Enzymic analysis of the structure of oxidized potato starches

The binding of TNP-ATP (2' or 3'-O-(2,4,6-trinitrophenyl)-ATP) to cytochrome c oxidase (COX) from bovine heart and liver and to the two-subunit COX of Paracoccus denitrificans was measured by its change of fluorescence. Three binding sites, two with high (dissociation constant Kd = 0.2 microM) and one with lower affinity (Kd = 0.9 microM), were found at COX from bovine heart and liver, while the Paracoccus enzyme showed only one binding site (Kd = 3.6 microM). The binding of [35S]ATP alpha S was measured by equilibrium dialysis and revealed seven binding sites at the heart enzyme (Kd = 7.5 microM) and six at the liver enzyme (Kd = 12 microM). The Paracoccus enzyme had only one binding site (Kd = 16 microM). The effect of variable intraliposomal ATP/ADP ratios, but at constant total concentration of [ATP + ADP] = 5 mM, on the H+/e- stoichiometry of reconstituted COX from bovine heart and liver were studied. Above 98% ATP the H+/e- stoichiometry of the heart enzyme decreased to about half of the value measured at 100% ATP. In contrast, the H+/e-stoichiometry of the liver enzyme was not influenced by the ATP/ADP ratio. It is suggested that high intramitochondrial ATP/ADP ratios, corresponding to low cellular work load, will decrease the efficiency of energy transduction and result in elevated thermogenesis for the maintenance of body temperature.     

Changes in overbite and face height from 5 to 45 years of age in normal subjects

Periodate-oxidized ADP (oADP)2 and periodate-oxidized ATP (oATP) stimulate the permeability transition in energized rat liver mitochondria measured as the Ca2+-efflux induced by Ca2+ and Pi. In the presence of Mg2+ and Pi, mitochondria lose intramitochondrial adenine nucleotides at a slow rate. oATP induces a strong decrease of the matrix adenine nucleotides which is inhibited by carboxyatractyloside. Under these conditions, Mg2+ prevents the opening of the permeability transition pore. EGTA prevents the Pi-induced slow efflux of adenine nucleotides, but is without effect on the oATP-induced strong decrease of adenine nucleotides. This oATP-induced strong adenine nucleotide efflux is inhibited by ADP. oATP reduces the increase of matrix adenine nucleotides occurring when the mitochondria are incubated with Mg2+ and ATP. This effect of oATP is also prevented by carboxyatractyloside. oATP is not taken up by the mitochondria. It is suggested that oATP induces a strong efflux of matrix adenine nucleotides by the interaction with the ADP/ATP carrier from the cytosolic side. The induction of the mitochondrial permeability transition by oADP and oATP is attributed to two mechanisms-a strong decrease in the intramitochondrial adenine nucleotide content, especially that of ADP, and a stabilization of the c-conformation of the ADP/ATP carrier.     

Cytochrome c oxidase from eucaryotes but not from procaryotes is allosterically inhibited by ATP

The activity of reconstituted cytochrome c oxidase from bovine heart but not from Rhodobacter sphaeroides is allosterically inhibited by intraliposomal ATP, which binds to subunit IV. The activity of cytochrome c oxidase of wild-type yeast and of a subunit VIa-deleted yeast mutant, measured with Tween 20-solubilized mitochondria in the presence of an ATP-regenerating system, was also allosterically inhibited by ATP, indicating the general validity of this mechanism of "respiratory control" in eucaryotic cytochrome c oxidases (Arnold and Kadenbach, Eur. J. Biochem. (1997) 249, 350-354). Deletion of subunit VIa changes the biphysic into monophysic kinetics of the yeast enzyme in the presence of ADP. A tenfold higher amount of horse heart cytochrome c, as compared to yeast cytochrome c, was required to relieve the ATP inhibition of the yeast enzyme.     

