ATP/ADP exchange activity of gastric (H+ + K+)-ATPase

The ATP/ADP exchange is shown to be a partial reaction of the $\text{(}\text{H}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to $\text{P}{}^1$, $\text{P}{}^5$ -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg²⁺ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration ($\text{K}{}_{0.5}\text{= 116 μ}\text{M}$) required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg²⁺ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K⁺ as a function of pH and Mg²⁺.     

Characterization of cytochrome P-450SCC-containing liposomes

Purified cytochrome $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}\text{-}\text{containing liposomes}$ showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was less stable than $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. Liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$ was subjected to $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ treatment. This reagent destroyed the liposomal $\text{P}{}_{\mathrm{<mtext>450</mtext><mtext>SCC</mtext>}}$. These results suggest that the heme is located in the proximity of the $\text{p-}\text{chloromercuriphenyl sulfonic acid}$ reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.     

catmap: Case-control And TDT Meta-Analysis Package

Background  

Risk for complex disease is thought to be controlled by multiple genetic risk factors, each with small individual effects. Meta-analyses of several independent studies may be helpful to increase the ability to detect association when effect sizes are modest. Although many software options are available for meta-analysis of genetic case-control data, no currently available software implements the method described by Kazeem and Farrall (2005), which combines data from independent family-based and case-control studies.     

AutoFACT: AnAutomaticFunctionalAnnotation andClassificationTool

Background  

Assignment of function to new molecular sequence data is an essential step in genomics projects. The usual process involves similarity searches of a given sequence against one or more databases, an arduous process for large datasets.     

Annular and non-annular binding sites on the (Ca2+ + Mg2+)-ATPase

Quenching of the fluorescence of the $\text{(}\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

No or little production of singlet molecular oxygen in HOCl or HOClH2O2 a model system for myeloperoxidase/H2O2/Cl−

The HOCl in chlorine-water oxidizes DPF to cis-DBE in parallel to the HOCl concentration. The addition of H₂O₂ produces singlet molecular oxygen, and a bimol collision above pH 6.0, but not in the pH region 3.0 to 4.0. The DPF conversion to cis-DBE is initiated by a 1,2-position attack of OH^? and Cl⁺, followed by the HCl elimination. The oxidation potency of HOCl is much greater than the singlet molecular oxygen generated in chlorine-water/H₂O₂ solution, on the pH range 6.0 to 8.0 where both HOCl and OCl^? are present.     

An electron spin probe study of (Na+ + K+)-ATPase-Containing membranes

The modulating effect of membrane lipids on enzyme function has been described by several investigators. We have used the spin probe $\text{N-}\text{oxyl}\text{-4′,4′-}\text{dimethyloxazolidine}\text{-12-}\text{keto}$ methyl stearate (M 12-NSE) to study this interaction in ox brain membranes enriched with $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. This methyl ester of stearic acid is practically insoluble in aqueous media, and consequently spectra of M 12-NSE-labelled preparations are free of “liquid lines”.At least two types of spectra may be obtained when ox brain microsomes are spin labelled with M 12-NSE, indicating the presence of two distinct binding sites. At one site the spin label is relatively unrestricted and gives rise to an isotropic spectrum. A second spectrum, which is obtained from spin label at another site, is similar to that which is observed after incorporation of M 12-NSE into phospholipid bilayers. This suggests that this latter site is within the core of the microsomal membrane.The two binding sites differ in their affinity for the spin probe. The low affinity site is both more abundant in crude preparations and is more easily removed by detergent treatment; spin labels at this site produce isotropic spectra. The high affinity sites are fewer in number and produce broad spectra. In addition these high affinity sites increase in concentration as the enzyme undergoes purification.The two sites are quite distinct in their sensitivity to ascorbic acid, the low affinity site showing a considerably greater rate of reduction by this agent.This study also demonstrates that the delipidation effects of sodium dodecyl sulfate and sodium deoxycholate on $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase-enriched}$ microsomes from ox brain are not identical.It is suggested that the two spin probe binding sites represent two different lipid domains, one of which is very closely associated with the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ enzyme and may reflect a protein-directed phospholipid specificity for this enzyme.     

