Inhibition of rat Na+/K+-ATPase isoforms by endogenous digitalis extracts from neonatal human plasma

An unique endogenous digitalis-like factor (EDLF) has been previously purified from human newborn cord plasma and its differential effects tested on the three well defined functional isoforms (alpha1, alpha2 and alpha3) of the alpha subunits of Na+/K+-ATPase in rat. EDLF specifically inhibits the enzymatic activity. It differs from ouabain by three criteria: a preincubation with the membranes is required for full activity, no effect on the rat cerebral alpha3 isoform and a steep dose-response curve with the same apparent potency for rat alpha2 and alpha1 isoforms of high (10(-7) M) and low affinity (3 x 10(-5) M) for ouabain. These results indicate that the Na+/K+-ATPase inhibitor involved in the regulation of sodium and body fluid volume and present in neonate and adult human plasmas is distinct from ouabain.     

Microenvironment of the high affinity ATP-binding site of Na+/K+-ATPase is slightly acidic

Fluorescein-5'-isothiocyanate (FITC) was used to study the high-affinity ATP-binding site of Na+/K+-ATPase. The molar ratio of specifically bound FITC per alpha-subunit of Na+/K+-ATPase was found to be 0.5 as followed from pretreatment experiments with another specific E1ATP-inhibitor Cr(H2O)4AdoPP[CH2]P. This indicated an existence of one high affinity ATP-binding site (E1ATP-binding site) in the native (alphabeta)2-diprotomer of Na+/K+-ATPase. Fluorescence dual-excitation ratio of specifically bound FITC revealed that at external pH 7.5, the pH value inside the E1ATP-binding site is 6.95 +/- 0.18. In addition, FITC fluorescence quenching by anti-fluorescein and by iodide choline indicated the limited access of water into the small pocket of the E1ATP-binding site.     

Distribution and elimination of RGH-5002 in rats

In this study we analysed the changes in the properties of rat cerebral cortex Na+K(+)-ATPase in streptozotocin induced diabetes (STZ-diabetes). Special attempt was made to determine whether insulin treatment of diabetic animals could restore the altered parameters of this enzyme. Na+/K(+)-ATPase activity was found to be decreased by 15% after 2 weeks, and by 37% after 4 weeks in diabetic rat brains with a parallel decrease in maximal capacity of low affinity ouabain binding sites. There was no significant change in the high affinity ouabain binding sites. The Kd values did not change significantly. Western blot analysis of brain Na+/K(+)-ATPase isoforms indicated a 61 +/- 5.8% and 20 +/- 2.8% decrease of the alpha 1 and alpha 3 isoforms, respectively in 4 weeks diabetic animals. Change in the amount of the alpha 2 isoform proved to be less characteristic. Both types of beta subunit isoform showed a significant decrease in four weeks diabetic rats. Our data indicate a good correlation in diabetic rats between changes in Na-/K(+)-ATPase activity, low affinity ouabain binding capacity and the level of alpha 1 isoform. While insulin treatment of diabetic animals restored the blood glucose level to normal, a complete reversal of diabetes induced changes in Na+/K(+)-ATPase activity, ouabain binding capacity and Na+/K(+)-ATPase isoform composition could not be achieved.     

Molecular distance measurements reveal an (alpha beta)2 dimeric structure of Na+/K+-ATPase. High affinity ATP binding site and K+-activated phosphatase reside on different alpha-subunits

