Electron beam dynamics in an ultrafast transmission electron microscope with Wehnelt electrode.

High temporal resolution transmission electron microscopy techniques have shown significant progress in recent years. Using photoelectron pulses induced by ultrashort laser pulses on the cathode, these methods can probe ultrafast materials processes and have revealed numerous dynamic phenomena at the nanoscale. Most recently, the technique has been implemented in standard thermionic electron microscopes that provide a flexible platform for studying material's dynamics over a wide range of spatial and temporal scales. In this study, the electron pulses in such an ultrafast transmission electron microscope are characterized in detail. The microscope is based on a thermionic gun with a Wehnelt electrode and is operated in a stroboscopic photoelectron mode. It is shown that the Wehnelt bias has a decisive influence on the temporal and energy spread of the picosecond electron pulses. Depending on the shape of the cathode and the cathode-Wehnelt distance, different emission patterns with different pulse parameters are obtained. The energy spread of the pulses is determined by space charge and Boersch effects, given by the number of electrons in a pulse. However, filtering effects due to the chromatic aberrations of the Wehnelt electrode allow the extraction of pulses with narrow energy spreads. The temporal spread is governed by electron trajectories of different length and in different electrostatic potentials. High temporal resolution is obtained by excluding shank emission from the cathode and aberration-induced halos in the emission pattern. By varying the cathode-Wehnelt gap, the Wehnelt bias, and the number of photoelectrons in a pulse, tradeoffs between energy and temporal resolution as well as beam intensity can be made as needed for experiments. Based on the characterization of the electron pulses, the optimal conditions for the operation of ultrafast TEMs with thermionic gun assembly are elaborated.

A cost-effective method for estimating di(2-ethylhexyl)phthalate in coastal sediments.

This study describes the development of a new method for the analysis of di(2-ethylhexyl)phthalate (DEHP) using 0.1-0.3 g of sediment sample, based on matrix solid phase dispersion (MSPD) using C18 as dispersant phase (0.4 g) and acetonitrile-water as eluting solvent (3.4 mL 1:3.25, v/v). No evaporation step is required. 3 mL of extracts were processed on-line by in-tube solid phase microextraction (IT-SPME) coupled to capillary liquid chromatography (CapLC) and diode array detector (DAD). A short analytical column Zorbax SB C18 (35×0.5 mm, 5 µm) provided suitable results. FTIR-ATR was employed for characterizing sediment samples and MSPD procedure. The total analysis time was less than 20 min (MSPD takes about 10 min). The utility of the described approach has been tested by analyzing several real samples. No matrix effect was found. Achieved precision was less than 10% for DEHP estimation. Detection limits in samples were 270 and 90 µg/kg for 0.1 and 0.3 g of taken sediment, respectively.

Hot electron cooling by acoustic phonons in graphene.

We have investigated the energy loss of hot electrons in metallic graphene by means of GHz noise thermometry at liquid helium temperature. We observe the electronic temperature T ∝ V at low bias in agreement with the heat diffusion to the leads described by the Wiedemann-Franz law. We report on T ∝ √V behavior at high bias, which corresponds to a T(4) dependence of the cooling power. This is the signature of a 2D acoustic phonon cooling mechanism. From a heat equation analysis of the two regimes we extract accurate values of the electron-acoustic phonon coupling constant Σ in monolayer graphene. Our measurements point to an important effect of lattice disorder in the reduction of Σ, not yet considered by theory. Moreover, our study provides a strong and firm support to the rising field of graphene bolometric detectors.

Toward an understanding of the unexpected regioselective hetero-Diels-Alder reactions of asymmetric tetrazines with electron-rich ethylenes: a DFT study.

The regioselective hetero-Diels-Alder (HDA) reaction of asymmetric tetrazines (TTZs) with electron-rich (ER) ethylenes has been studied with use of DFT methods at the B3LYP/6-31G* level of theory. The reaction is a domino process that comprises three consecutive reactions: (i) a HDA reaction between the TTZ and the ER ethylene; (ii) a retro-Diels-Alder reaction with loss of nitrogen; and (iii) a beta-hydrogen elimination with formation of the final pyridazines. The first polar HDA reaction, which is associated to the nucleophilic attack of the ER ethylene to the electrophilically activated TTZ, is the rate and regioselectivity determining step of the domino process. The unexpected regioselectivity of these HDA reactions is explained within the polar cycloaddition model by using the conceptual DFT. Although the nucleophilic attack of the ER ethylene over the para position relative to the methylsulfinyl substituent could favor the charge transfer, it is energetically more unfavorable because it diminishes the electron density at the electronegative TTZ core.