Characterization of partially purified (Na+ + K+)-ATPase from porcine lens

The partial purification of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from pig lens has been achieved by treatment with deoxycholate followed by density gradient centrifugation. The specific activity of the final preparation, ranging from 300 to 500 nmol/h per mg protein, is increased approx. 100-fold compared to the homogenate. A parallel increase in $\text{p-}\text{nitrophenylphosphatase}$ activity is also observed. Sodium dodecyl sulfate (SDS) gel electrophoresis reveals six major protein bands, one of which is the 93 kDa α subunit of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ which can be phosphorylated by reaction with [γ-³²P]ATP. A second band contains a glycoprotein which displays an apparent molecular weight of 51 000 and thus appears to be the β subunit of the enzyme. The enzyme is sensitive to ouabain with the $\text{I}{}_{50}$ for $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ and $\text{p-}\text{nitrophenylphosphatase}$ inhibition being 1.2 and 1.3 μM, respectively. Several agents which inhibit $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from other tissues such as oligomycin, Ca²⁺, vanadate, $\text{N-}\text{ethylmaleimide}$, $\text{p-}\text{chloromercuribenzenesulfonic acid}$ (PCMBS) and 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) also inhibit the lens enzyme. Monovalent cations other than K⁺ are partially effective in activating the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$)-ATPase and $\text{p-}\text{nitrophenylphosphatase}$ activities. The K⁺ congeners were relatively more effective in supporting $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ compared to $\text{p-}\text{nitrophenylphosphatase}$ activity. Other kinetic properties of the lens enzyme are also comparable to those of the enzyme from other tissues. Utilizing the partially purified membrane bound enzyme, discontinuities in Arrhenius plots of $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, $\text{p-}\text{nitrophenylphosphatase}$ activity and fluoresence polarization of the fluidity probe, 1,6-diphenyl-1,3,5-hexatriene (DPH), are observed near the physiological temperature of lens. The possible significance of these observations for the mechanism of cataract formation are discussed.     

Reconstitution of (Na+ + K+)-ATPase into phospholipid vesicles with full recovery of its specific activity

(1) ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ from rectal glands of the spiny dogfish has been reconstituted into phospholipid vesicles. The nonionic detergent octaethyleneglycoldodecyl monoether (C₁₂E₈) is used to dissolve both the enzyme and the lipids and reconstitution is accomplished by subsequent removal of the detergent by adsorption to polystyrene beads. (2) About 60% of the enzyme incorporates in the right-side-out orientation (r/o). The fraction of molecules in the inside-out orientation (i/o) increases from about 10% to about 30% with a parallel decrease in the fraction of ‘non-oriented’ (n-o) molecules (both sides exposed) when the protein/lipid ratio decreases from 1:10 to 1:75. (3) The orientation of enzyme molecules detected from vanadate binding is the same as measured from activity, i.e., the turnover of the enzyme molecule in the diffrent orientations is the same. (4) The recovery of the specific activity of the incorporated enzyme increases with an increase in the protein/lipid ratio and is 100% with a protein/lipid ration of about 1:20 or higher. Full recovery is only obtained provided a proper lipid composition is chosen which includes both negatively charged phospholipids, preferably phosphatidylinositol, and cholesterol. (5) The ATP-dependent, K⁺-stimulated Na⁺-influx is found to be about 35 μmol Na⁺ per mg (i/o)-protein per min at 22°C in 1:10 protein/lipid liposomes. The specific activity corresponds to 3 Na⁺ transported per ATP molecule hydrolyzed.     

Eosin,a fluorescent probe of ATP binding to the (Na+ + K+)-ATPase

(1) Eosin bound to the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the presence of K⁺ has practically the same fluorescence as eosin without enzyme while in the presence of Na⁺ the fluorescence is higher, the excitation maximum is shifted from 518 to 524 nm, the emission maximum from 538 to 542 nm, and a shoulder appears at about 490 nm on the excitation curve. (2) The amount of eosin bound increases with the K⁺ concentration but with a low affinity. With equal concentrations of Na⁺ and K⁺ more is bound in the presence of Na⁺, and the difference between 150 mM Na⁺ and 150 mM K⁺ shows one high-affinity eosin binding site per ³²P-labelling site ($\text{K}{}_{\mathrm{D}}$ 0.45 μM). With lower concentrations of the cations there are between one and two Na⁺-dependent high-affinity eosin binding sites per ³²P-labelling site. (3) ATP (and ADP) prevents the hig-affinity Na⁺-dependent eosin binding and there is competition between eosin and ATP for the hydrolysis in the presence of Na⁺ (+Mg²⁺). (4) Eosin, like ATP, increases the Na⁺ relative to K⁺ affinity $\text{(}\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{= 150}\text{mM}$) for Na⁺ activation of hydrolysis and for Na⁺ protection against inactivation by $\text{N-}\text{ethylmaleimide}$. (5) The results suggest that the high affinity eosin binding site is an ATP binding site and that it is located on the enzyme in an environment with a low polarity, i.e., the conformational change induced by Na⁺ opens a high-affinity site for ATP while K⁺ closes the site (or decreases the affinity to a low level). The experiments suggest, furthermore, that the ATP which increases the Na⁺ relative to K⁺ affinity of the internal sites is not the ATP which is hydrolyzed, i.e., in a turnover cycle in the presence of $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}$ the system reacts with two different ATP molecules.     