ATP hydrolysis by Na+/K+-ATPase proceeds via the interaction of simultaneously existing and cooperating high (E1ATP) and low (E2ATP) substrate binding sites. It is unclear whether both ATP sites reside on the same or on different catalytic alpha-subunits. To answer this question, we looked for a fluorescent label for the E2ATP site that would be suitable for distance measurements by F?rster energy transfer after affinity labeling of the E1ATP site by fluorescein 5'-isothiocyanate (FITC). Erythrosin 5'-isothiocyanate (ErITC) inactivated, in an E1ATP site-blocked enzyme (by FITC), the residual activity of the E2ATP site, namely K+-activated p-nitrophenylphosphatase in a concentration-dependent way that was ATP-protectable. The molar ratios of FITC/alpha-subunit of 0.6 and of ErITC/alpha-subunit of 0.48 indicate 2 ATP sites per (alpha beta)2 diprotomer. Measurements of F?rster energy transfer between the FITC-labeled E1ATP and the ErITC-labeled or Co(NH3)4ATP-inactivated E2ATP sites gave a distance of 6.45 +/- 0.64 nm. This distance excludes 2 ATP sites per alpha-subunit since the diameter of alpha is 4-5 nm. F?rster energy transfer between cardiac glycoside binding sites labeled with anthroylouabain and fluoresceinylethylenediamino ouabain gave a distance of 4.9 +/- 0.5 nm. Hence all data are consistent with the hypothesis that Na+/K+-ATPase in cellular membranes is an (alpha beta)2 diprotomer and works as a functional dimer (Thoenges, D., and Schoner, W. (1997) J. Biol. Chem. 272, 16315-16321).     

Bepridil reducing levothyroxine-induced enhancement of mitochondria Ca2+ Mg(2+)-ATPase activity in rat cerebrum

AIM: To study if bepridil (Bep) could affect the enhancement of activity of cerebral mitochondria Ca2+ Mg(2+)-ATPase caused by levothyroxine (Lev) in relation to ischemic overload calcium cerebrum injury. METHODS: The experimental hyperthyroidism model with ischemic cerebrum was developed in rats by ig Lev 1 mg.kg-1.d-1 for 7 d. Ca2+ Mg(2+)-ATPase activity and its kinetic parameters were assayed. RESULTS: The activity, Vmax and Km of cerebral mitochondria Ca2+ Mg(2+)-ATPase in control rats were 3.1 +/- 0.8, 5.1 +/- 2.3 mmol.P(i).h-1/g protein and 0.81 +/- 0.08 mmol.L-1 (ATP) respectively, whereas those of hyperthyroid rats were significantly altered to 4.6 +/- 0.5, 8.5 +/- 1.9 mmol.P(i).h-1/g protein and 0.49 +/- 0.11 mmol.L-1 (ATP) respectively. After treated with Bep 10 or 20 mg.kg-1.d-1 ig for 3 d, allabove 3 parameters of the enzyme were very significantly reduced vs those of either control or hyperthyroid. CONCLUSION: Bep, via decreasing Ca2+ Mg(2+)-ATPase activity and increasing the affinity of Ca2+ Mg(2+)-ATPase to ATP, could prevent rat cerebrum from ATP depletion and ischemic overload calcium injury.     

The influence of beta subunit structure on the stability of Na+/K(+)-ATPase complexes and interaction with K+

Heterologous expression of the beta subunit of H+/K(+)-ATPase (HK beta) with alpha subunits of Na+/K(+)-ATPase (NK alpha) in yeast leads to the formation of ouabain binding complexes, indicating assembly of the two subunits into active ion pumps (Eakle, K. A., Kim, K. S., Kabalin, M. A., and Farley, R. A. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2834-2838). Complexes of NK alpha and HK beta are less sensitive to inhibition of ouabain binding by K+, suggesting that HK beta lowers the affinity of K+ binding sites. This effect is particularly pronounced when HK beta is combined with the alpha 3 isoform of NK alpha. In this case, titration with K+ yields a biphasic curve, suggesting that there are two nonequivalent sites for K+ binding. Attempts at purifying complexes formed with either alpha 1 + HK beta or alpha 3 + HK beta using SDS extraction of microsomal membranes resulted in the loss of ouabain binding. Controls show that alpha 1 + beta 1 and alpha 3 + beta 1 complexes still retain ouabain binding after SDS extraction under the same conditions. This suggests that the HK beta subunit forms a less stable complex with NK alpha subunits. We have created chimeric beta subunits comprised of the amino-terminal cytoplasmic and transmembrane regions of HK beta combined with the carboxyl-terminal extracellular region of Na+/K(+)-ATPase beta 1 (HN beta 1) and the complementary chimera with amino-terminal cytoplasmic and transmembrane regions of beta 1 combined with the carboxyl-terminal extracellular region of HK beta (NH beta 1). When NH beta 1 is combined with either alpha 1 or alpha 3, the complexes show profiles of K+ inhibition of ouabain binding that are very similar to HK beta combined with either alpha 1 or alpha 3. The data suggest that the extracellular region of HK beta is primarily responsible for the effect on apparent K+ affinity. When the HN beta 1 subunit is expressed with the alpha 3 subunit, less than 5% of the amount of ouabain binding complexes are formed compared with HN beta 1 + alpha 1. This observation suggests that the HN beta 1 subunit either assembles poorly or forms an unstable complex with alpha 3. After SDS extraction, complexes of alpha 1 + NH beta 1 and alpha 3 + NH beta 1 retain ouabain binding, while alpha 1 + HN beta 1 complexes are sensitive to SDS extraction.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spectroscopic properties of Escherichia coli UDP-N-acetylenolpyruvylglucosamine reductase

