Who will be lost? Identifying patients at risk of loss to follow-up in Malawi. The DREAM Program Experience.

OBJECTIVES: Retention of subjects in HIV treatment programmes is crucial for the success of treatment. We evaluated retention/loss to follow-up (LTFU) in subjects receiving established care in Malawi. METHODS: Data for HIV-positive patients registered in Drug Resource Enhancement Against AIDS and Malnutrition centres in Malawi prior to 2014 were reviewed. Visits entailing HIV testing/counselling, laboratory evaluations, nutritional evaluation/supplementation, community support, peer education, and antiretroviral (ART) monitoring/pharmacy were noted. LTFU was defined as > 90 days without an encounter. Parameters potentially associated with LTFU were explored, with univariate/multivariate logistic regression analyses being performed. RESULTS: Fifteen thousand and ninety-nine patients registered before 2014; 202 (1.3%) were lost to follow-up (LTFU) (1.3%). Nine (0.5%) of 1744 paediatric patients were LTFU vs. 1.4% (n = 193) of 13 355 adults (P < 0.001). Subjects who were LTFU had fewer days in care than retained subjects (1338 vs. 1544, respectively; P < 0.001) and a longer duration of ART (1530 vs. 1300 days, respectively; P < 0.001). Subjects who were LTFU had higher baseline HIV viral loads (P = 0.016) and higher body mass indexes (P < 0.001), were more likely to live in urban settings (88% of patients who were LTFU lived in urban settings) with better housing [relative risk (RR) 2.3; 95% confidence interval (CI) 1.67-3.09; P < 0.001], and were more likely to be educated (RR 1.88; 95% CI 1.42-2.50; P < 0.001). Distance to the centre and cost of transportation were associated with LTFU (RR 3.4; 95% CI 2.84-5.37; P < 0.001), as was absence of a maternal figure (RR 1.57; 95% CI 1.17-2.09; P < 0.001). Viral load, distance index, education and a maternal figure were predictive of LTFU. CONCLUSIONS: Educated, urbanized HIV-infected adults living far from programme centres are at high risk of LTFU, particularly if there is no maternal figure in the household. These variables must be taken into consideration when developing retention strategies.

Modulation of neuronal plasticity following chronic concomitant administration of the novel antipsychotic lurasidone with the mood stabilizer valproic acid.

RATIONALE: Combinatory therapy is widely used in psychiatry owing to the possibility that drugs with different mechanisms of action may synergize to improve functions deteriorated in schizophrenia, bipolar disorders, and major depression. While combinatory strategies rely on receptor and synaptic mechanisms, it should also be considered that two drugs may also "interact" on the long-term to determine more robust changes in neuronal plasticity, which represents a downstream target important for functional recovery. OBJECTIVE: The aim of the study is to investigate neuroadaptive changes set in motion by chronic concomitant administration of the novel antipsychotic lurasidone and the mood stabilizer valproate. METHODS: Animals were chronically treated with lurasidone, valproate, or the combination of the two drugs and killed 24 h after the last injection to evaluate alterations of different measures of neuronal plasticity such as the neurotrophin brain-derived neurotrophic factor (BDNF), the immediate early gene Activity-regulated cytoskeletal associated protein, and the epigenetic regulators HDAC 1, 2, and 5 in dorsal and ventral hippocampus. RESULTS: The results suggest that coadministration of lurasidone and valproate produces, when compared to the single drugs, a larger increase in the expression of BDNF in the ventral hippocampus, through the regulation of specific neurotrophin transcripts. We also found that the histone deacetylases were regulated by the drug combination, suggesting that some of the transcriptional changes may be sustained by epigenetic mechanisms. CONCLUSIONS: Our results suggest that the beneficial effects associated with combinatory treatment between a second-generation antipsychotic and a mood stabilizer could result from the ability to modulate neuroplastic molecules, whose expression and function is deteriorated in different psychiatric conditions.

Seroprevalence of HHV8 in a cohort of HIV-negative and HIV-positive patients in Mozambique.

