The myosin converter domain modulates muscle performance.

Myosin is the molecular motor that powers muscle contraction as a result of conformational changes during its mechanochemical cycle. We demonstrate that the converter, a compact structural domain that differs in sequence between Drosophila melanogaster myosin isoforms, dramatically influences the kinetic properties of myosin and muscle fibres. Transgenic replacement of the converter in the fast indirect flight muscle with the converter from an embryonic muscle slowed muscle kinetics, forcing a compensatory reduction in wing beat frequency to sustain flight. Conversely, replacing the embryonic converter with the flight muscle converter sped up muscle kinetics and increased maximum power twofold, compared to flight muscles expressing the embryonic myosin isoform. The substitutions also dramatically influenced in vitro actin sliding velocity, suggesting that the converter modulates a rate-limiting step preceding cross-bridge detachment. Our integrative analysis demonstrates that isoform-specific differences in the myosin converter allow different muscle types to meet their specific locomotion demands.

The GDA1_CD39 superfamily: NTPDases with diverse functions.

The first comprehensive review of the ubiquitous "ecto-ATPases" by Plesner was published in 1995. A year later, a lymphoid cell activation antigen, CD39, that had been cloned previously, was shown to be an ecto-ATPase. A family of proteins, related to CD39 and a yeast GDPase, all containing the canonical apyrase conserved regions in their polypeptides, soon started to expand. They are now recognized as members of the GDA1_CD39 protein family. Because proteins in this family hydrolyze nucleoside triphosphates and diphosphates, a unifying nomenclature, nucleoside triphosphate diphopshohydrolases (NTPDases), was established in 2000. Membrane-bound NTPDases are either located on the cell surface or membranes of intracellular organelles. Soluble NTPDases exist in the cytosol and may be secreted. In the last 15 years, molecular cloning and functional expression have facilitated biochemical characterization of NTPDases of many organisms, culminating in the recent structural determination of the ecto-domain of a mammalian cell surface NTPDase and a bacterial NTPDase. The first goal of this review is to summarize the biochemical, mutagenesis, and structural studies of the NTPDases. Because of their ability in hydrolyzing extracellular nucleotides, the mammalian cell surface NTPDases (the ecto-NTPDases) which regulate purinergic signaling have received the most attention. Less appreciated are the functions of intracellular NTPDases and NTPDases of other organisms, e.g., bacteria, parasites, Drosophila, plants, etc. The second goal of this review is to summarize recent findings which demonstrate the involvement of the NTPDases in multiple and diverse physiological processes: pathogen-host interaction, plant growth, eukaryote cell protein and lipid glycosylation, eye development, and oncogenesis.

The stability of chicken nucleoside triphosphate diphosphohydrolase 8 requires both of its transmembrane domains.

Chicken nucleoside triphosphate diphosphohydrolase 8 (NTPDase8) is a cell surface ectonucleotidase with a large extracellular domain (ECD) containing the active site and is anchored to the membrane by two transmembrane domains (TMDs) at the N- and C-termini. Unlike other cell surface NTPDases that have been characterized, the chicken NTPDase8 is not susceptible to substrate inactivation or agents that cause membrane perturbation. To determine if the stability of the enzyme is inherent in its ECD, the cDNA construct of the soluble chicken NTPDase8 was expressed and the protein purified. The ATPase activity of the purified soluble chicken NTPDase8 was less than 15% of that of the purified full-length enzyme. Strikingly, in contrast to the membrane-bound enzyme, the activity of the soluble chicken NTPDase8 decreased with time in a temperature-dependent manner as a result of inactivation by ATP, ADP, and P(i). Truncated mutants in which the ECD is anchored by a single TMD at either the N- or the C-terminus by the native chicken NTPDase TMDs or a TMD from a different NTPDase, human NTPDase2, also displayed a nonlinear time course of ATP hydrolysis. While removal of the N- or C-terminal TMD affected protein expression differently, the truncated mutants were generally similar to the soluble chicken NTPDase8 with respect to ATP, ADP, and P(i) inactivation. Other biochemical characteristics, e.g., ATPase/ADPase ratios, inhibition by azide, and affinity for ATP, were also altered when one or both of the TMDs were removed from the chicken NTPDase8. These results indicate that (1) both TMDs of the chicken NTPDase8 are required to maintain stability of the enzyme and maximal catalytic activity and (2) the conformations of the ectodomain in the soluble enzyme and the truncated mutants differ from that of the full-length chicken NTPDase8.