Androgen synthesis in a songbird: a study of cyp17 (17alpha-hydroxylase/C17,20-lyase) activity in the zebra finch

Extracellular nucleotides, e.g., ATP, ADP, and UTP, are important signaling molecules which elicit various physiological responses in different tissues. Their degradation is catalyzed by ectonucleotidases which are located on cell surfaces. Most tissues have a mixed population of ectonucleotidases. In this report, the ATP and ADP hydrolyzing ectonucleotidases of chicken gizzard smooth muscle and liver plasma membranes were studied. The two membranes exhibited marked differences in the ratio of ATPase/ADPase activities, activation by divalent cations, thermal stability, responses to detergents and cross-linking agents, and sensitivity to several enzyme inhibitors. The ATPase activity of chicken gizzard membranes is (i) labile to heat and detergents; (ii) activated by concanavalin A and disuccinimidyl suberate, both cross-linking agents; (iii) inhibited by mercurials; and (iv) insensitive to high concentrations of azide, a known inhibitor of ecto-ATP diphosphohydrolases (ecto-ATP/Dase). In contrast, the liver membrane ATPase and ADPase activities are more stable to treatment by heat and detergents and insensitive to cross-linking agents and mercurials, but are inhibited by azide. A low ADP hydrolase activity in the gizzard membranes could be distinguished from both the gizzard ATPase and the liver ATPase/ADPase. This ADP hydrolase, which is markedly stimulated by NBD-Cl, accounts for most of the ADP hydrolysis activity in gizzard membranes. It is concluded that the major ectonucleotidase in the gizzard membranes is an ecto-ATPase whereas that in the liver membranes is an ecto-ATP/Dase. That both membranes contain a mixed population of the ecto-ATPase and ecto-ATP/Dase, but in different proportions, is further demonstrated by immunochemical characterization. The different composition of ectonucleotidases in the two membranes is expected to have an important effect on the regulation of hydrolysis of extracellular ATP as well as the concentration of extracellular adenine nucleotides in the gizzard and liver tissues.     

The state of ADP or ATP fixed to the mitochondria by bongkrekate

The reported studies are intended to clarify the binding state of ADP fixed to mitochondria under the influence of bongkrekate, and thus to discern between the affinity increase and reorientation mechanism proposed for the bongkrekate effect. (a) The composition of the intramitochondrial adenine nucleotide pool is not changed under the influence of bongkrekate with and without added nucleotides. (b) The added ADP and ATP fixed by bongkrekate can be identified as AMP, ADP and ATP in the same proportions as in the endogenous pool. (c) The bound nucleotides respond to oxidative phosphorylation or uncoupler stimulated dephosphorylation similar as endogenous nucleotides. It can be concluded that the ADP or ATP fixed under the influence of bongkrekate to the mitochondria are equilibrated with the intramitochondrial adenine nucleotide pool and are active in intramitochondrial phosphate transfer reactions. The results disagree with the affinity increase mechanism but support the reorientation mechanism which postulates that ADP and ATP are trapped in the mitochondria under the influence of bongkrekate in the same amount as there are carrier sites available outside before bongkrekate addition.     

Correlated effluxes of adenine nucleotides, Mg2+ and Ca2+ induced in rat-liver mitochondria by external Ca2+ and phosphate

The presence of inorganic phosphate and Ca2+ in the external medium induces a closely parallel efflux of both endogenous adenine nucleotides and Mg2+ from rat liver mitochondria. These effluxes are (a) pH-dependent and inhibited by uncouplers, respiration inhibitors and external Mg2+; (b) completely prevented by bongkrekate, but stimulated by atractylate. ATP, ADP or AMP each inhibit the release of Mg2+ promoted by Ca2+ and phosphate; however, in the presence of oligomycin and P1,P5-di(adenosine-5')-pentaphosphate (an inhibitor of adenylate kinase) only ADP is effective. Also the release of accumulated Ca2+ observed when approximately 50% Mg2+ is discharged is retarded by bongkrekate and added Mg2+ whereas it is accelerated by atractylate. All adenine nucleotides have a significant effect in retarding the efflux of accumulated Ca2+ but, in the presence of oligomycin and P1,P5-di(adenosine-5')-pentaphosphate, only ADP is active. From these results we conclude that effluxes of Mg2+, Ca2+ and adenine nucleotide from rat liver mitochondria induced by external phosphate are interconnected and regulated by external ADP and Mg2+ levels.     