Kaposi Sarcoma shows several different clinical and epidemiological patterns. In Sub-Saharan Africa, where the HIV achieves an high prevalence of infection, the KS can be found both in HIV positive than in HIV negative patients, and the diffusion of the HHV8 virus is endemic. The aim of the work is to evaluate the HHV8 seroprevalence in Mozambique. Moreover the relationship of some main indicators, as CD4 and CD8 cells count, HIV viral load, Body Mass Index and haemoglobin values have been calculated in a part of the DREAM Cohort, (HIV positive patients enrolled in the Community of Sant'Egidio program to fight AIDS in the Sub-Saharan Africa). In the HIV positive cohort HHV8 negative and HHV8 positive groups show statistical significance (p < 0.05) in CD4 cells count, a strong significance (p = 0.01) in CD8 cells count and a significance also in Haemoglobin levels (p = 0.35). The difference in Haemoglobin levels (0.5 g/dl) is related more to a statistical than a clinical significance. The study confirms the free circulation of the HHV8 virus in the Mozambican population, with a prevalence rate of 51.1%, similar than that measured in bordering countries. Considering the CD8 value within the HIV positive sub-cohort a strong correlation with the positivity for HHV8 and the immunological status is suggested.

Safety of nevirapine-containing antiretroviral triple therapy regimens to prevent vertical transmission in an African cohort of HIV-1-infected pregnant women.

OBJECTIVE: To assess the incidence and consequences of adverse reactions among African HIV-positive pregnant women treated with fixed-dose combinations of a nevirapine-containing antiretroviral (ARV) triple therapy. METHODS: A retrospective analysis of the clinical files of 703 HIV-1-positive pregnant women treated with a nevirapine-containing regimen between May 2002 and July 2004 was conducted. Selection criteria for inclusion in the analysis were: (a) taking ARV for more than 14 days; (b) baseline values of transaminases below the threshold of 2.5 times the upper limit of normal (ULN). The women were on a nevirapine-containing regimen for a median of 127 days [interquartile range (IQR) 86-190 days], starting on average at the 27th week of gestation (standard deviation+/-9.5) and continuing up to a maximum of 6 months after delivery. All women were offered formula milk to feed the babies. Highly active antiretroviral therapy (HAART) was continued beyond 6 months only if the patient qualified on the first visit. The main outcome measures were incidence of hepatotoxicity, skin rashes and Stevens-Johnson syndrome. Multivariate analysis to assess the impact of several factors on the adverse reaction rate was performed. RESULTS: As of 1 August 2004, 554 pregnancies reached term, 96 women were still pregnant, and 53 women dropped out of the programme before giving birth. After 2 months of therapy the percentage of patients with a viral load less than 1000 HIV-1 RNA copies/mL increased to 78.6%; average CD4 cell counts increased from 490 cells/microL before therapy to 630 after therapy. The incidence of grade 3-4 adverse reactions (hepatotoxicity, skin rashes and Stevens-Johnson syndrome) was 6.5, 2.4 and 1.1%, respectively. Five women died during pregnancy (0.88%). Only one of the deaths could be associated with ARV treatment. CONCLUSION: Nevirapine-containing regimens in pregnant woman, at all CD4 cell count levels, appear to be safe in African settings.

Pediatric highly active antiretroviral therapy in Mozambique: an integrated model of care.

AIM: To test the impact of a public health model to implement HIV pediatric care in Limited Resource Settings. METHODS: A retrospective study on the clinical files of 679 Mozambican children (mean age 4.4 years; SD 3.2), has been carried out. The pediatric patients received HAART (Highly Active Anti-Retroviral Therapy) in the framework of DREAM, a nationwide public health program offering an integrated model of care to HIV patients including free-of-charge HAART and monitoring, nutritional assessment and supplementation, peer-to-peer education, active tracing of the dropped out patients. RESULTS: HAART was started in 297 subjects out of 679. The median time of treatment was 286 (IQ 25-75:125-465). Mortality rate was lower in the sub-sample receiving HAART (8.4%; CI 95%: 5.2-11.6 vs 13.1%; CI 95%: 9.7-16.5). After 6 months of treatment the percentage of viral load lower than 400 copies/mL rose from 4.9% to 46.3%. The percentage of patients with less than 15% of CD4 cells out of the total lymphocyte count and the percentage of patients below the 2 z-score decreased from 56.4% and 58.1% to 8.8% and 38.1% respectively. CONCLUSIONS: Pediatric HAART in limited resource settings often face difficulties to handle complex treatment schemes, but the implemented model seems to be an effective tool to reduce mortality rate in HIV positive pediatric population.