Alternative S2 hinge regions of the myosin rod affect myofibrillar structure and myosin kinetics.

The subfragment 2/light meromyosin "hinge" region has been proposed to significantly contribute to muscle contraction force and/or speed. Transgenic replacement of the endogenous fast muscle isovariant hinge A (exon 15a) in Drosophila melanogaster indirect flight muscle with the slow muscle hinge B (exon 15b) allows examination of the structural and functional changes when only this region of the myosin molecule is different. Hinge B was previously shown to increase myosin rod length, increase A-band and sarcomere length, and decrease flight performance compared to hinge A. We applied additional measures to these transgenic lines to further evaluate the consequences of modifying this hinge region. Structurally, the longer A-band and sarcomere lengths found in the hinge B myofibrils appear to be due to the longitudinal addition of myosin heads. Functionally, hinge B, although a significant distance from the myosin catalytic domain, alters myosin kinetics in a manner consistent with this region increasing myosin rod length. These structural and functional changes combine to decrease whole fly wing-beat frequency and flight performance. Our results indicate that this hinge region plays an important role in determining myosin kinetics and in regulating thick and thin filament lengths as well as sarcomere length.

The single NTPase gene of Drosophila melanogaster encodes an intracellular nucleoside triphosphate diphosphohydrolase 6 (NTPDase6).

I report here the cloning and characterization of a nucleoside triphosphate diphosphohydrolase 6 (NTPDase6) encoded by the single Dmel/NTPase gene of Drosophila melanogaster. S2 cells stably transfected with the Drosophila NTPDase6 cDNA displayed strong UDPase activity only after addition of NP-40, indicating the intracellular location of the enzyme. The enzyme hydrolyzed UDP, GDP, and IDP equally well whereas other NDP and NTP were poor substrates. It was not or only partially inhibited by several modulators of the cell surface NTPDases, but was strongly inhibited upon oxidative cross-linking by copper phenanthroline. The decrease of activity correlated with dimer formation. Mutagenesis studies indicated that dimer formation required C42 in the transmembrane domain and C447 in the exoplasmic domain. Fluorescence microscopy revealed that the protein was located primarily in the ER. The substrate specificity and cellular localization of the Drosophila NTPDase6 suggest that it participates in Drosophila glycoprotein processing.

Inhibition of human NTPDase 2 by modification of an intramembrane cysteine by p-chloromercuriphenylsulfonate and oxidative cross-linking of the transmembrane domains.

Human NTPDase 2 is a cell surface integral membrane glycoprotein that is anchored to the membranes by two transmembrane domains while the bulk of the protein containing the active site faces the extracellular milieu. It contains 10 conserved cysteine residues in the extracellular domain that are involved in disulfide bond formation and one free cysteine residue, C26, which is located in the N-terminal transmembrane domain. The human NTPDase 2 activity is inactivated by membrane perturbation that disrupts interaction of the transmembrane domains and is inhibited by p-chloromercuriphenylsulfonate (pCMPS), a sulfhydryl reagent. In this report, we show that C26 is the target of pCMPS modification, since a mutant in which C26 was replaced with a serine was no longer inhibited by pCMPS. Mutants in which cysteine residues are placed in the C-terminal transmembrane domain near the extracellular surface were still modified by pCMPS, but the degree of inhibition of their ATPase activity was lower than that of the wild-type enzyme. Thus, loss of the ATPase activity of human NTPDase 2 in the presence of pCMPS probably results from the disturbance of both transmembrane domain interaction and its active site. Inhibition of human NTPDase 2 activity by pCMPS and membrane perturbation is attenuated when the enzyme is cross-linked by glutaraldehyde. On the other hand, NTPDase 2 dimers formed from oxidative cross-linking of the wild-type enzyme and mutants containing a single cysteine residue in the C-terminal transmembrane domain displayed reduced ATPase activity. A similar reduction in activity was also obtained upon intramolecular disulfide formation in mutants that contain a cysteine residue in each of the two transmembrane domains. These results indicate that the mobility of the transmembrane helices is necessary for maximal catalysis.