Role of reactive oxygen species and poly-ADP-ribose polymerase in the development of AZT-induced cardiomyopathy in rat

The short term cardiac side-effects of AZT (3'-azido-3'-deoxythymidine, zidovudine) was studied in rats to understand the biochemical events contributing to the development of AZT-induced cardiomyopathy. Developing rats were treated with AZT (50 mg/kg/day) for 2 wk and the structural and functional changes were monitored in the cardiac muscle. AZT treatment provoked a surprisingly fast appearance of cardiac malfunctions in developing animals characterized by prolonged RR, PR and QT intervals and J point depression. Electron microscopy showed abnormal mitochondrial structure but the cardiomyocyte had normal myofibers. The AZT treatment of rats significantly increased ROS and peroxynitrite formation in heart tissues as determined by the oxidation of nonfluorescent dihydrorhodamine123 and dichlorodihydro-fluorescein diacetate (H2DCFDA) to fluorescent dyes, and induced single-strand DNA breaks. Lipid peroxidation and oxidation of cellular proteins determined from protein carbonyl content were increased as a consequence of AZT treatment. Activation of the nuclear poly-ADP-ribose polymerase and the accelerated NAD+ catabolism were also observed in AZT-treated animals. Western blot analysis showed that mono-ADP-ribosylation of glucose regulated protein (GRP78/BIP) was enhanced by AZT treatment, that process inactivates GRP78. In this way moderate decrease in the activity of respiratory complexes was detected in the heart of AZT-treated animals indicating a damaged mitochondrial energy production. There was a significant decrease in creatine phosphate concentration resulting in a decrease in creatine phosphate/creatine ratio from 2.08 to 0.58. ATP level remained close to normal but the total extractable ADP increased with 45%. The calculated free ATP/ADP ratio decreased from 340 to 94 in the heart of AZT-treated rats as a consequence of increased free ADP concentration. It was assumed that the increased free ADP in AZT-treated cardiomyocyte may help cells to compensate the defective ATP production in damaged mitochondria by activating the ATP synthesis in undamaged mitochondria. Southern blot analysis did not show decreased quantity of mtDNA deriving from AZT-treated rat hearts indicating that under our experimental conditions AZT-induced heart abnormalities are not the direct consequence of the mtDNA depletion. These data show that ROS-mediated oxidative damages, activated ADP-ribosylation reactions and accelerated NAD+ catabolism play basic roles in the development of AZT-induced cardiomyopathy in our animal model and indicated that these ROS-mediated processes can be important factors in the development of myopathy and cardiomyopathy in zidovudine-treated AIDS patients.     

On the protection by inorganic phosphate of calcium-induced membrane permeability transition

The role of inorganic phosphate as inhibitor of mitochondrial membrane permeability transition was studied. It is shown that in mitochondria containing a high phosphate concentration, i.e., 68 nmo/mg, Ca2+ did not activate the pore opening. Conversely, at lower levels of matrix phosphate, i.e., 38 nmol/mg, Ca2+ was able to induce subsequent pore opening. The inhibitory effect of phosphate was apparent in sucrose-based media, but it was not achieved in KCI media. The matrix free Ca2+ concentration and matrix pH were lowered by phosphate, but they were always higher in K+-media. In the absence of ADP, phosphate strengthened the inhibitory effect of cyclosporin A on carboxyatractyloside-induced Ca2+ efflux. Acetate was unable to replace phosphate in the induction of the aforementioned effects. It is concluded that phosphate preserves selective membrane permeability by diminishing the matrix free Ca2+ concentration.     