A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

Modulation of Escherichia coliN-acetylmuramoyl-l-alanine amidase activity by phosphatidylglycerol

The activity of pure Escherichia coli murein (peptidoglycan) amidase ($\text{N-}\text{acetylmuramoyl-}\text{l}\text{-alanine}$ amidase, EC 3.5.1.28) was measured after preincubation with E. coli phosphatidylglycerol microdispersions in final concentration ranging over micro- and millimolarities. The enzyme activity was increased up to 160% of the control for phosphatidylglycerol concentrations increasing from 2 to 50 μM. After a plateau extending from 0.05 to 0.3 mM, higher phosphatidylglycerol concentrations inactivated the enzyme down to 15% of initial activity for concentrations of 2 mM. Positive kinetic cooperativity was observed for the activation as well as for the inactivation processes. Cardiolipin (or diphosphatidylglycerol) from the same origin and under same conditions had no significant effect. Molecular sieving experiments have shown that, when inactivated, the enzyme remained firmly bound to the phosphatidylglycerol vesicles, whereas the activated phosphatidylglycerol-enzyme complex was totally dissociable by dilution. Activated phosphatidylglycerol complexes were recovered by gel exclusion chromatography at equilibrium in 40 μM phosphatidylglycerol. Possible physiological meaning of the results is briefly discussed in the context of our work and that done previously by others.     

Decrease of apparent calmodulin affinity of erythrocyte (Ca2+ + Mg2+)-ATPase at low Ca2+ concentrations

The calmodulin activation of the ($\text{Ca}{}^{\mathrm{2+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{)-}\text{ATPase}$ (ATP phosphohydrolase, EC 3.6.1.3) in human erythrocyte membranes was studied in the range of 1 nM to 40 μM of purified calmodulin. The apparent calmodulin-affinity of the ATPase was strongly dependent on Ca²⁺ and decreased approx. 1000-times when the Ca²⁺ concentration was reduced from 112 to 0.5 μM. The data of calmodulin (Z) activation were analyzed by the aid of a kinetic enzyme model which suggests that 1 molecule of calmodulin binds per ATPase unit and that the affinities of the calcium-calmodulin complexes (Ca_iZ) decreases in the order of $\text{Ca}{}_3\text{Z}\text{>}\text{Ca}{}_4\text{Z}\text{>}\text{Ca}{}_2\text{Z}\text{?}\text{CaZ}$. Furthermore, calmodulin dissociates from the calmodulin-saturated Ca²⁺-ATPase in the range of 10^?7–10^?6 M Ca²⁺, even at a calmodulin concentration of 5 μM. The apparent concentration of calmodulin in the erythrocyte cytosol was determined to be 3 to 5 μM, corresponding to 50–80-times the cellular concentration of Ca²⁺-ATPase, estimated to be approx. 10 nmol/g membrane protein. We therefore conclude that most of the calmodulin id dissociated from the Ca²⁺-transport ATPase in erythrocytes at the prevailing Ca²⁺ concentration (probably 10^?7 – 10^?8 M) in vivo, and that the calmodulin-binding and subsequent activation of the Ca²⁺-ATPase requires that the Ca²⁺ concentration rises to 10^?6 – 10^?5 M.     

The distribution of (Na++K+)-ATPase along the villus crypt-axis in the rabbit small intestine

The migration of intestinal epithelial cells from the crypts to the tips of villi is associated with progressive cell differentiation. The changes in Na⁺-pump levels during migration have been measured in epithelial cells isolated from rabbit small intestine. A significant proportion of ouabain-sensitive ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in the cell homogenates was latent but could be unmasked by detergent treatment. Highest detergent activation was observed in villus cells. The distribution of pumping sites was also assessed by measuring ouabain binding to intact cells. The kinetics of specific binding was consistent with the interaction of the cardiac glycoside with a single population of binding sites with an apparent $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ of around 10^?7 M. Both enzyme assay and ouabain-binding measurements suggest that a 2–3-fold increase in the number of Na⁺-pumping sites accompanies cell differentiation in rabbit jejunal epithelium. This increase in pumping capacity might be an adaptation of the cells to their absorptive function.     

Occurrence of N-malonyl-D-alanine in pea seedlings

One of the ninhydrin-negative alanine conjugates isolated from pea seedlings was identified as $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$.The identification of this conjugate was carried out by a comparison of its gas-liquid chromatographic and mass spectrometric properties, and its nuclear magnetic resonance and infrared spectra with those of synthetic $\text{N-}\text{malonyl}\text{-}\text{D}\text{-}\text{alanine}$. The alanine in the conjugate was shown to be present as the D-isomer by enzymatic and chromatographic analyses.     