Transmembrane domain interactions affect the stability of the extracellular domain of the human NTPDase 2.

Human NTPDase2 and chicken NTPDase8 are cell surface nucleotidases that contain two transmembrane domains (TMD) and five apyrase conserved regions (ACRs). ACR1 is located near the N-terminal TMD whereas ACR5 is located near the C-terminal TMD. The human NTPDase2 activity is decreased by low concentration of NP-40 and at temperatures higher than 37 degrees C, and undergoes substrate inactivation, whereas the chicken NTPDase8 activity is not. When freed from membrane anchorage, the soluble human NTPDase2 is no longer inactivated by detergents, high temperature, and substrate. These characteristics are retained in the hu-ck ACR1,5 chimera in which the extracellular domain is anchored to the membrane by the two TMDs of the chicken NTPDase8. The hu-ck ACR1,5 chimera is the first chimeric NTPDase reported that shows a resistance to membrane perturbation and substrate inactivation. Our results indicate that the strengths of interaction of the respective TMD pairs of the human NTPDase2 and chicken NTPDase8 determine their different responses to membrane perturbation and substrate.

An alternative domain near the nucleotide-binding site of Drosophila muscle myosin affects ATPase kinetics.

In Drosophila melanogaster expression of muscle myosin heavy chain isoforms occurs by alternative splicing of transcripts from a single gene. The exon 7 domain is one of four variable regions in the catalytic head and is located near the nucleotide-binding site. To ascribe a functional role to this domain, we created two chimeric myosin isoforms (indirect flight isoform-exon 7a and embryonic-exon 7d) that differ from the native indirect flight muscle and embryonic body-wall muscle isoforms only in the exon 7 region. Germline transformation and subsequent expression of the chimeric myosins in the indirect flight muscle of myosin-null Drosophila allowed us to purify the myosin for in vitro studies and to assess in vivo structure and function of transgenic muscles. Intriguingly, in vitro experiments show the exon 7 domain modulates myosin ATPase activity but has no effect on actin filament velocity, a novel result compared to similar studies with other Drosophila variable exons. Transgenic flies expressing the indirect flight isoform-exon 7a have normal indirect flight muscle structure, and flight and jump ability. However, expression of the embryonic-exon 7d chimeric isoform yields flightless flies that show improvements in both the structural stability of the indirect flight muscle and in locomotor abilities as compared to flies expressing the embryonic isoform. Overall, our results suggest the exon 7 domain participates in the regulation of the attachment of myosin to actin in order to fine-tune the physiological properties of Drosophila myosin isoforms.

Extracellular ATP and adenosine induce cell apoptosis of human hepatoma Li-7A cells via the A3 adenosine receptor.