Purified uridine diphosphate N-acetylenolpyruvylglucosamine reductase (E.C. 1.1.1.158) was analyzed by circular dichroism (CD) and UV-visible spectroscopy to establish the spectral properties of its tightly bound flavin adenine dinucleotide (FAD) cofactor. The polypeptide backbone displayed a single circular dichroic minimum at 208 nm and a single maximum at 193 nm. The CD spectrum of bound flavin exhibited a single major negative Cotton peak at 364 nm and two minor negative Cotton peaks at 464 and 495 nm. The protein was reversibly unfolded in 9.8 M urea and refolded in buffer in the presence of excess FAD. The refolded enzyme incorporated FAD and catalyzed full activity. The bound FAD displayed an absorption maximum at 464 nm with an extinction coefficient of epsilon 464 = 11700 M-1 cm-1. Anaerobic reduction with dithionite was complete at 1 equiv. Anaerobic reduction with nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), also was essentially complete at 1 equiv and produced a long-wavelength absorbance band characteristic of an FAD-pyridine nucleotide charge transfer complex. Photochemical bleaching in the presence of ethylenediaminetetraacetic acid (EDTA) followed exponential kinetics. None of the anaerobic reductive titrations produced a spectral intermediate characteristic of a flavin semiquinone, and all reduced enzyme species could be fully reoxidized by oxygen, with full recovery of catalytic activity. Photochemically reduced enzyme was reoxidized by titration with either NADP+ or uridine diphospho N-acetylglucosamine enolpyruvate (UNAGEP). Reoxidation by NADP+ reached a chemical equilibrium, whereas reoxidation by UNAGEP was stoichiometric. Binding of NADP+ or UNAGEP to the oxidized form of the enzyme produced a dead-end complex that could be titrated by following a 10-nm red shift in the absorption spectrum of the bound FAD. The Kd of NADP+ for oxidized enzyme was 0.7 +/- 0.3 microM and the Kd of UNAGEP was 2.7 +/- 0.3 microM. Solvent deuterium isotope effects on binding were observed for both NADP+ and UNAGEP, depending on the pH. At pH 8.5, the HKd/DKd was 2.2 for NADP+ and 3.9 for UNAGEP. No spectral changes were observed in the presence of a 40-fold excess of uridine diphospho N-acetylmuramic acid (UNAM) either aerobically or anaerobically. These studies have identified spectral signals for five steps in the kinetic mechanism, have indicated that product formation is essentially irreversible, and have indicated that hydrogen bonding or protonation contributes significantly to ground-state complex formation with the physiological substrate.     