Evaluation of birth cohort patterns in population disease rates

1. The effects of piroxicam, a nonsteroidal anti-inflammatory drug, on rat liver mitochondria were investigated in order to obtain direct evidence about a possible uncoupling effect, as suggested by a previous work with the perfused rat liver. 2. Piroxicam increased respiration in the absence of exogenous ADP and decreased respiration in the presence of exogenous ADP, the ADP/O ratios and the respiratory control ratios. 3. The ATPase activity of intact mitochondria was increased by piroxicam. With 2,4-dinitrophenol uncoupled mitochondria, inhibition was observed. The ATPase activity of freeze-thawing disrupted mitochondria was insensitive to piroxicam. 4. Swelling driven by the oxidation of several substrates and safranine uptake induced by succinate oxidation were inhibited. 5. The results of this work represent a direct evidence that piroxicam acts as an uncoupler, thus, decreasing mitochondrial ATP generation.     

Fluctuation of the first loop facing the matrix of the mitochondrial ADP/ATP carrier deduced from intermolecular cross-linking of Cys56 residues by bifunctional dimaleimides

The effects of six thiol-specific cross-linker dimaleimides, in which the distance of the two maleimide groups ranged from 7.7 to 16. 8 A, on bovine heart mitochondria were studied at pH 7.2 and 0 degrees C. None of the dimaleimides affected mitochondrial proteins, but they caused significantly specific intermolecular cross-linking of the 30 kDa ADP/ATP carrier in submitochondrial particles. All the cross-links were found to be formed specifically between two Cys56 residues in the first loop facing the matrix, as we observed previously in intermolecular disulfide bridge formation catalyzed by copper o-phenanthroline [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J.Biol. Chem. 270, 29548-29554]. The dimerization was dependent on the cross-linking span of the dimaleimides, being maximum with the dimaleimide having a span of about 12 A. Cross-linking took place in the m-state carrier, but not in the c-state carrier, and inhibited ADP transport via the ADP/ATP carrier. We suggest that a pair of first loops with Cys56 residues in the dimer form of the m-state carrier fluctuates widely with a most probable distance between them of about 12 A, and that this fluctuation modulates the transport activity of the ADP/ATP carrier.     

Clinical study of living-related liver transplantation

OBJECTIVE: To investigate the effect of ONO-5046, an elastase inhibitor, on liver mitochondrial dysfunction after endotoxin administration. DESIGN: Prospective, randomized, controlled animal study. SETTING: Research laboratory. SUBJECTS: Male Hartley guinea pigs. INTERVENTIONS: Endotoxin shock was induced by intravenous infusion of Escherichia coli lipopolysaccharide endotoxin (50 mg/kg). Six guinea pigs were treated with endotoxin and saline. Twenty-four guinea pigs received 5, 10, and 30 mg/kg/hr of ONO-5046 after endotoxin administration. Six guinea pigs received only saline. MEASUREMENTS AND MAIN RESULTS: We measured oxygen uptake in state 3 (substrate and adenosine 5'-diphosphate [ADP]) and state 4 (excess substrate, no ADP), as well as the respiratory control ratio (state 3/state 4), adenosine 5'-diphosphate/oxygen ratio (ADP/O), and arterial ketone body ratio. ONO-5046 was dose dependently effective in liver mitochondrial oxidative phosphorylation, such as oxygen uptake in stage 4, respiratory control ratio, adenosine triphosphate synthesis, ADP/O, and arterial ketone body ratio when ONO-5046 was started 30 mins after endotoxin. The administration of 30 mg/kg/hr of ONO-5046 improved mean blood pressure, which had decreased after endotoxin. CONCLUSION: ONO-5046 attenuates the endotoxin-induced liver mitochondrial dysfunctions that may be related to increased liver blood flow.     