1. Extracellular ATP is a potent signaling molecule that modulates a myriad of cellular functions through the activation of P2 purinergic receptors and is cytotoxic to a variety of cells at higher concentrations. The mechanism of ATP-elicited cytotoxicity is not fully understood. In this study, we investigated the effect of extracellular ATP on the human hepatoma Li-7A cells. 2. We observed a time- and dose-dependent growth inhibition of Li-7A cells by ATP, which is accompanied by an increase in the active form of caspase-3 as well as increased cleavage of its substrate, poly (ADP-ribose) polymerase. The cytotoxic effect of extracellular ATP was not mediated by the P2X7 receptor, since (1).the effect was not abolished by the P2X7 receptor antagonists oxidized ATP and KN-62, and (2).extracellular ADP, AMP, and adenosine were also cytotoxic. 3. We found that ATP and ADP were degraded to adenosine by Li-7A cells and that treatment of Li-7A cells by adenosine resulted in growth inhibition and caspase-3 activation, indicating that adenosine is the apoptotic agent. Using adenosine receptor agonists and antagonists, as well as inhibitors of adenosine transport and deamination, we showed that the cytotoxic effect of adenosine is specifically mediated by the A3 receptor even though transcripts of A1, A2A, A2B, and a splice variant of the P2X7 receptors were detected in Li-7A cells by RT-PCR. 4. Cytotoxicity caused by exogenous ATP and adenosine was completely abolished by the caspase-3 inhibitor Z-DEVD-FMK, demonstrating the central role of caspase-3 in apoptosis of Li-7A cells.

Ectonucleotidase NTPDase2 is selectively down-regulated in biliary cirrhosis.

BACKGROUND: Portal fibroblasts are newly identified, potentially fibrogenic liver cells that are distinct from hepatic stellate cells. The ectonucleotidase* nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) is restricted to portal fibroblasts in the normal liver. However, the fate of NTPDase2 after bile duct ligation (BDL) is unknown. AIMS: The aim of this study was to assess the effect of experimental rat and disease-mediated human biliary cirrhosis on NTPDase2 expression in the liver. METHODS: Cirrhosis was induced in experimental rats via BDL and carbon tetrachloride (CCl4) administration. Archived human liver biopsy specimens from normal liver, primary biliary cirrhosis, or hepatitis C cirrhosis were examined. Changes in expression of NTPDase2 were determined using confocal immunofluorescence, immunoblot, and real-time polymerase chain reaction. RESULTS: Confocal immunofluorescence demonstrated a decrease in NTPDase2 expression after BDL. Immunoblot and real-time polymerase chain reaction demonstrated a decrease in NTPDase2 expression by portal fibroblasts after BDL. No decrease in NTPDase2 protein was noted after CCl4 administration, and NTPDase2 messenger ribonucleic acid was markedly up-regulated after CCl4 administration. Confocal immunofluorescence demonstrated a shift of NTPDase2 expression from portal areas to central areas that colocalized with alpha-smooth muscle actin after CCl4 administration. In human biopsy specimens, NTPDase2 expression was lost in cirrhosis owing to primary biliary cirrhosis, whereas NTPDase2 expression was shifted to bridging fibrous bands in cirrhosis owing to hepatitis C. CONCLUSIONS: Loss of NTPDase2 is a common pathway in both rat and human manifestations of biliary cirrhosis. Conversely, in non-biliary cirrhosis, NTPDase2 is shifted from the portal area to bridging fibrous bands. Elucidations of the mechanisms regulating NTPDase2 expression may lead to new therapeutic approaches to fibrotic liver disease.

Development of a microbioreactor with ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) immobilized on a polyacrylamide-coated capillary at the outlet.