Improving adherence to highly active anti-retroviral therapy in Africa: the DREAM programme in Mozambique.

Ensuring high levels of adherence to highly active anti-retroviral therapy (HAART) is a priority in treating people living with AIDS. This study reports the rates of adherence of patients served by DREAM (Drug Resource Enhancement against AIDS and Malnutrition) in the city of Matola, Mozambique. DREAM, an innovative programme tailored for Africa, was implemented by the Community of Sant'Egidio in August 2001. DREAM provides patients with anti-retroviral drugs and laboratory tests at no charge, and is based on a particular strategy of health education and organization of services designed for a population that is predominantly poor and has a low level of formal education. This study analyzes the adherence of 154 patients over a period of 6 months. In evaluating adherence, two indicators were used: (1) the percentage of appointments kept for check-ups, tests and the collection of medicine, and (2) the overall change in the patients' blood chemistry over the 6-month period. Of the 154 patients, 127 (82.5%) kept more than 90% of their appointments. Adherence to the programme was further confirmed by a relevant increase of hemoglobin levels and CD4 counts, and a significant decrease in the viral loads among the 154 patients.

Cell size reduction induced by inhibition of the mTOR/S6K-signaling pathway protects Jurkat cells from apoptosis.

In Jurkat cells, the decreased cell growth rate associated with a long-lasting deactivation of the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (S6K)-signaling pathway generates a cell population of progressively reduced cellular mass and size. When promoted by rapamycin as prototype inhibitor, the mTOR deactivation-dependent cell size reduction was associated with slowed, but not suppressed, proliferation. Small-size cells were significantly protected from apoptosis induced by Fas/Apo-1 death-receptor activation (as shown by reduced procaspase cleavage and decreased catalytic activity of relevant caspases) or by stress signals-dependent mitochondrial perturbation (as shown by reduced cleavage of caspase-2, lower dissipation of mitochondrial membrane potential and decreased release of cytochorome c and apoptosis-inducing factor from mitochondria). Protection faded when reactivation of the mTOR/S6K pathway promoted the cell recovery to normal size. These results suggest that cells induced to reduce their mass by the mTOR deactivation-dependent inhibition of cell growth become more resilient to lethal assaults by curbing the cell's suicidal response.

Stress-induced apoptosis: toward a symmetry with receptor-mediated cell death.

Apoptosis is a form of programmed cell death executed by caspases activated along signalling pathways initiated by ligation of cell-surface death receptors ( extrinsic pathway ) or by perturbation of the mithocondrial membrane promoted by physical or chemical stress agents ( intrinsic pathway ). In metazoans, this evolutionary conserved, genetically controlled process has a role in a variety of physiological settings, as development, homeostasis of tissues and maintenance of the organism integrity. When deranged by impaired regulation or inappropriate activation apoptosis contributes to the pathogenesis of diseases as autoimmunity, cancer, restenosis, ischaemia, heart failure and neurodegenerative disorders. In this review we will present a survey of the stress-induced intrinsic, mithochondrial, pathway and, based on recent experimental data, we will propose a view compatible with an emergent conceptual symmetry between the two apoptogenic extrinsic and intrinsic pathways. Elements of symmetry present in both the apoptogenic signalling pathways include: early activation of initiator caspases (feed-forwarded by a direct or post-mitocondrial effector caspase-mediated amplification loop in some cell types) and mitochondrial membrane permeabilization with required release of antagonists of active caspase inhibitors (IAPs) in high-level IAPs-expressing cells and apoptosome-mediated amplification of the caspase cascade more or less needed in different cell types.

The metal coordination of sCD39 during ATP hydrolysis.