Hepatic energy charge and adenine nucleotide status in rats anesthetized with halothane, isoflurane or enflurane

BACKGROUND: Volatile anesthetics are known to have varying effects on hepatic oxygen supply in vivo and have been shown to depress hepatic mitochondrial respiration and so energy charge in vitro. However, the effect of halothane, isoflurane and enflurane on hepatic adenine nucleotide status in vivo has not been evaluated. METHODS: Ninety male rats were exposed to 40% oxygen (n = 22) or 40% oxygen in equipotent (1 MAC) concentrations of halothane (1%) (n = 23), isoflurane (1.4%) (n = 22) or enflurane (2%) (n = 23) for 2 hours. All animals were then administered intraperitoneal pentobarbital and anesthesia continued and laparotomy was performed. A liver biopsy was taken for determination of hepatocellular adenosine-5-triphosphate (ATP), adenosine-5-diphosphate (ADP) and adenosine-5-monophosphate (AMP) and computation of energy charge (EC) from ?(ATP + 1/2ADP)+(ATP + ADP + AMP)? and total adenine nucleotides (TAN) from (ATP + ADP + AMP). After the biopsy the aorta was cannulated for blood sampling. RESULTS: Rats in each group were similar in weight, as well as acid base and blood gas status just after liver biopsy. Hepatic energy charge, ATP, ADP, AMP, and TAN levels were not different in animals receiving either halothane, isoflurane or enflurane when compared with those receiving only oxygen. CONCLUSION: One MAC of anesthesia for a period of 2 hours with the described volatile anesthetic agents did not affect adenine nucleotide status in vivo in rats.     

Unusual properties of mitochondria from the human term placenta are caused by alkaline phosphatase

Human placental mitochondria prepared by a new isolation procedure exhibit low but well coupled rates of state 3 respiration with different substrates (succinate: 32.3 nmol O2/mg/min, RCI = 4.4; pyruvate: 12.6 nmol O2/mg/min, RCI R = 4.2; palmitoylcarnitine: 16.6 nmol O2/mg/min, RCI R = 4.9). The addition of the uncoupler FCCP increased the respiratory rates (succinate: 40.7 nmol O2/mg/min; pyruvate: 21.2 nmol O2/mg/min: palmitoylcarnitine: 25.4 nmol O2/mg/min). The low respiratory rates correlate well with a low capacity of the respiratory chain as shown by the specific contents of cytochrome c (0.15 nmol/mg), cytochrome b (0.19 nmol/mg) and cytochrome oxidase (0.14 nmol/mg) as well as with the low content of adenine nucleotides (2.71 nmol/mg). These data together with the finding of high activities of alkaline phosphatase (2.2 U/mg) support the view that human placental mitochondria are contaminated with nonmitochondrial membranes. Since it was not possible to obtain functionally intact mitochondria with negligible activities of alkaline phosphatase the influence of this enzyme on the extramitochondrial adenine nucleotide turnover was investigated. Alkaline phosphatase splits phosphate from ATP, ADP and AMP with different rates resulting in an intermediate accumulation of AMP. Mitochondrial adenylate kinase (0.16 U/mg) regenerated ADP from AMP and ATP resulting in drastically decreased ADP/O ratios and prolonged state 3 respirations. Inhibiting the adenylate kinase with diadenosine pentaphosphate the ADP regeneration from AMP and ATP was suppressed which, in turn, enhanced the ADP/O ratios. In the absence of magnesium ions, if both the alkaline phosphatase and the adenylate kinase are inhibited normal ADP/O ratios and state 3-state 4 transitions can be observed. Under these conditions, human placental mitochondria showed normal properties comparable to those of mitochondria from other tissues with the only exception of low specific activities.     

Defects at center P underlie diabetes-associated mitochondrial dysfunction

Detailed respiration studies on isolated liver mitochondria from streptozotocin-induced diabetic Sprague-Dawley rats revealed a disease-associated decrease in the ADP/O ratio, a marker for mitochondrial ability to couple the consumption of oxygen to the phosphorylation of ADP. This decrease was observed following induction of respiration with glutamate/malate, succinate, or duroquinol, which enter the electron transport chain selectively at complexes I (NADH dehydrogenase), II (succinate dehydrogenase), or III (cytochrome bc1 complex), respectively. These data, coupled with studies using respiratory inhibitors (most importantly antimycin A and myxothiazol), localize at least a portion of this defect to a single site within the electron transport chain (center P in the Q-cycle portion of complex III). These results suggest that liver mitochondria from diabetic animals may generate increased levels of reactive oxygen species at the portion of the electron transport chain already established as the major site of mitochondrial free radical generation. The reduction in the ADP/O ratio occurred in mitochondria that do not have overt defects in the respiratory control ratio or in State 3 and State 4 respiration. The data in this paper suggest that defects in center P of the electron transport chain likely increase mitochondrial exposure to oxidants in the diabetic. This data may partially explain the evidence of altered exposure and/or response to reactive species in mitochondria from diabetics. This work thus provides further clues to the interaction between oxidative stress and diabetes-associated mitochondrial dysfunction.     