A simple and fast method of immobilization of cell membrane suspension containing human ecto-nucleoside triphosphate diphosphohydrolase 2 (NTPDase2) on a polyacrylamide-coated capillary was developed. The enzyme microbioreactor was prepared by hydrodynamic injection of a small plug of the polycationic electrolyte hexadimethrine bromide (HDB) followed by a suspension of an enzyme-containing membrane preparation. In order to shorten the enzyme assay time and to increase the throughput of the assay, the capillary was coated from the outlet end and all injections were performed from the outlet end of the capillary. For the monitoring of the enzymatic reaction, the substrate ATP dissolved in reaction buffer (140 mM NaCl, 5mM KCl, 1mM MgCl(2), 2mM CaCl(2), and 10mM Hepes, pH 7.4, internal standard: 10 microM UMP) in the absence or presence of inhibitor was injected electrokinetically and incubated in the microbioreactor for 1 min with 1 kV of applied voltage. Then, the electrophoretic separation of the reaction products was initiated by applying a constant current of 60 microA. A 50mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by UV absorbance at 260 nm. The new method was compared with an at-capillary-inlet method without immobilization of the enzyme. The results (K(m) values, K(i) values for inhibitor) obtained with both methods were similar and comparable with literature data. The developed outlet immobilized enzyme microreactor using a coated capillary is very fast, simple and most economic allowing multiple use of the enzyme.

Alternative exon 9-encoded relay domains affect more than one communication pathway in the Drosophila myosin head.

We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide-binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced actomyosin dissociation rate constant (K(1)k(+2)) and ADP affinity (K(AD)), measured for the chimeric constructs IFI-9b and EMB-9a, compared to wild-type IFI and EMB values. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide-binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50-kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of actomyosin cross-bridge kinetics.

Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.

Alternative relay domains of Drosophila melanogaster myosin differentially affect ATPase activity, in vitro motility, myofibril structure and muscle function.

The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.

Selective nucleoside triphosphate diphosphohydrolase-2 (NTPDase2) inhibitors: nucleotide mimetics derived from uridine-5'-carboxamide.

Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases, subtypes 1, 2, 3, 8 of NTPDases) dephosphorylate nucleoside tri- and diphosphates to the corresponding di- and monophosphates. In the present study we synthesized adenine and uracil nucleotide mimetics, in which the phosphate residues were replaced by phosphonic acid esters attached to the nucleoside at the 5'-position by amide linkers. Among the synthesized uridine derivatives, we identified the first potent and selective inhibitors of human NTPDase2. The most potent compound was 19a (PSB-6426), which was a competitive inhibitor of NTPDase2 exhibiting a K i value of 8.2 microM and selectivity versus other NTPDases. It was inactive toward uracil nucleotide-activated P2Y 2, P2Y 4, and P2Y 6 receptor subtypes. Compound 19a was chemically and metabolically highly stable. In contrast to the few known (unselective) NTPDase inhibitors, 19a is an uncharged molecule and may be perorally bioavailable. NTPDase2 inhibitors have potential as novel cardioprotective drugs for the treatment of stroke and for cancer therapy.

Mutagenesis of lysine 62, asparagine 64, and conserved region 1 reduces the activity of human ecto-ATPase (NTPDase 2).

The human ecto-ATPase (NTPDase 2) contains conserved motifs including five apyrase conserved regions (ACRs) and four conserved regions (CRs) as well as conserved lysine and arginine residues that are also present in other cell surface E-NTPDases. Some of the positively charged amino acids may be involved in ATP binding. The protein also contains six potential N-linked glycosylation sites. Results obtained with seven lysine and six arginine mutants indicate the importance of K62 that is located in CR1, K182, which is downstream of ACR3, and R155, which immediately follows CR3. Mutation of asparagine at the six potential N-linked glycosylation sites individually to glutamine established the importance of N64 in CR1 and N443 in ACR5 in protein function and expression. Mutation of N64, which is conserved in all cell surface NTPDases, results in the expression of an unstable protein, the activity of which is only manifested in the presence of concanavalin A. Both K62 and N64 reside in CR1 that is conserved in all cell surface NTPDases. In the sequence of the CR1 of human ecto-ATPase, 58WPADKENDTGIV69, 65DTG67 is similar to the phosphate-binding motif (DXG) in ACR1 and 4. The D65A and G67A mutants have reduced protein expression and activity. Mutations of other residues in CR1 to alanine led to partial to complete loss of protein expression and activity except for P59. The alanine mutants of the three acidic amino acid residues, D61, E63, and D65, all have decreased affinity for divalent ions. D61 can be substituted by glutamate, but E63 appears to be invariable. Taken together, these results indicate that CR1, which follows ACR1 in the cell surface NTPDases, is an essential structural element in these enzymes.