1,3-Dipolar cycloadditions of electrophilically activated benzonitrile N-oxides. Polar cycloaddition versus oxime formation.

The reactions of electrophilically activated benzonitrile N-oxides (BNOs) toward 3-methylenephthalimidines (MPIs) have been studied using density functional theory (DFT) at the B3LYP/6-31G* level. For these reactions, two different channels allowing the formation of the [3 + 2] cycloadducts and two isomeric (E)- and (Z)-oximes have been characterized. The 1,3-dipolar cycloadditions take place along concerted but highly asynchronous transition states, while formation of the oximes is achieved through a stepwise mechanism involving zwitterionic intermediates. Both reactions are initiated by the nucleophilic attack of the methylene carbon of the MPIs to the carbon atom of the electrophilically activated BNOs. The analysis based on the natural bond orbital (NBO) and the topological analysis of the electron localization function (ELF) at the transition structures and intermediates explains correctly the polar nature of these reactions. Solvent effects considered by the PCM model allow explaining the low incidence of the solvent polarity on the rate and composition of the reactions.

Understanding the nature of the molecular mechanisms associated with the competitive Lewis acid catalyzed [4+2] and [4+3] cycloadditions between arylidenoxazolone systems and cyclopentadiene: a DFT analysis.

The molecular mechanisms of the reactions between aryliden-5(4H)-oxazolone 1, and cyclopentadiene (Cp), in presence of Lewis acid (LA) catalyst to obtain the corresponding [4+2] and [4+3] cycloadducts are examined through density functional theory (DFT) calculations at the B3LYP/6-31G* level. The activation effect of LA catalyst can be reached by two ways, that is, interaction of LA either with carbonyl or carboxyl oxygen atoms of 1 to render [4+2] or [4+3] cycloadducts. The endo and exo [4+2] cycloadducts are formed through a highly asynchronous concerted mechanism associated to a Michael-type addition of Cp to the beta-conjugated position of alpha,beta-unsaturated carbonyl framework of 1. Coordination of LA catalyst to the carboxyl oxygen yields a highly functionalized compound, 3, through a domino reaction. For this process, the first reaction is a stepwise [4+3] cycloaddition which is initiated by a Friedel-Crafts-type addition of the electrophilically activated carbonyl group of 1 to Cp and subsequent cyclization of the corresponding zwitterionic intermediate to yield the corresponding [4+3] cycloadduct. The next rearrangement is the nucleophilic trapping of this cycloadduct by a second molecule of Cp to yield the final adduct 3. A new reaction pathway for the [4+3] cycloadditions emerges from the present study.

Pharmacology of INS37217 [P(1)-(uridine 5')-P(4)- (2'-deoxycytidine 5')tetraphosphate, tetrasodium salt], a next-generation P2Y(2) receptor agonist for the treatment of cystic fibrosis.

INS37217 [P(1)-(uridine 5')-P(4)-(2'-deoxycytidine 5')tetraphosphate, tetrasodium salt] is a deoxycytidine-uridine dinucleotide with agonist activity at the P2Y(2) receptor. In primate lung tissues, the P2Y(2) receptor mRNA was located by in situ hybridization predominantly in epithelial cells and not in smooth muscle or stromal tissue. The pharmacologic profile of INS37217 parallels that of UTP, leading to increased chloride and water secretion, increased cilia beat frequency, and increased mucin release. The combined effect of these actions was confirmed in an animal model of tracheal mucus velocity that showed that a single administration of INS37217 significantly enhanced mucus transport for at least 8 h after dosing. This extended duration of action is consistent with the ability of INS37217 to resist metabolism by airway cells and sputum enzymes. The enhanced metabolic stability and resultant increased duration of improved mucociliary clearance may confer significant advantages to INS37217 over other P2Y(2) agonists in the treatment of diseases such as cystic fibrosis.

The CF salt controversy: in vivo observations and therapeutic approaches.

