrophotometric methods. Measurement of ATPase activity using the new method can be accomplished in a final volume of 0.25 ml, allowing the assay to be conducted in individual wells of 96-well microplates commonly used for the enzyme-linked immunosorbent assay (ELISA). The microassay is performed by adding purified myosin to microplate wells followed by addition of ATP to initiate the enzymatic reaction. The reaction is subsequently terminated by addition of an acidic solution containing malachite green and ammonium molybdate. The level of inorganic phosphate produced by enzymatic hydrolysis of ATP is measured by scanning the microplates using a microELISA plate reader. An entire 96-well microplate can be scanned in less than 2 min, and data from the microassay can be transferred directly to a microprocessor for statistical analysis. The microassay is capable of detecting between 0.2 and 3 nmol of inorganic phosphate in a reaction volume of 50 microliter, and the ATPase activity of as little as 10 ng of rat cardiac myosin can be measured. The increased sensitivity compared with that of other spectrophotometric assays and ease of performing the microassay enable a detailed analysis of the enzymatic properties of cardiac myosin to be conducted on large numbers of small tissue specimens. Several kinetic properties of rat cardiac myosin were determined using this technique.     

Salivary apyrase of Rhodnius prolixus. Kinetics and purification.

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.     

Gadolinium Inhibition of Ecto-Nucleoside Triphosphate Diphosphohydrolase Activity in Torpedo Electric Organ

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) are widely expressed enzymes implicated in the modulation of nucleotide cell signaling. They dephosphorylate either ATP or ADP in the presence of divalent cations, and efforts have been made to identify efficient inhibitors. E-NTPDase activity has been described in Torpedo electric organ electrocytes. We show here that gadolinium, an established blocker of stretch-activated channels, efficiently inhibits E-NTPDase activity of Torpedo electric organ (Ki = 3 microM for ATPase) as well as apyrase from potato tuber, frequently used in inhibition experiments. To our knowledge, gadolinium is the most potent inhibitor described to date for both membrane-bound and soluble E-NTPDases.     

Cross-immunoreactivity between anti-potato apyrase antibodies and mammalian ATP diphosphohydrolases: potential use of the vegetal protein in experimental schistosomiasis

We have previously showed that Schistosoma mansoni ATP-diphosphohydrolase and Solanum tuberosum potato apyrase share epitopes and the vegetable protein has immunostimulatory properties. Here, it was verified the in situ cross-immunoreactivity between mice NTPDases and anti-potato apyrase antibodies produced in rabbits, using confocal microscopy. Liver samples were taken from Swiss Webster mouse 8 weeks after infection with S. mansoni cercariae, and anti-potato apyrase and TRITC-conjugated anti-rabbit IgG antibody were tested on cryostat sections. The results showed that S. mansoni egg ATP diphosphohydrolase isoforms, developed by anti-potato apyrase, are expressed in miracidial and egg structures, and not in granulomatous cells and hepatic structures (hepatocytes, bile ducts, and blood vessels). Therefore, purified potato apyrase when inoculated in rabbit generates polyclonal sera containing anti-apyrase antibodies that are capable of recognizing specifically S. mansoni ATP diphosphohydrolase epitopes, but not proteins from mammalian tissues, suggesting that autoantibodies are not induced during potato apyrase immunization. A phylogenetic tree obtained for the NTPDase family showed that potato apyrase had lower homology with mammalian NTPDases 1-4, 7, and 8. Further analysis of potato apyrase epitopes could implement their potential use in schistosomiasis experimental models.     

Characterization of an ATP diphosphohydrolase activity (APYRASE,EC 3.6.1.5) in rat blood platelets

In the present report we describe an apyrase (ATP diphosphohydrolase, EC 3.6.1.5) in rat blood platelets. The enzyme hydrolyses almost identically quite different nucleoside di- and triphosphates. The calcium dependence and pH requirement were the same for the hydrolysis of ATP and ADP and the apparent Km values were similar for both Ca²⁺-ATP and Ca²⁺-ADP as substrates. Ca²⁺-ATP and Ca²⁺-ADP hydrolysis could not be attributed to the combined action of different enzymes because adenylate kinase, inorganic pyrophosphatase and nonspecific phosphatases were not detected under our assay conditions. The Ca²⁺-ATPase and Ca²⁺-ADPase activity was insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors, thus excluding these enzymes as contaminants. The results demonstrate that rat blood platelets contain an ATP diphosphohydrolase involved in the hydrolysis of ATP and ADP which are vasoactive and platelet active adenine nucleotides.     

The Na(+)-translocating F₁F₀-ATPase from the halophilic, alkalithermophile Natranaerobius thermophilus

Natranaerobius thermophilus is an unusual anaerobic extremophile, it is halophilic and alkalithermophilic; growing optimally at 3.3-3.9M Na(+), pH(50°C) 9.5 and 53°C. The ATPase of N. thermophilus was characterized at the biochemical level to ascertain its role in life under hypersaline, alkaline, thermal conditions. The partially purified enzyme (10-fold purification) displayed the typical subunit pattern for F-type ATPases, with a 5-subunit F(1) portion and 3-subunit-F(O) portion. ATP hydrolysis by the purified ATPase was stimulated almost 4-fold by low concentrations of Na(+) (5mM); hydrolysis activity was inhibited by higher Na(+) concentrations. Partially purified ATPase was alkaliphilic and thermophilic, showing maximal hydrolysis at 47°C and the alkaline pH(50°C) of 9.3. ATP hydrolysis was sensitive to the F-type ATPase inhibitor N,N'-dicylohexylcarbodiimide and exhibited inhibition by both free Mg(2+) and free ATP. ATP synthesis by inverted membrane vesicles proceeded slowly and was driven by a Na(+)-ion gradient that was sensitive to the Na(+)-ionophore monensin. Analysis of the atp operon showed the presence of the Na(+)-binding motif in the c subunit (Q(33), E(66), T(67), T(68), Y(71)), and a complete, untruncated ε subunit; suggesting that ATP hydrolysis by the enzyme is regulated. Based on these properties, the F(1)F(O)-ATPase of N. thermophilus is a Na(+)-translocating ATPase used primarily for expelling cytoplasmic Na(+) that accumulates inside cells of N. thermophilus during alkaline stress. In support of this theory are the presence of the c subunit Na(+)-binding motif and the low rates of ATP synthesis observed. The complete ε subunit is hypothesized to control excessive ATP hydrolysis and preserve intracellular Na(+) needed by electrogenic cation/proton antiporters crucial for cytoplasmic acidification in the obligately alkaliphilic N. thermophilus.     

Use of helper enzymes for ADP removal in infrared spectroscopic experiments: application to Ca2+-ATPase

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca(2+)-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free P(i), which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca(2)E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca(2)E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.