pH dependence of corneal oxygen consumption

PURPOSE: To determine whether corneal acidosis, which occurs during contact lens wear, alters corneal O2 consumption (QO2) and if so, whether increased ion transport activity could contribute to altered QO2 during acidosis. METHODS: PO2 was measured, using the phosphorescence quenching of Pd-meso-tetra-(4-carboxyphenyl) porphine, in an airtight chamber that held a trephined rabbit cornea. The rate of change in chamber PO2 was used as a measure of QO2. QO2 was measured at pH 7.5 and then at either pH 6.7, 7.1, or 7.3. Measurements of QO2 at pHs 7.5 and 6.7 were repeated in the presence of 0.5 mM amiloride and 0.5 mM ouabain. RESULTS: When pH was changed from 7.5 to 6.7, 7.1, or 7.3, O2 consumption increased by a factor of 1.80+/-0.11 (+/-SE), 1.65+/-0.12, and 1.44+/-0.06, respectively. Amiloride (0.5 mM) and ouabain (0.5 mM) inhibited 50% and 65%, respectively, of the increase in QO2 at pH 6.7. CONCLUSIONS: Corneal acidosis leads to increased QO2 in a dose-dependent manner. The increased QO2 is in part secondary to the activation of pH regulatory mechanisms, including Na+/H+ exchange, which then stimulates Na+/ K+-ATPase activity. These findings indicate that contact lens-induced acidosis can exacerbate corneal hypoxia and related complications.     

Activation of soluble guanylyl cyclase by the nitrovasodilator 3-morpholinosydnonimine involves formation of S-nitrosoglutathione

Microenvironment and conformation of the active site of xylanase from an extremophilic Bacillus was deciphered for the first time using fluorescence spectroscopy. NBS modified enzyme showed complete inactivation and the kinetic analysis implicated the presence of an essential tryptophan at the active site of xylanase. Xylan (0.5%) protected the enzyme completely from inactivation with NBS, whereas it afforded 35% protection against the loss of fluorescence, suggesting that not all the tryptophans are involved at the substrate binding site. Quenching studies revealed that acrylamide was more efficient than KI and CsCl as indicated by the higher Stern-Volmer quenching constants (Ksv). The steric factor represented by the percentage accessibility of the tryptophan residues of XylII was higher with the positively charged Cs+ (80) than with the negatively charged I- (10), suggesting that the tryptophan residues are located in a relatively electronegative environment. In the presence of 6 M Gdn HCl the fluorescence shifted to 350 nm with increased accessibility of the fluorophore to the quenchers. The proximity of the essential carboxyl groups with a high pKa value of 6.9 [Chauthaiwale and Rao (1994) Biochim. Biophys. Acta] probably contributes to the electronegative environment of the tryptophan residue. Our results on sequence analysis of the gene encoding for XylII (Accession Number U83602 in the GenBank database) have shown that Trp 61 is highly conserved and may play a role in the structure-function relationship of the enzyme.     

Double immunofluorescence staining of Fos and Jun in the hypothalamus of the rat

Epidermal growth factor (EGF) triggers rapid and delayed effects on gluconeogenesis, cytosolic (lactate/pyruvate ratio) and mitochondrial (3-hydroxybutyrate/acetoacetate ratio) redox states (Soler, C. and Soley, M., Biochem. J., 294 (1993) 865-872). This study attempts to determine whether the mechanism by which EGF modulates any of these parameters is dependent on the regulation of Na+/H+ exchange and/or Na+/K(+)-ATPase activities. The Na+/H+ exchange was inhibited by either amiloride or the analogue 5-(N,N-hexamethylene)amiloride (HMA), and the Na+/K(+)-ATPase activity was inhibited by ouabain. The delayed EGF inhibition of gluconeogenesis, increase of the lactate/pyruvate ratio and decrease in the 3-hydroxybutyrate/acetoacetate ratio were unaltered in the presence of amiloride, HMA or ouabain. The rapid EGF stimulation of gluconeogenesis was also observed in the presence of HMA or ouabain. Although Na+/H+ exchange and/or Na+/K(+)-ATPase are regulated by EGF, our results indicate that these activities are not required for the effects of EGF on gluconeogenesis and/or cytosolic and mitochondrial redox state.     