There is controversy over whether abnormalities in the salt concentration or volume of airway surface liquid (ASL) initiate cystic fibrosis (CF) airway disease. In vivo studies of CF mouse nasal epithelia revealed an increase in goblet cell number that was associated with decreased ASL volume rather than abnormal [Cl(-)]. Aerosolization of osmolytes in vivo failed to raise ASL volume. In vitro studies revealed that osmolytes and pharmacological agents were effective in producing isotonic volume responses in human airway epithelia but were typically short acting and less effective in CF cultures with prolonged volume hyperabsorption and mucus accumulation. These data show that (1) therapies can be designed to normalize ASL volume, without producing deleterious compositional changes in ASL, and (2) therapeutic efficacy will likely depend on development of long-acting pharmacologic agents and/or an increased efficiency of osmolyte delivery.

Biochemical evidence for an ecto alkaline phosphodiesterase I in human airways.

Because dinucleotides are signaling molecules that can interact with cell surface receptors and regulate the rate of mucociliary clearance in lungs, we studied their metabolism by using human airway epithelial cells. A membrane-bound enzyme was detected on the mucosal surface of polarized epithelia that metabolized dinucleotides with a broad substrate specificity (diadenosine polyphosphates and diuridine polyphosphates [Up(n)U], n = 2 to 6). The enzymatic reaction yielded nucleoside monophosphates (NMP) and Np(n)(-)(1) (N = A or U), and was inhibited by nucleoside 5'-triphosphates (alpha,betamet adenosine triphosphate [ATP] > ATP >/= uridine triphosphate > guanidine triphosphate > cytidine triphosphate). The apparent Michaelis constant (K(m,app)) and apparent maximal velocity (V(max,app)) for [(3)H]Up(4)U were 22 +/- 4 microM and 0.24 +/- 0.05 nmoles. min(-)(1). cm(-)(2), respectively. Thymidine 5'-monophosphate p-nitrophenyl ester and adenosine diphosphate (ADP)- ribose, substrates of ecto alkaline phosphodiesterase I (PDE I) activities, were also hydrolyzed by the apical surface of airway epithelia. ADP-ribose competed with [(3)H]Up(4)U, with a K(i) of 23 +/- 3 microM. The metabolism of ADP-ribose and Ap(4)A was not affected by inhibitors of cyclic nucleotide phosphodiesterases (3-isobutyl-1-methylxanthine, Ro 20-1724, and 1,3-dipropyl-8-p-sulfophenylxanthine), but similarly inhibited by fluoride and N-ethylmaleimide. These results suggest that a PDE I is responsible for the hydrolysis of extracellular dinucleotides in human airways. The wide substrate specificity of PDE I suggests that it may be involved in several signaling events on the luminal surface of airway epithelia, including purinoceptor activation and cell surface protein ribosylation.

Basal nucleotide levels, release, and metabolism in normal and cystic fibrosis airways.

BACKGROUND: Cystic fibrosis (CF) is a syndrome caused by mutations in the cystic fibrosis transmembrane regulator (CFTR) gene. Despite advances in our understanding of the molecular pathogenesis of CF, the link between CFTR gene mutations and the pathogenesis of CF lung disease remains poorly defined. CFTR has been assigned a number of putative functions that may contribute to innate airway defense, including the regulation of adenosine 5'-triphosphate (ATP) release into the extracellular environment. Because extracellular ATP and uridine 5'-triphosphate (UTP) may regulate airway mucociliary clearance via interaction with luminal P2Y2 receptors, the loss of CFTR-mediated nucleotide release could explain the defect in CF airway defense. MATERIALS AND METHODS: We tested the physiologic importance of CFTR-mediated nucleotide release in vivo by directly measuring levels of ATP and UTP in nasal airway surface liquid from normal and CF subjects. Because these basal nucleotide levels reflect the net activities of nucleotide release and metabolic pathways, we also measured constitutive rates of nucleotide release and metabolism on well-differentiated normal and CF airway cultures in vitro. The measurement of ATP release rates were paralleled by in vivo studies employing continuous nasal perfusion in normal and CF subjects. Finally, the regulation of ATP release by isoproterenol and methacholine-stimulated submucosal gland secretion was tested. RESULTS: These studies revealed that steady-state ATP and UTP levels were similar in normal (470 +/- 131 nM and 37 +/- 7 nM, respectively) and CF (911 +/- 199 nM and 33 +/- 12 nM, respectively) subjects. The rates of both ATP release and metabolism were also similar in normal and CF airway epithelia both in vitro and in vivo. Airway submucosal glands did not secrete nucleotides, but rather, secreted a soluble nucleotidase in response to cholinergic stimuli. CONCLUSION: The concentration of ATP in airway surface liquid is in a range that is relevant for the activation of airway nucleotide receptors. However, despite this finding that suggests endogenous nucleotides may be important for the regulation of mucociliary clearance, our data do not support a role for CFTR in regulating extracellular nucleotide concentrations on airway surfaces.