Influence of ADP, AMP-PNP and of depletion of nucleotides on the structural properties of F1ATPase: a Fourier transform infrared spectroscopic study

Mitochondrial F1ATPase from beef heart was treated with different buffers in order to modulate the nucleotide content of the enzyme and then analysed by FT-IR spectroscopy. Treatment of F1ATPase with a buffer lacking nucleotides and glycerol led to the formation of two fractions consisting of an inactive aggregated enzyme deprived almost completely of bound nucleotides and of an active enzyme containing ATP only in the tight sites and having a structure largely accessible to the solvent and a low thermal stability. Treatment of F1ATPase with saturating ADP, which induced the hysteretic inhibition during turnover, or AMP-PNP did not affect remarkably the secondary structure of the enzyme complex but significantly increased its compactness and thermal stability. It was hypothesised that the formation of the inactive aggregated enzyme was mainly due to the destabilisation of the alpha-subunits of F1ATPase and that the induction of the hysteretic inhibition is related to a particular conformation of the enzyme, which during turnover becomes unable to sustain catalysis.     

Alterations in energy metabolism of hypertrophied rat cardiomyocytes: influence of propionyl-L-carnitine

Alterations in energy metabolism, reduced fatty acid oxidation, and cardiac carnitine content have been implicated in the evolution from compensated to decompensated cardiac hypertrophy. We determined high-energy nucleotide levels in hypertrophied quiescent cardiomyocytes isolated from rat hearts 4 weeks after banding of abdominal aorta. In hypertrophied quiescent cardiomyocytes, a decrease in ATP content (p = 0.03), and ratios of ATP/total adenine nucleotides and of ATP/ADP were observed, together with an increase in ADP. In addition, palmitate, but not glucose oxidation, was markedly reduced in hypertrophied myocytes. In the presence of 25 microM propionyl-L-carnitine (PLC) or L-carnitine (LC), palmitate oxidation was significantly stimulated in hypertrophied myocytes. The ATP/ADP ratio was significantly increased only with PLC. This effect was not due to an enhanced PLC uptake, since total PLC uptake was 50% lower than that of LC. Changes in the energy generating system of quiescent myocytes occur early in pressure overload hypertrophy, and these alterations can be attenuated by PLC.     

ATP and ADP bind to cytochrome c oxidase and regulate its activity

By equilibrium dialysis of cytochrome c oxidase from bovine heart with [35S]ATPalphaS and [35S]ADPalphaS, seven binding sites for ATP and ten for ADP were determined per monomer of the isolated enzyme. The binding of ATP occurs in a time-dependent manner, as shown by a filtration method, which is apparently due to slow exchange of bound cholate. In the crystallized enzyme 10 mol of cholate were determined and partly identified in the high resolution crystal structure. Binding of ADP leads to conformational changes of the Tween 20-solubilized enzyme, as shown by a 12% decrease of the gamma-band. The conformational change is specific for ADP, since CDP, GDP and UDP showed no effects. The spectral changes are not obtained with the dodecylmaltoside solubilized enzyme. The polarographically measured activity of cytochrome c oxidase is lower after preincubation with high ATP/ADP-ratios than with low, in the presence of Tween 20. This effect of nucleotides is due to interaction with subunit IV, because preincubation of the enzyme with a monoclonal antibody to subunit IV released the inhibition by ATP. In the presence of dodecylmaltoside the enzyme had a 2 to 3-fold higher total activity, but this activity was not influenced by preincubation with ATP or ADP.     