Serotonin modulation of low-affinity ouabain binding in rat brain determined by quantitative autoradiography

Previous results showed that Na+/K+-ATPase may have a functional relationship with the neurotransmitter serotonin which activates the glial sodium pump in the rat brain. Both the reaction rate (V) of Na+/K+-ATPase activity and [3H]ouabain binding were significantly increased in the presence of serotonin. It is not known, however, which alpha isoform is involved in the Na+/K+-ATPase response to serotonin and its regional distribution. Quantitative autoradiography of [3H]ouabain binding to rat brain slices was employed at different [3H]ouabain concentrations in order to gain information on both the distribution and the possible isoform involved. The results showed that 1500 nM [3H]ouabain binding was sensitive to serotonin 10(-3) M and significantly increased in the following brain regions: frontal cortex, areas CA1, CA2, and CA3 of the hippocampus, presubiculum, zona incerta, caudate putamen and the amygdaloid area, confirming and extending previous results. An effect of serotonin on brain but not kidney tissue at high, 1500 nM, and the lack of effect at low, 50 nM [3H]ouabain concentrations, strongly suggests the participation of the alpha2 isoform in the response of the pump to the neurotransmitter. Glial cells showed stimulation of ouabain binding by serotonin at ouabain concentrations above 350 nM. The present results open interesting questions related to the brain regions involved and the K+ handling by the glial alpha2 isoform of the pump.     

Sodium, PMA and calcium play an important role on intracellular pH modulation in rat mast cells

In a series of experiments aimed to understand the signaling pathways that regulate intracellular pH (pHi) in rat mast cells, the effect of different intracellular mechanisms on the activity of the Na+/H+ exchanger was studied. After promoting an artificial acidification with sodium propionate we determined the variations on pHi rate recovery. pHi was measured with the dye 2, 7-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. We studied the effect that the inhibition of some cellular exchangers with different drugs induced on pHi. When the Na+/H+ exchanger was inhibited in the presence of amiloride, the recovery rate constant was twofold smaller than the control value. After the recovery, the final pH was lower than the initial value when the cells were treated either with amiloride or with 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (an anionic antiport inhibitor). No effect was observed when the Na+/K+-ATPase or the Na+/Ca2+ exchanger were inhibited. The suppression of intracellular and extracellular calcium did not induced any change in pHi. The addition of thapsigargin, an activator of capacitative calcium influx, or the phorbol esther 12-O-tetradecanoylphorbol-13-acetate (PMA), a protein kinase C (PKC) activator, increased the activity of the antiporter. Both effects were abrogated by inhibition of the Na+/K+-ATPase with ouabain. The increase in cAMP levels did not affect the effect of PMA on pHi recovery, but it blocked the effect of thapsigargin. Our results indicate that rat mast cells regulate pHi by the combination of some anionic exchanger and the Na+/H+ antiporter. And also that the modulation of this exchanger is the consequence of the connection between different intracellular mechanisms, Na+/K+-ATPase-PKC-calcium, among which cAMP seems not to have a direct role.     

Kinetic uniformity of the ATP hydrolysis reaction catalyzed by Mg2+-ATPase in smooth muscle cell membrane

The kinetic properties of Mg(2+)-ATPase (EC 3.6.1.3) from myometrium cell plasma membranes have been studied. Under conditions of enzyme saturation with ATP (0.5-1.0 mM) or Mg2+ (1.0-5.0 mM) the initial maximal rates of the Mg(2+)-dependent enzymatic ATP hydrolysis, V0 ATP and V0 Mg, are 27.4 +/- 3.3 and 25.2 +/- 4.1 mumol Pi/hour/mg of protein, respectively. The apparent Michaelis constant, Km, for ATP and of the apparent activation constant, K alpha, for Mg2+ are equal to 28.1 +/- 2.6 and 107.0 +/- 26.0 microM, respectively. The bivalent metal ions used at 1.0 mM suppress the Mg(2+)-dependent hydrolysis of ATP whose efficiency decreases in the following order: Cu2+ > Zn2+ = Ni2+ > Mn2+ > Ca2+ > Co2+. Alkalinization of the incubation medium from pH 6.0 to pH 8.0 stimulates the Mg(2+)-dependent hydrolysis of ATP. It has been found that Mg(2+)-ATPase has the properties of an H(+)-sensitive enzymatic sensor which is characterized by a linear dependence between the initial maximal rate of the reaction, V0, and the pH value. The feasible role of plasma membrane Mg(2+)-ATPase in some reactions responsible for the control of proton and Ca2+ homeostasis in myometrium cells has been investigated.     