BACKGROUND: The hydrolysis of ATP and ADP by ecto-nucleoside triphosphate diphosphohydrolase 1 (CD39) requires divalent cations, like Ca2+ and Mg2+. In spite of considerable work, it is not clear whether divalent cations bind to the enzyme in the absence of nucleotide or only as nucleotide-Me+2 complex. Here we study the protein ligands for Me+2. RESULTS: When VO2+ was used as a substitute for Ca2+, the ATPase activity of soluble CD39 was 25% of that with Ca2+ as cofactor. Protein ligands of the VO2+-nucleotide complex bound to the catalytic site of soluble CD39 were characterized by electron paramagnetic resonance (EPR) spectroscopy. The EPR spectrum contained one species designated T with VO2+-AMPPNP as ligand. Two species D1 and D2 were observed when VO2+-AMPCP was bound to soluble CD39. The results suggest that species D1 and D2 represent the metal-ADP complexes at the catalytic site of soluble CD39 corresponding to the intermediate formed during ATP hydrolysis and the substrate for further hydrolysis, respectively. CONCLUSIONS: VO2+ can functionally substitute for Ca2+ as a cofactor of sCD39, and it produces four different EPR features when bound in the presence of different nucleotides or in the absence of nucleotide. The metal coordination for each conformation corresponding to each EPR species is proposed, and the mechanism of sCD39 catalysis is discussed.

Glutamine deprivation-mediated cell shrinkage induces ligand-independent CD95 receptor signaling and apoptosis.

Cell shrinkage and loss of cell viability by apoptosis have been examined in cultured CD95(Fas/Apo-1)-expressing leukemia-derived CEM and HL-60 cells subjected to acute deprivation of glutamine, a major compatible osmolyte engaged in cell volume control. Glutamine deprivation-mediated cell shrinkage promoted a ligand-independent activation of the CD95-mediated apoptotic pathway. Cell transfection with plasmids expressing FADD-DN or v-Flip viral proteins pointed to a functional clustering of CD95 receptors at the cell surface with activation of the 'extrinsic pathway' caspase cascade. Accordingly, cell shrinkage did not induce apoptosis in CD95 receptor-negative lymphoma L1210 cells. Replacement of glutamine with surrogate compatible osmolytes counteracted cell volume decrement and protected the CD95-expressing cells from apoptosis. A glutamine deprivation-dependent cell shrinkage with activation of the CD95-mediated pathway was also observed when asparaginase was added to the medium. Asparagine depletion had no role in this process. The cell-size shrinkage-dependent apoptosis induced by glutamine restriction in CD95-expressing leukemic cells may therefore be of clinical relevance in amidohydrolase enzyme therapies.

Batten disease and the control of the Fo subunit c pore by cGMP and calcium.

Subunit c of ATP synthase functions as a high conductance ion channel, tightly regulated by calcium. We have suggested that the pathogenesis of Batten syndromes involving overaccumulation of subunit c are linked to the protein's ion channel function. In normal electrically excitable tissue the channel could act as a pacer setting nodal voltage via control of cation entry. The channel conductance is controlled by voltage, calcium, cyclic nucleotides and polyamines. We discuss the pathogenic role that subunit c could play in the electrically excitable tissues of retina, brain and heart where Batten neurodegeneration is seen. Focus is given to potential links between subunit c and the known mutant gene products in the Batten diseases, the process of apoptosis, and the requirement of the growing brain for gradients of cGMP, a ligand of the subunit c channel.

Mammalian plasma membrane ecto-nucleoside triphosphate diphosphohydrolase 1, CD39, is not active intracellularly. The N-glycosylation state of CD39 correlates with surface activity and localization.

CD39 is a member of the membrane-bound ecto-nucleoside triphosphate diphosphohydrolase family. The active site for native CD39 is located on the outer surface of the cellular plasma membrane; however, it is not yet known at what stage this enzyme becomes active along the secretory pathway to the plasma membrane. In this study, sucrose density fractionations performed on CD39-transfected COS-7 cell membranes suggest that CD39 activity resides primarily in the plasma membrane. Furthermore, we have created recombinant, soluble versions of CD39, one that is secreted and others that are retained in the endoplasmic reticulum, to demonstrate that CD39 is not active until it reaches the plasma membrane both in yeast and COS-7 cells. Moreover, the secreted active soluble CD39 in COS-7 cells is found to receive a higher degree of N-glycan addition than the inactive form retained intracellularly. When COS-7 cells were treated with tunicamycin to prevent N-glycosylation, soluble CD39 was not detected in the extracellular medium and remained inactive intracellularly. Surface biotinylation analysis also revealed that surface-expressed wild type CD39 receives a higher degree of N-glycosylation than intracellular forms and that inhibition of N-glycosylation prevents its plasma membrane localization. In addition, both intact and digitonin-permeablized COS-7 cells transfected with CD39 possess similar ecto-ATPase activities, further supporting the conclusion that only surface-expressed CD39 is enzymatically active. All of these data suggest that intracellular CD39 is inactive and that only a fully glycosylated CD39 has apyrase activity and is localized at the cell surface.