The ROSA26 LacZ-neo R insertion confers resistance to mammary tumors in Apc Min/+ mice

B6.129S7-Gtrosa26 (ROSA26) mice carry a LacZ-neo ^R insertion on Chromosome (Chr) 6, made by promoter trapping with AB1 129 ES cells. Female C57BL/6J Apc ^Min /+ (B6 Min/+) mice are very susceptible to the induction of mammary tumors after treatment with ethylnitrosourea (ENU). However, ENU-treated B6 mice carrying both Apc ^Min and ROSA26 are resistant to mammary tumor formation. Thus, ROSA26 mice carry a modifier of Min-induced mammary tumor susceptibility. We have previously mapped the modifier to a 4-cM interval of 129-derived DNA that also contains the ROSA26 insertion. Here we report additional evidence for the effect of the ROSA26 insertion on mammary tumor formation. To test the hypothesis that the resistance was due to a linked modifier locus, we utilized two approaches. We have derived and tested two lines of mice that are congenic for 129-derived DNA within the minimal modifier interval and show that they are as susceptible to mammary tumors as are B6 mice. Additionally, we analyzed a backcross population segregating for the insertion and show that mice carrying the insertion are more resistant to mammary tumor development than are mice not carrying the insertion. Thus, the resistance is not due to a 129-derived modifier allele, but must be due to the ROSA26 insertion. In addition, the effect of the ROSA26 insertion can be detected in a backcross population segregating for other mammary modifiers. Received: 29 December 2000 / Accepted: 4 April 2001     

Inhibition of the (Na+ + K+)-dependent ATPase by inorganic phosphate

Inhibition of the $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase by inorganic phosphate, P_i, was examined in terms of product inhibition of the various activities catalyzed by an enzyme preparation from rat brain, and considered in terms of the specific transport processes of the membrane Na⁺,K⁺-pump that these activities reflect. The K⁺-dependent phosphatase activity of the enzyme was most sensitive to P_i, and inhibition was competitive toward the substrate, nitrophenyl phosphate, as would be expected if P_i were released from the same enzyme form that bound substrate. However, this enzymatic activity does not seem to represent a transport process, and thus a cyclical discharge of K⁺ may not be involved. The Na⁺-dependent exchange activity was unaffected by P_i, in accord with the absence of P_i release in the reaction sequence. For the corresponding Na⁺/Na⁺ exchange function of the pump, which reportedly does not involve ATP hydrolysis either, prior release of P_i obviously cannot be required for Na⁺ discharge. With the Na⁺-dependent ATPase activity, measured using micromolar concentrations of ATP, P_i inhibited, but far less than with the phosphatase activity, and inhibition was not competitive toward ATP. Moreover, inhibition decreased as the Na⁺ concentration was raised from 10 to 100 mM. This elevated concentration of Na⁺ also led to substrate inhibition. For this ATPase activity, and the corresponding transport process, uncoupled Na⁺ efflux, the findings suggest that Na⁺ discharge follows P_i release, in contrast to Na⁺/Na⁺ exchange. The $\text{(}\text{Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-dependent}$ ATPase activity, measured with millimolar concentrations of ATP and reflecting the coupled Na⁺,K⁺-transport function, was similarly sensitive to P_i, and again inhibition was not competitive toward ATP. However, in this case inhibition did not increase as the Na⁺ concentration was lowered. For this activity, and the associated transport process, the site of Na⁺ discharge in the overall reaction sequence remains unresolved.     

The insect brain (Na+ + K+)-ATPase Binding of ouabain in the hawk moth, Manduca sexta

(1) A quantitative study has been made of the binding of ouabain to the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ in homogenates prepared from brain tissue of the hawk moth, Manduca sexta. The results have been compared to those obtained in bovine brain microsomes. (2) The insect brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ will bind ouabain either in the presence of Mg²⁺ and P_i, (‘Mg²⁺, P_i’ conditions) or in the presence of Na⁺, Mg²⁺, and an adenine nucleotide (‘nucleotide’ conditions) as is the case for the bovine brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$. The binding conditions did not alter the total number of receptor sites measured at high ouabain concentrations in either tissue. (3) Potassium ion decreases the affinity (increases the $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$) of ouabain to the M. sexta brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ under both binding conditions. However, ouabain binding is more sensitive to K⁺ inhibition under the nucleotide conditions. In bovine brain ouabain binding is equally sensitive to K⁺ inhibition under the both conditions. (4) The enzyme-ouabain complex has a rate of dissociation that is 10-fold faster in the M. sexta preparation than in the bovine brain preparation. Because of this, the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ has a higher $\text{K}{}_{\mathrm{<mtext>D</mtext>}}$ for ouabain binding and is less sensitive to inhibition by ouabain than the bovine brain enzyme. (5) This data supports the hypothesis that two different conformational states of the M. sexta ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ can bind ouabain.