Energy characteristics of an ATP-hydrolase reaction catalyzed by solubilized Ca2+,Mg2+-ATPase from smooth muscle cell membrane

The effects of temperature, dielectric permeability and ionic strength on the activity of purified Ca2+, Mg(2+)-ATPase solubilized from myometrial sarcolemma have been studied under saturation of the enzyme with Ca2+, Mg2+ and ATP. The values of activation energy calculated from Arrhenius plots for both ATP hydrolase reactions catalysed by solubilized and reconstituted into azolectin liposomes Ca2+, Mg(2+)-ATPase and Mg2+, ATP-dependent Ca2+ transport by the reconstituted enzyme were 56.4 +/- 1.5, 68.0 +/- 5.1 and 63.1 +/- 2.9 kJ/mol, respectively. Analysis of experimental data in terms of the Laidler-Scatchard and Bronsted-Bjerrum theories revealed that the separation of the reaction products--the chelate MgADP complex--from the active site of the enzyme bearing one unity positive charge is the limiting step of the Ca2+, Mg(2+)-dependent enzymatic ATP-hydrolysis under conditions of substrate saturation. The values of the electrostatic components of the free energy, enthalpy and entropy of activation of the ATP hydrolase reaction were 46.6 +/- 0.3 kJ/mol, -(20.5 +/- 0.4) kJ/mol and -(214.2 +/- 4.3) J/(mol.degrees K), respectively. The nonelectrostatic component of activation enthalpy was 76.9 kJ/mol. The results obtained suggest that changes in polarity of the incubation medium markedly affect the activity of transport Ca2+, Mg(2+)-ATPase solubilized from smooth muscle cell plasma membranes and that the electrostatic interactions between the enzyme active site and specific reagents (MgADP, in particular) significantly contribute to the energetics of the ATP hydrolase reaction.     

应用V-H_2O_2-TADAP三元配合物光度法测定钒

在pH5.6～6.0时钒(V)与2-(2-噻吩偶氮)-5-二乙氨基酚(TADAP)形成λmax位于590nm的红色配合物。当加入过氧化氢时则形成组成为1:1:1的三元配合物,反应pH值向酸性方向移动,适宜的pH值为1.10～1.50,λmax移至600nm,显色反应具有较高的灵敏度(ε=5.1×10_4L·mol~(-1)·cm~(-1)和良好的选择性,方法可用于钢铁和其他样品中钒的直接分光光度测定。     

Metmyoglobin/fluoride: effect of distal histidine protonation on the association and dissociation rate constants

The kinetics of formation and dissociation of the horse metmyoglobin/fluoride complex has been investigated between pH 3.4 and 11. The ionic strength dependence of the reaction has been measured at integral pH values between pH 5 and 10. Hydrofluoric acid, HF, binds to metmyoglobin with a rate constant of (4.7 +/- 0. 7) x 10(4) M-1 s-1. An apparent ionization in metmyoglobin with a pKa of 4.4 +/- 0.5 influences the rate of HF binding and is attributed to the distal histidine, His-64. Protonation of His-64 increases the HF binding rate by a factor of 2.6. The fluoride anion, F-, binds to metmyoglobin with a rate constant of (5.6 +/- 1.4) x 10(-2) M-1 s-1, about 10(6) times slower than HF. Binding of either HF or F- to hydroxymetmyoglobin cannot be detected. Protonation of the distal histidine facilitates HF dissociation from the metmyoglobin/fluoride complex. HF dissociates with a rate constant of 1.9 +/- 0.3 s-1. The fluoride anion dissociates 2000 times more slowly, with a rate constant of (8.7 +/- 1.6) x 10(-4) s-1. The apparent pKa for His-64 ionization in the fluorometmyoglobin complex is 5.7 +/- 0.1. The association and dissociation rate constants are relatively independent of ionic strength with secondary kinetic salt effects sufficient to account for the ionic strength variation of both, consistent with the idea that association and dissociation of neutral HF dominate the kinetics of fluoride binding to metmyoglobin.     