Regulation of resting and IL-2-activated human cytotoxic lymphocytes by exogenous nucleotides: role of IL-2 and ecto-ATPases

In this investigation we studied the modulation of human NK- and CTL-mediated cytotoxicity in response to extracellular nucleotides. NK cell-mediated cytotoxicity (CMC) was inhibited in a dose-dependent manner by ATP/ADP, GTP/GDP, and by pentasodium triphosphate (PST), whereas MHC-restricted CTL were inhibited by GTP/GDP and PST, but not by ATP/ADP. Triphosphates were the most potent inhibitors, followed by diphosphates and monophosphates which were the least effective, suggesting that the inhibition was not due to the sugars nor adenosine and guanosine nucleotides, but rather to the increasing negative charges. Cultured CTL, fresh NK cells that had been incubated with IL-2 for 18 hr and IL-2-dependent NK 3.3 cells were all inhibited by GTP, but not by ATP. This differential regulation of fresh NK cells and CTL by exogenous nucleotides is dependent upon the presence of IL-2, but IL-4, IL-6, and IL-8 did not have any effect. Mouse CTL are resistant to ATP presumably because they contain high levels of ecto-ATPases. Different levels of ecto-ATPase activity in human CTL and NK cells may therefore explain the difference in the responses of these effector cells to extracellular nucleotides. To test this possibility we determined the levels of ecto-ATPases in human CTL and NK cells and showed that CTL contained five times more ecto-ATPases than NK cells. Incubation of NK cells with IL-2 or IL-4 did not significantly change the level of ecto-ATPase activity on NK cells. Treatment of NK cells with IL-2 also did not significantly change the substrate specificity of NK-ecto-ATPases toward the extracellular ATP and GTP. Furthermore, treatment of CTL and NK cells with a potent ecto-ATPase inhibitor, 5'-fluorosulfonylbenzoyladenosine (FSBA), did not significantly alter the effect of exogenous nucleotides on the lytic potential of CTL and NK cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1)

The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped-flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph-ADP. The CF1 was in a state in which two of the three binding sites on the beta subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the alpha subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772-3777)]. Thus, it was possible to characterise a single nucleotide-binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph-ADP binding to the remaining site on the third beta subunit was characterised by a high dissociation rate of 15 s(-1), leading to a very low affinity (dissociation constant higher than 150 microM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre-loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide-binding site on a beta subunit. Pre-loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a beta subunit, while the other was probably located on an alpha subunit.     

Structure and expression of a murine homologue of sky receptor tyrosine kinase gene

We examined effects of adenine nucleotide on ischemic myocardial stunning in dogs. Pentobarbitalanesthetized open-chest dogs were subjected to 20-min ligation of the left anterior descending coronary artery (LAD), followed by reperfusion for 30 min. Either saline, 5 mM 8-bromo-5'-AMP (tributyryl-AMP), or 30 mM N6, 2', 3'-tributyryl-5'-AMP (tributyryl-AMP), 5 mM 5-amino-4-imidazole carboxamide riboside (AICAr) as a positive reference, was infused at 0.1 ml/kg/min in the left femoral vein throughout the experiment. The myocardial contractile function was measured by ultrasonometry. The tissue levels of high-energy phosphates in the reperfused heart were determined. Myocardial contractile function assessed by % segment shortening (%SS) in the saline-infused group decreased during ischemia and returned toward the preischemic level during reperfusion but incompletely. A significant improvement in the %SS during reperfusion was observed in the 8-bromo-AMP- and AICAr-infused groups but not in the tributyryl-AMP-infused group. The magnitude of the protective effect of the drugs on myocardial contractility during reperfusion was 8-bromo-AMP > AICAr > tributyryl-AMP = saline. Only in the 8-bromo-AMP-infused group were the levels of ATP, ADP, and total adenine nucleotides in the reperfused heart significantly higher than those in the saline-infused group. The present result indicates that 8-bromo-AMP improves the ability of the heart to recover from ischemia and reperfusion associated with a significant restoration of ATP.