Characterization of a diadenosine tetraphosphate-receptor distinct from the ATP-purinoceptor in human tracheal gland cells.

Human submucosal tracheal glands are now believed to play a major role in the physiopathology of cystic fibrosis, a genetic disease in which ATP is used as a therapeutic agent. However, actions of ATP on tracheal gland cells are not well known. ATP binds to P2 receptors and induced secretory leucocyte protease inhibitor (SLPI) secretion through formation of cyclic adenosine monophosphate and mobilization of intracellular [Ca(2+)]. Since diadenosine polyphosphates (ApnA) are also endogenous effectors of P2 receptors, we investigated their effects in a cell line (MM39) of human tracheal gland cells. Diadenosine tetraphosphates (Ap4A) induced significant stimulation (+50+/-12%) of SLPI secretion and to a similar extent to that of ATP (+65+/-10%). No significant effects were observed with diadenosine triphosphate (Ap3A), diadenosine pentaphosphate (Ap5A), ADP and 2-methylthio-adenosine triphosphate (2-MeS-ATP). Since Ap4A was weakly hydrolyzed (<2% of total), and the hydrolysis product was only inosine which is ineffective on cells, this Ap4A effect was not due to Ap4A hydrolysis in ATP and adenosine monophosphate (AMP). A mixture of Ap4A and ATP elicited only partial additive effects on SLPI secretion. ADP was shown to be a potent antagonist of ATP and Ap4A receptors, with IC(50)s of 0.8 and 2 microM, respectively. 2-MeS-ATP also showed antagonistic properties with IC(50)s of 20 and 30 microM for ATP- and Ap4A-receptors, respectively. Single cell intracellular calcium ([Ca(2+)](i)) measurements showed similar transient increases of [Ca(2+)](i) after ATP or Ap4A challenges. ATP desensitized the cell [Ca(2+)](i) responses to ATP and Ap4A, and Ap4A also desensitized the cell response to Ap4A. Nevertheless, Ap4A did not desensitize the cell [Ca(2+)](i) responses to ATP. In conclusion, both P2Y2-ATP-receptors and Ap4A-P2D-receptors seem to be present in tracheal gland cells. Ap4A may only bind to P2D-receptors whilst ATP may bind to both Ap4A- and ATP-receptors.

Theoretical Study on the Molecular Mechanism of the Domino Cycloadditions between Dimethyl Acetylenedicarboxylate and Naphthaleno- and Anthracenofuranophane.

AM1, B3LYP/6-31G//AM1, and B3LYP/6-31G computational studies were performed to select the reaction pathway controlling the reactions between dimethyl acetylenedicarboxylate (DMAD) and two furanophanes, naphthalenofuranophane and anthracenofuranophane. For these domino reactions, several pathways have been characterized on the potential energy surface corresponding to two consecutive cycloadditions. The first step corresponds to a [4 + 2] intermolecular cycloaddition of DMAD with the furan ring or with the naphthalene or anthracene ring of both furanophane systems to yield an oxabicyclo[2.2.1]heptadiene or a bicyclo[2.2.2]octadiene intermediate, respectively. The second step corresponds to [4 + 2] intramolecular cycloadditions of these intermediates. For the naphthalenofuranophane, the most favorable reaction pathway takes place along the initial [4 + 2] intermolecular cycloaddition involving the nonsubstituted ring of the naphthalene system to give a benzobicyclo[2.2.2]octadiene intermediate, which by a [4 + 2] intramolecular cycloaddition between the substituted double bond of this intermediate and the furan ring affords the final cycloadduct. For the anthracenofuranophane, the most favorable reaction pathway takes place along the initial [4 + 2] intermolecular cycloaddition involving the furan ring to give an oxabicyclo[2.2.1]heptadiene intermediate, which by a [4 + 2] intramolecular cycloaddition between the nonsubtituted double bond of the bicyclic system and the naphthalene system affords the final cycloadduct. An analysis of energetic contributions to the potential energy barriers identifies the different factors controlling the competitive reaction pathways. The present theoretical results are able to explain the available experimental data.