Molecular cloning and characterization of expressed human ecto-nucleoside triphosphate diphosphohydrolase 8 (E-NTPDase 8) and its soluble extracellular domain.

An ecto-nucleoside triphosphate diphosphohydrolase (ecto-NTPDase) has been cloned from human liver RNA by RT-PCR. The 1.5 kb cDNA codes for a protein of 495 amino acids. Sequence analysis indicated that it is most closely related to a chicken ecto-ATPDase previously cloned in our laboratory [Knowles et al. (2002) Eur. J. Biochem. 269, 2373-2382] and a mouse homologue that has been designated as E-NTPDase 8 [Bigonnesses et al. (2004) Biochemistry 43, 5511-5519]. The human E-NTPDase 8 has similar topology as the avian and mouse E-NTPDase 8 but has fewer potential N-glycosylation sites and only two amino acid residues in the cytoplasm at its C-terminus. Despite 52% identity in primary structures, enzymatic properties of human E-NTPDase 8 expressed in HEK293 cells differ from that of the chicken E-NTPDase 8. In contrast to the chicken E-NTPDase 8, the human E-NTPDase 8 hydrolyzes MgADP poorly and is inhibited by several detergents as well as benzyl alcohol; the latter attribute may be related to weaker interaction of the transmembranous domains of the human E-NTPDase 8. To demonstrate that inhibition by detergents is mediated by the transmembranous domains, a recombinant pSecTag2 plasmid containing the extracellular domain (ECD) of the human E-NTPDase 8 was constructed. The soluble human E-NTPDase 8 which was secreted into the culture media of transfected HEK293 cells was purified by ammonium sulfate fractionation and nickel affinity chromatography. Besides becoming resistant to detergent inhibition, the soluble human E-NTPDase 8 ECD displays greater activity with Ca nucleotide substrates, an increased affinity for ATP, different pH dependence, and a decreased sensitivity to azide inhibition when compared to the membrane-bound enzyme. These differences may result from the different conformations that the ECD assume without or with constraints exerted by the transmembranous domains. These results indicate that the transmembranous domains are important in regulating enzyme activity as well as in determining the structure of human E-NTPDase 8.

Either the carboxyl- or the amino-terminal region of the human ecto-ATPase (E-NTPDase 2) confers detergent and temperature sensitivity to the chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8).

Human ecto-ATPase (E-NTPDase 2) and chicken ecto-ATP-diphosphohydrolase (E-NTPDase 8) are cell surface nucleotidases with two transmembranous domains, one each at the N- and C-termini. Hydrolysis of substrates occurs in active sites residing in their extracellular domains. Human ecto-ATPase activity is decreased by NP-40 and at temperatures higher than 37 degrees C. Reduction of activity is abolished by prior cross-linking of the ecto-ATPase by lectin and chemical cross-linking agents [Knowles, A. F., and Chiang, W.-C. (2003) Arch. Biochem. Biophys. 418, 217-227]. In contrast, the chicken ecto-ATP-diphosphohydrolase is not inhibited by NP-40, and activity is approximately 2-fold higher at 55 degrees C. To determine if the transmembranous domains of the two E-NTPDases mediate their respective responses to detergents and high temperature, we first constructed a chimera (ck-hu ACR5) in which the C-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase. While this chimera displays many similar enzymatic characteristics as the parental chicken ecto-ATP-diphosphohydrolase, its inhibition by NP-40, high temperature, and substrate resemble that of the human ecto-ATPase, which donates the C-terminus including the C-terminal transmembranous domain. Additionally, comparison of the effects of ConA, disuccinimidyl suberate, and glutaraldehyde on the parental enzymes and the chimera indicated that catalysis which occurs in the extracellular domains of the two E-NTPDases responds differently to conformational constraints. Enzyme activity of a second chimera (ck-hu ACR1) in which the N-terminus of the chicken ecto-ATP-diphosphohydrolase is substituted by the corresponding region of the human ecto-ATPase is also inhibited by NP-40 and is less active at 55 degrees C; however, its temperature dependence differs from that of ck-hu ACR5. These results indicate that (1) the C- and N-termini of the two E-NTPDases encompassing the two transmembranous domains are important elements in determining the sensitivity of the human ecto-ATPase to NP-40 and high temperatures; (2) incorporation of either the C- or N-terminus of the human ecto-ATPase alone in the chicken ecto-ATP-diphosphohydrolase is sufficient to impart negative regulation on ATP hydrolysis due to membrane perturbation; and (3) interactions of the two sets of heterologous transmembranous domains are not equivalent, which are most likely related to their different amino acid sequences.