Bioluminometric method for real-time detection of ATPase activity.

We have developed a real-time, simple, and sensitive method for the detection of ATP hydrolysis activity (ATPase) of apyrase (EC 3.6.1.5). The assay is based on the continuous monitoring of the ATP hydrolysis reaction using the firefly luciferase system. The method is sensitive and yields linear responses between 0.7 and 70 mU for the Solanum tuberosum apyrase. The detection limit was found to be 0.7 mU apyrase. We used the method to study the inhibitory effects of various compounds on the ATPase activity of potato apyrase, measured with 500 nM ATP. The concentrations of azide, AMP, Pi, fluoride, and ADP, which inhibit the ATPase activity by 50% (IC50), were found to be approximately 100, 0.25, 0.125, 0.04, and 0.035 mM, respectively. Under our assay conditions, vanadate inhibited about 98% of the ATPase activity of the potato apyrase at a concentration of 250 microM. The possibility of using the new method for other applications is discussed.

Cellular and biophysical evidence for interactions between adenosine triphosphate and P-glycoprotein substrates: functional implications for adenosine triphosphate/drug cotransport in P-glycoprotein overexpressing tumor cells and in P-glycoprotein low-level expressing erythrocytes.

P-glycoprotein is involved with the removal of drugs, most of them cations, from the plasma membrane and cytoplasm. Pgp is also associated with movement of ATP, an anion, from the cytoplasm to the extracellular space. The central question of this study is whether drug and ATP transport associated with the expression of Pgp are in any way coupled. We have measured the stoichiometry of transport coupling between drug and ATP release. The drug and ATP transport that is inhibitable by the sulfonylurea compound, glyburide (P. E. Golstein, A. Boom, J. van Geffel, P. Jacobs, B. Masereel, and R. Beauwens, Pfluger's Arch. 437, 652, 1999), permits determination of the transport coupling ratio, which is close to 1:1. In view of this result, we asked whether ATP interacts directly with Pgp substrates. We show by measuring the movement of Pgp substrates in electric fields that ATP and drug movement are coupled. The results are compatible with the view that substrates for Pgp efflux are driven by the movement of ATP through electrostatic interaction and effective ATP-drug complex formation with net anionic character. This mechanism not only pertains to drug efflux from tumor cells overexpressing Pgp, but also provides a framework for understanding the role of erythrocytes in drug resistance. The erythrocyte consists of a membrane surrounding a millimolar pool of ATP. Mammalian RBCs have no nucleus or DNA drug/toxin targets. From the perspective of drug/ATP complex formation, the RBC serves as an important electrochemical sink for toxins. The presence in the erythrocyte membrane of approximately 100 Pgp copies per RBC provides a mechanism for eventual toxin clearance. The RBC transport of toxins permits their removal from sensitive structures and ultimate clearance from the organism via the liver and/or kidneys.

Soluble apyrases release adp during ATP hydrolysis.

A soluble form of CD39 was expressed and purified from High-Five insect cells. The soluble CD39 is a monomer with a molecular weight of 54,000. The k(cat) and K(m) of the purified soluble CD39 were 4.6 s(-1) and 12 microM for ATP and 1.3 s(-1) and 7 microM for ADP as substrates, respectively. One nucleotide binding site was detected on the monomer only in the presence of Ca(2+). In contrast to the membrane bound CD39, soluble CD39 released ADP as an intermediate during ATP hydrolysis, as did the soluble potato apyrase.

Interaction of the alpha subunit of Na,K-ATPase with cofilin.