Conducting and voltage-dependent behaviors of the native and purified SR Ca2+-release channels from the canine diaphragm.

The ryanodine-sensitive Ca2+-release channel of the canine diaphragm sarcoplasmic reticulum (SR) was characterized using biochemical assays and the planar lipid bilayer technique. Diaphragm SR membranes have a [3H]ryanodine-binding capacity (Bmax) of 1.2 pmol/mg protein and a binding affinity (K(D)) of 6.3 nM. The conductance of the native channel was 330 pS in 50 mM/250 mM trans/cis CsCH3SO3 and was reduced to 71 pS by 10 mM Ca2+ trans. The Ca2+-release channel was purified as a 400 kDa protein on SDS-PAGE and displayed a conductance of 715 pS in 200 mM KCl. The native and purified Ca2+ channels were activated by micromolar Ca2+ and ATP and inhibited by Mg2+, ryanodine and ruthenium red. Although diaphragm muscle contraction was shown to depend on extracellular Ca2+ like cardiac muscles, we provide evidence that the diaphragm SR Ca2+-release channel may be classified as a skeletal ryanodine receptor isoform. First, the IC50 for [3H]ryanodine binding was in the same range as estimated for skeletal SR, with 20 nM. Second, the channel was maximally activated by 10-30 microM cytoplasmic Ca2+ and inhibited at higher concentrations. Third, ryanodine binding to the diaphragm SR was less sensitive to Ca2+ than cardiac SR, with EC50, values of 50 and 1 microM, respectively. Finally, Ca2+-release activity and [3H]ryanodine binding capacity of the diaphragm and skeletal SR were similarly more sensitive to Mg2+ than cardiac SR. Together, these results suggest a predominantly skeletal-type of excitation-contraction coupling in the diaphragm.

Purification and immunohistochemical localization of the ATP diphosphohydrolase in bovine lungs.

We have recently described different isoforms of mammalian ATP diphosphohydrolase (ATPDase; EC 3.6.1.5). In the present study, we purified the lung ATPDase by column chromatographies followed by polyacrylamide gel electrophoresis under nondenaturing conditions. The active polypeptide that has a molecular mass of 78 kDa was identified by affinity labeling to the ATP analog 5'-p-fluorosulfonylbenzoyladenosine (FSBA), followed by detection on Western blot with an antibody specific for FSBA. N-glycosidase F treatment shifted the molecular mass of the 78-kDa polypeptide down to 54 kDa, indicating that the enzyme bears approximately 6-12 NH2-linked oligosaccharide chains. A polyclonal antibody raised against the pancreas ATPDase, which specifically recognized the 78-kDa glycoprotein on Western blot, was used to carry out an immunological survey of the enzyme distribution in bovine lungs. Immunoreactivity was detected on airway epithelia from the trachea down to alveolar cells, airway and vascular smooth muscle cells, submucous glands, chondrocytes, leucocytes, as well as endothelial and mesothelial cells. Such a wide distribution suggests that the ATPDase may affect a variety of physiological effects mediated by extracellular nucleotides, such as airway smooth muscle tone, surfactant secretion, platelet aggregation, and inflammation.

Hydrolysis of P2-purinoceptor agonists by a purified ectonucleotidase from the bovine aorta, the ATP-diphosphohydrolase.