Extensive random mutagenesis analysis of the Na+/K+-ATPase alpha subunit identifies known and previously unidentified amino acid residues that alter ouabain sensitivity--implications for ouabain binding

Random mutagenesis with ouabain selection has been used to comprehensively scan the extracellular and transmembrane domains of the alpha1 subunit of the sheep Na+/K+-ATPase for amino acid residues that alter ouabain sensitivity. The four random mutant libraries used in this study include all of the transmembrane and extracellular regions of the molecule as well as 75% of the cytoplasmic domains. Through an extensive number of HeLa cell transfections of these libraries and subsequent ouabain selection, 24 ouabain-resistant clones have been identified. All previously described amino acids that confer ouabain resistance were identified, confirming the completeness of this random mutagenesis screen. The amino acid substitutions that confer the greatest ouabain resistance, such as Gln111-->Arg, Asp121-->Gly, Asp121-->Glu, Asn122-->Asp, and Thr797-->Ala were identified more than once in this study. This extensive survey of the extracellular and transmembrane regions of the Na+/K+-ATPase molecule has identified two new regions of the molecule that affect ouabain sensitivity: the H4 and the H10 transmembrane regions. The new substitutions identified in this study are Leu330-->Gln, Ala331-->Gly, Thr338-->Ala, and Thr338-->Asn in the H4 transmembrane domain and Phe982-->Ser in the H10 transmembrane domain. These substitutions confer modest increases in the concentration of cardiac glycoside needed to produce 50% inhibition of activity (IC50 values), 3.1-7.9-fold difference. The results of this extensive screening of the Na+/K+-ATPase alpha1 subunit to identify amino acids residues that are important in ouabain sensitivity further supports our hypothesis that the H1-H2 and H4-H8 regions represent the major binding sites for the cardiac glycoside class of drugs.     

A model of the quaternary structure of the Escherichia coli F1 ATPase from X-ray solution scattering and evidence for structural changes in the delta subunit during ATP hydrolysis

The shape and subunit arrangement of the Escherichia coli F1 ATPase (ECF1 ATPase) was investigated by synchrotron radiation x-ray solution scattering. The radius of gyration and the maximum dimension of the enzyme complex are 4.61 +/- 0.03 nm and 15.5 +/- 0.05 nm, respectively. The shape of the complex was determined ab initio from the scattering data at a resolution of 3 nm, which allowed unequivocal identification of the volume occupied by the alpha3beta3 subassembly and further positioning of the atomic models of the smaller subunits. The delta subunit was positioned near the bottom of the alpha3beta3 hexamer in a location consistent with a beta-delta disulfide formation in the mutant ECF1 ATPase, betaY331W:betaY381C:epsilonS108C, when MgADP is bound to the enzyme. The position and orientation of the epsilon subunit were found by interactively fitting the solution scattering data to maintain connection of the two-helix hairpin with the alpha3beta3 complex and binding of the beta-sandwich domain to the gamma subunit. Nucleotide-dependent changes of the delta subunit were investigated by stopped-flow fluorescence technique at 12 degrees C using N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) as a label. Fluorescence quenching monitored after addition of MgATP was rapid [k = 6.6 s-1] and then remained constant. Binding of MgADP and the noncleavable nucleotide analog AMP . PNP caused an initial fluorescent quenching followed by a slower decay back to the original level. This suggests that the delta subunit undergoes conformational changes and/or rearrangements in the ECF1 ATPase during ATP hydrolysis.     

The reaction of NO. with O2.- and HO2.: a pulse radiolysis study

The reactions of NO. with O2.- and with HO2. were studied using the pulse radiolysis technique under pseudo first order conditions where ([O2.-]o + [HO2.]o) > [NO.]o at pH 3.3-10.0. The rate constant of the reaction of NO. with O2.- was determined both by monitoring the decay of O2.- at 250 nm and the formation of ONOO- at 302 nm to be (4.3 +/- 0.5) x 10(9) M-1s-1, independent of ionic strength and pH in the range of 6.1-10.0. The rate constant of the reaction of NO. with HO2.- was determined by following the decay of HO2. at 250 nm to be (3.2 +/- 0.3) x 10(9) M-1s-1 at pH 3.3.