The alpha1 subunit of rat Na,K-ATPase, composed of 1018 amino acids, is arranged in the membrane so that the middle third of the polypeptide forms a large cytoplasmic loop bordered on both sides by multiple transmembrane segments. To identify proteins that might interact with the large cytoplasmic loop of Na,K-ATPase and potentially affect the function and/or the disposition of the pump in the cell, the yeast two-hybrid system was used to screen a rat skeletal muscle cDNA library. Several cDNA clones were isolated, some of which coded for cofilin, an actin-binding protein. Cofilin was co-immunoprecipitated with the alpha subunit of Na,K-ATPase from extracts of COS-7 cells transiently transfected with haemagglutinin-epitope-tagged cofilin cDNA as well as from yeast extracts. By means of deletion analysis we showed that the segment of cofilin between residues 45 and 99 is essential for functional association with the large cytoplasmic loop of Na,K-ATPase. Recombinant cofilin was shown to bind to the membrane-bound Na,K-ATPase; the association between the two proteins was demonstrated by confocal microscopy. The increased level of cofilin in transfected COS-7 cells caused an increase in the rate of ouabain-sensitive (86)Rb(+) uptake, indicating that cofilin elicits, either directly or indirectly, enhanced Na,K-ATPase activity and that the interaction occurs in vivo.

Regulation of yeast ectoapyrase ynd1p activity by activator subunit Vma13p of vacuolar H+-ATPase.

CD39-like ectoapyrases are involved in protein and lipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae. By using a two-hybrid screen, we found that an activator subunit (Vma13p) of yeast vacuolar H(+)-ATPase (V-ATPase) binds to the cytoplasmic domain of Ynd1p, a yeast ectoapyrase. Interaction of Ynd1p with Vma13p was demonstrated by direct binding and co-immunoprecipitation. Surprisingly, the membrane-bound ADPase activity of Ynd1p in a vma13Delta mutant was drastically increased compared with that of Ynd1p in VMA13 cells. A similar increase in the apyrase activity of Ynd1p was found in a vma1Delta mutant, in which the catalytic subunit A of V-ATPase is missing, and the membrane peripheral subunits including Vma13p are dissociated from the membranes. However, the E286Q mutant of VMA1, which assembles inactive V-ATPase complex including Vma13p in the membrane, retained wild type levels of Ynd1p activity, demonstrating that the presence of Vma13p rather than the function of V-ATPase in the membrane represses Ynd1p activity. These results suggest that association of Vma13p with the cytoplasmic domain of Ynd1p regulates its apyrase activity in the Golgi lumen.

Opposing actions of cGMP and calcium on the conductance of the F(0) subunit c pore.

Subunit c of ATP synthase can be purified from neuronal plasma membrane and from the inner mitochondrial membrane. In the latter location the hydrophobic 75 amino acid protein is one component of the F(1) F(0) ATP synthase complex but in the former it is alone as a pore that is capable of generating spontaneous electrical oscillations. Pure mammalian subunit c when reconstituted in lipid bilayers and voltage clamped, yields a voltage sensitive pore that conducts a cation current regulated by calcium. The current is here found to be activated by cGMP with a K(M) ranging from 14 nM to 19 microM depending on calcium and temperature. It is sensitively inhibited by a number of ligands. The K(I) for calcium ranges from 100 nM to 100 microM depending on cGMP and temperature. DCCD inhibits with a K(app) of 100 nM. The polyamine nicotine inhibits at 84 nM. The pore has properties that would allow it to deliver sodium or calcium through the cell membrane in a controlled manner while maintaining membrane polarization.

Biological-to-electronic interface with pores of ATP synthase subunit C in silicon nitride barrier.

An oscillator pore is identified that generates intermittent, large amplitude, ionic current in the plasma membrane. The pore is thought to be composed of 10-12 units of subunit c of ATP synthase. Pore opening and closing is a co-operative process, dependent on the release, or binding, of as many as six calcium ions. This mechanism suggests a more general method of co-operative threshold detection of chemical agents via protein modification, the output being directly amplified in a circuit. Here the authors describe steps in the development of a sensor of chemical agents. The subunit c pore in a lipid bilayer spans a nanometer-scale hole in a silicon nitride barrier. Either side of the barrier are electrolyte solutions and current through the pore is amplified by circuitry. The techniques of laser ablation, electron beam lithography and ion beam milling are used to make successively smaller holes to carry the lipid patch. Holes of diameter as small as 20 nm are engineered in a silicon nitride barrier and protein activity in lipid membranes spanning holes as small as 30 nm in diameter is measured. The signal-to-noise ratio of the ionic current is improved by various measures that reduce the effective capacitance of the barrier. Some limits to scale reduction are discussed.

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.