Pharmacologists are becoming more and more aware of the possibility that certain ATP analogues currently used to classify the P2-purinoceptors are dephosphorylated by ectonucleotidases. In this study, we provide evidence that in the vascular system, these purine analogues are hydrolysed by an ATP-diphosphohydrolase (ATPDase). This enzyme is known as the major plasma membrane nucleotidase of endothelial and smooth muscle cells, and is believed to dephosphorylate extracellular triphospho- and diphosphonucleosides. Assays were conducted with a purified ATPDase from smooth muscle cells of bovine aorta. At a concentration of 250 microM, adenosine 5'-(alpha,beta-methylene) triphosphonate (alpha,beta-metATP), adenosine 5'-(beta,gamma-methylene) triphosphonate (beta,gamma-metATP), adenosine 5'-(alpha,beta-methylene) disphosphonate (alpha,beta-metADP), adenylyl 5'-(beta,gamma-imido) diphosphonate (beta,gamma-imidoATP) and adenosine 5'-O-(2-thiodiphosphate) (ADP beta S) all resisted dephosphorylation, whereas 2-chloroadenosine triphosphate (2-chloroATP), 2-methylthioadenosine triphosphate (2-MeSATP) and 8-bromoadenosine triphosphate (8-bromo-ATP) were hydrolysed at 99, 63, and 20% of the rate of ATP hydrolysis, respectively. All the non-hydrolysable analogues tested, except alpha,beta-metADP, competed with ATP and ADP for the ATPDase catalytic site, reducing their hydrolysis by 35-50%. Apparent Km values for ATP and ADP were estimated at 14.1 and 12.0 microM, respectively, whereas apparent Km and Ki values for the purine analogues ranged from 12 to 28 microM. These results strongly support the view that (1) the ATPDase is expected to reduce substantially the P2-response induced by ATP, ADP, and some hydrolysable agonists; and (2) by competing with the hydrolysis of endogenously released ATP and ADP, non-hydrolysable analogues could alter the amplitude or direction of the cellular response induced by these natural substrates.

Conducting and voltage-dependent behaviors of potassium ion channels reconstituted from diaphragm sarcoplasmic reticulum: comparison with the cardiac isoform.

Sarcoplasmic reticulum (SR) K+ channels from canine diaphragm were studied upon fusion of longitudinal and junctional membrane vesicles into planar lipid bilayers (PLB). The large-conductance cation selective channel (gamma(max) = 250 pS; Km = 33 mM) displays long-lasting open events which are much more frequent at positive than at negative voltages. A major subconducting state about 45% of the fully-open state current amplitude was occasionally observed at all voltages. The voltage-dependence of the open probability displays a sigmoid relationship that was fitted by the Boltzmann equation and expressed in terms of thermodynamic parameters, namely the free energy (delta Gi) and the effective gating charge (Zs): delta Gi = 0.27 kcal/mol and Zs = -1.19 in 250 mM potassium gluconate (K-gluconate). Kinetic analyses also confirmed the voltage-dependent gating behavior of this channel, and indicate the implication of at least two open and three closed states. The diaphragm SR K+ channel shares several biophysical properties with the cardiac isoform: g = 180 pS, delta Gi = 0.75 kcal/mol, Zs = -1.45 in 150 mM K-gluconate, and a similar sigmoid P(o)/voltage relationship. Little is known about the regulation of the diaphragm and cardiac SR K+ channels. The conductance and gating of these channels were not influenced by physiological concentrations of Ca2+ (0.1 microM-1 mM) or Mg2+ (0.25-1 mM), as well as by cGMP (25-100 microM), lemakalim (1-100 microM), glyburide (up to 10 microM) or charybdotoxin (45-200 nM), added either to the cis or to the trans chamber. The apparent lack of biochemical or pharmacological modulation of these channels implies that they are not related to any of the well characterized surface membrane K+ channels. On the other hand, their voltage sensitivity strongly suggests that their activity could be modulated by putative changes in SR membrane potential that might occur during calcium fluxes.

Demonstration of an ectoATP-diphosphohydrolase (E.C.3.6.1.5.) in non-vascular smooth muscles of the bovine trachea.

An ectoATP-diphosphohydrolase (ATPDase) is put in evidence in non-vascular smooth muscles of the bovine trachea. The enzyme has an optimum pH of 7.0 and catalyzes the hydrolysis of the gamma- and beta-phosphate residues from extracellular triphospho- and diphosphonucleosides. It requires either Ca2+ or Mg2+ and is insensitive to ouabain, oligomycin and Ap5A. Sodium azide (20 mM), mercuric chloride (10 microM) and gossypol (35 microM) inhibit the enzyme activity by more than 45%. Polyacrylamide gel electrophoresis under non-denaturing conditions and kinetic properties, namely pH dependency profiles, heat inactivation and 60Co gamma-irradiation-inactivation curves, support the view that the same catalytic site is responsible for the hydrolysis of ATP and ADP to AMP. Accordingly, when both ATP and ADP were combined, reaction rates were not additive. With ATP, Km,app and Vmax,app were estimated at 15 +/- 2 microM and 1.9 +/- 0.1 mumol inorganic phosphate/min per mg of protein, respectively. From 60Co gamma-irradiation-inactivation curves, the molecular mass of the enzyme was estimated at 71 +/- 5 kDa. Enzyme markers indicate that the ATPDase is associated with the plasma membrane. Enzyme assays on trachea smooth muscle cells in suspension confirm that the catalytic site of this ATPDase is localized on the outer surface of the plasma membrane. Analysis of the biochemical properties shows many points of similarity between the tracheal ATPDase and the ATPDase recently described in the bovine lung.