Enzymatic and transcriptional regulation of human ecto-ATPase/E-NTPDase 2.

We have characterized the regulation of expressed human ecto-ATPase (E-NTPDase 2), a cell surface integral membrane glycoprotein. Ecto-ATPase activity is inhibited by parameters that decrease membrane protein interaction, i.e., detergents and high temperatures. These inhibitory effects are overcome when membranes are pretreated with concanavalin A or chemical cross-linking agents that increase the amounts of ecto-ATPase oligomers. Cross-linking agents also abrogate substrate inactivation of the ecto-ATPase, a unique characteristic of the enzyme. These effects indicate that the magnitude of negative substrate regulation is dependent on quaternary structures of the protein, which likely involves interaction of transmembrane domains. The importance of transmembrane domains of ecto-ATPase in activity modulation is demonstrated further by the stimulatory effect of digitonin, a steroid glycoside that preferentially interacts with cholesterol in the membranes but does not promote oligomer formation. These results indicate that ecto-ATPase activity is regulated by a multitude of mechanisms, some of which may have physiological significance. Ecto-ATPase is also susceptible to transcriptional regulation. Ecto-ATPase gene expression is increased in a human hepatoma whereas it is undetectable in the normal liver.

Poly(ADP-ribose) polymerase activation and changes in Bax protein expression associated with extracellular ATP-mediated apoptosis in human embryonic kidney 293-P2X7 cells.

Extracellular ATP is a potent signaling factor that modulates a variety of cellular functions through the activation of P2 purinergic receptors. Extracellular ATP at higher concentrations exerts cytostatic as well as cytotoxic effects in a variety of cell systems, the mechanism of which is not fully understood. In this study, we used cultured human embryonic kidney (HEK) cells stably transfected with human P2X(7) receptors (HEK-P2X(7)) to investigate the mechanism of ATP-induced cell death. The cytotoxic effects of ATP in HEK-P2X(7) cells were dose- and time-dependent, whereas ADP, AMP, and UTP had no effect. ATP treatment induced a significant increase in apoptotic HEK-P2X(7) cells as ascertained by the terminal deoxynucleotidyl transferase dUTP nick-end labeling technique and flow cytometry. An ATP-induced decrease in the pro-apoptotic bax gene expression was detected by apoptosis-related cDNA microarray analysis, which correlated with a decrease of Bax protein expression. Western blot analysis revealed that ATP treatment resulted in the processing of pro-caspase 3 to its active form and cleavage of the nuclear enzyme, poly(ADP-ribose) polymerase (PARP). Both ATP-induced molecular alterations in HEK-P2X(7) cells (i.e., decrease of Bax expression and increase of PARP cleavage) were blocked by the purinergic P2X(7) receptor antagonist oxidized ATP. In conclusion, we demonstrated the importance of the P2X(7) receptor in ATP induced cell death of HEK-P2X(7) cells, which seems to be independent of bax expression; however, the activation of caspases is required.