Demonstration of a novel type of ATP-diphosphohydrolase (EC 3.6.1.5) in the bovine lung.

A novel type of ATP-diphosphohydrolase (ATPDase) is demonstrated in bovine lung. The enzyme has an optimum pH of 7.5 and catalyzes the hydrolysis of the beta- and gamma-phosphate residues from diphospho- and triphosphonucleosides. It requires Ca2+ or Mg2+ and is insensitive to ouabain, an inhibitor of Na+/K(+)-ATPase, P1,P5-di(adenosine 5')-pentaphosphate, an inhibitor of adenylate kinase, and tetramisole, an inhibitor of alkaline phosphatase. In contrast, sodium azide (10 mM), a known inhibitor of ATPDases and mitochondrial ATPases, as well as mercuric chloride (10 microM) and gossypol (2,2'-bis[8-formyl-1,6,7-trihydroxy-5-isopropyl-3-methylnaphthalene]) (35 microM) are powerful inhibitors of this enzyme. The same inhibition profile is obtained with ATP or ADP as substrate, thereby supporting the concept of a common catalytic site for these substrates. This is further confirmed by enzyme localization after polyacrylamide gel electrophoresis under nondenaturing conditions and by kinetic properties, namely pH dependence profiles, heat inactivation, and 60Co irradiation-inactivation curves. The native molecular mass of the enzyme calculated from 60Co gamma-irradiation-inactivation curves is estimated at 70 +/- 3 kDa, whereas Km,app and Vmax,app of the ATPDase are evaluated at 7 +/- 2 microM and 1.1 +/- 0.3 mumol of Pi/min/mg protein, respectively. A comparison of the kinetic properties of this ATPDase with those of pig pancreas (Type I) and bovine aorta (Type II) lead us to believe that this enzyme is an hitherto undescribed type of ATPDase. By reference to the previously described ATPDase, we propose to identify this enzyme as ATPDase Type III (EC 3.6.1.5).

Identification and localization of ATP-diphosphohydrolase (apyrase) in bovine aorta: relevance to vascular tone and platelet aggregation.

In this work, we confirm the existence of an ATP-diphosphohydrolase (apyrase) in bovine aorta and we show that its properties are different from the previously described pancreas ATP-diphosphohydrolase. Hence the aorta enzyme should be considered as a novel type of apyrase. The demonstration is based on pH dependency profiles, heat denaturation curves, 60Co irradiation-inactivation curves and enzyme localization after polyacrylamide gel electrophoresis under non-denaturing conditions. In addition, the irradiation-inactivation curves clearly showed that for both pancreas and aorta enzymes preparations, the same catalytic site is responsible for the hydrolysis of ATP and ADP. The molecular masses of enzymes calculated with this method are 132 +/- 19 kDa (mean +/- S.D.) and 189 +/- 30 kDa (mean +/- S.D.) for the pancreas and aorta enzymes, respectively. Preliminary observations on isolated bovine brain capillaries revealed a high level of enzyme activity strongly suggesting that an ATP-diphosphohydrolase is associated with endothelial cells. The presence of the enzyme on this type of cells was confirmed with pulmonary endothelial cells in culture. Considering the high proportions of smooth muscle cells relative to endothelial cells and the high level of enzyme activity in the aorta preparation, an ATP-diphosphohydrolase activity is definitely present in smooth muscle cells. The ATP-diphosphohydrolase activities described above could regulate the relative concentrations of purine nucleotides both in the plasma and within the vascular wall and hence could play a role both in platelet aggregation and in the control of vascular tone.