Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.     

Glutaraldehyde crosslinking analysis of the C12E8 solubilized H,K-ATPase

A soluble porcine H,K-ATPase preparation was obtained with the nonionic detergent, C12E8. ATP hydrolysis by the soluble H,K-ATPase was stimulated with respect to the native preparation at pH 6.1, while the K(+)-phosphatase activity was comparable to the native enzyme. The soluble enzyme demonstrated characteristic ligand-dependent effects on ATP hydrolysis, including ATP activation of K(+)-stimulated hydrolysis with a K0.5 of 28 +/- 4 microM ATP, and inhibition with an IC50 of 2.1 mM ATP. The activation and inhibition of ATP hydrolysis by K+ was also observed with a K0.5 for activation of 2.8 +/- 0.4 mM KCl at 2.0 mM ATP (pH 6.1) and inhibition with an IC50 of 135 mM KCl at 0.05 mM ATP. 2-Methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080), a specific inhibitor of the native H,K-ATPase, competitively inhibited the K(+)-stimulated activity with a Ki of 0.035 microM. The soluble enzyme was stable with a t0.5 for ATPase activity of 6 h between 4 and 11 degrees C. The demonstration of these related ligand responses in the catalytic reactions of the soluble preparation indicates that it is an appropriate medium for investigation of the subunit associations of the functional H,K-ATPase. Subunit associations of the active soluble enzyme were assessed following treatment with the crosslinking reagent, glutaraldehyde. The distribution of crosslinked particles was independent of the soluble protein concentration in the crosslinking buffer within the protein range 0.3 to 2.0 mg/ml or the detergent to protein ratio varied from 1 to 15 (w/w). The crosslinked pattern was unaffected by the presence or absence of K during crosslinking or nucleotide concentration. These observations suggest that crosslinking occurs in associated subunits that do not undergo rapid associations dependent upon enzyme turnover. Phosphorylation of the soluble enzyme with 0.1 mM MgATP produced a phosphoprotein at 94 kDa. A phosphoprotein obtained after glutaraldehyde treatment exhibited identical electrophoretic mobility to the crosslinked particle identified by silver stain. Glutaraldehyde treatment of soluble protein fractions resolved on a linear 10-35% glycerol gradient revealed several smaller peptides partially resolved from the crosslinked pump particle, but no active fraction enriched in the monomeric H,K-ATPase. This data indicates that the functional porcine gastric H,K-ATPase is organized as a structural dimer.     

Glu-857 moderates K+-dependent stimulation and SCH 28080-dependent inhibition of the gastric H,K-ATPase.

The rabbit H,K-ATPase alpha- and beta-subunits were transiently expressed in HEK293 T cells. The co-expression of the H,K-ATPase alpha- and beta-subunits was essential for the functional H,K-ATPase. The K+-stimulated H,K-ATPase activity of 0.82 +/- 0.2 micromol/mg/h saturated with a K0.5 (KCl) of 0.6 +/- 0.1 mM, whereas the 2-methyl-8-(phenylmethoxy)imidazo[1,2a]pyridine-3-acetonitrile (SCH 28080)-inhibited ATPase of 0.62 +/- 0.07 micromol/mg/h saturated with a Ki (SCH 28080) of 1.0 +/- 0.3 microM. Site mutations were introduced at the N,N-dicyclohexylcarbodiimide-reactive residue, Glu-857, to evaluate the role of this residue in ATPase function. Variations in the side chain size and charge of this residue did not inhibit the specific activity of the H,K-ATPase, but reversal of the side chain charge by substitution of Lys or Arg for Glu produced a reciprocal change in the sensitivity of the H,K-ATPase to K+ and SCH 28080. The K0.5 for K+stimulated ATPase was decreased to 0.2 +/-.05 and 0.2 +/-.03 mM, respectively, in Lys-857 and Arg-857 site mutants, whereas the Ki for SCH 28080-dependent inhibition was increased to 6.5 +/- 1.4 and 5.9 +/- 1.5 microM, respectively. The H,K-ATPase kinetics were unaffected by the introduction of Ala at this site, but Leu produced a modest reciprocal effect. These data indicate that Glu-857 is not an essential residue for cation-dependent activity but that the residue influences the kinetics of both K+ and SCH 28080-mediated functions. This finding suggests a possible role of this residue in the conformational equilibrium of the H,K-ATPase.     

Cation selectivity of gastric H,K-ATPase and Na,K-ATPase chimeras.

Chimeras of the catalytic subunits of the gastric H,K-ATPase and Na, K-ATPase were constructed and expressed in LLC-PK1 cells. The chimeras included the following: (i) a control, H85N (the first 85 residues comprising the cytoplasmic N terminus of Na,K-ATPase replaced by the analogous region of H,K-ATPase); (ii) H85N/H356-519N (the N-terminal half of the cytoplasmic M4-M5 loop also replaced); and (iii) H519N (the entire front half replaced). The latter two replacements confer a decrease in apparent affinity for extracellular K+. The 356-519 domain and, to a greater extent, the H519N replacement confer increased apparent selectivity for protons relative to Na+ at cytoplasmic sites as shown by the persistence of K+ influx when the proton concentration is increased and the Na+ concentration decreased. The pH and K+ dependence of ouabain-inhibitable ATPase of membranes derived from the transfected cells indicate that the H519N and, to a lesser extent, the H356-519N substitution decrease the effectiveness of K+ to compete for protons at putative cytoplasmic H+ activation sites. Notable pH-independent behavior of H85N/H356-519N at low Na+ suggests that as pH is decreased, Na+/K+ exchange is replaced largely by (Na+ + H+)/K+ exchange. With H519N, the pH and Na+ dependence of pump and ATPase activities suggest relatively active H+/K+ exchange even at neutral pH. Overall, this study provides evidence for important roles in cation selectivity for both the N-terminal half of the M4-M5 loop and the adjacent transmembrane helice(s).     

The beta-subunits of Na+,K+-ATPase and gastric H+,K+-ATPase have a high preference for their own alpha-subunit and affect the K+ affinity of these enzymes

The alpha- and beta-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the beta-subunit of H+,K+-ATPase (NaKalphaHKbeta) showed an ATPase activity, which was only 12 +/- 4% of the activity of the Na+,K+-ATPase with its own beta-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the beta-subunit of Na+,K+-ATPase (HKalphaNaKbeta) showed an ATPase activity which was 9 +/- 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKalphaHKbeta was decreased, while the apparent K+ affinity of the opposite hybrid HKalphaNaKbeta was increased. The hybrid NaKalphaHKbeta could be phosphorylated by ATP to a level of 21 +/- 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKalphabeta and NaKalphaHKbeta of 8800 +/- 310 min-1 and 4800 +/- 160 min-1, respectively. Measurements of phosphorylation of the HKalphaNaKbeta and HKalphabeta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.     

Vanadate binding to the gastric H,K-ATPase and inhibition of the enzyme's catalytic and transport activities

Vanadate inhibition of the catalytic and transport activities of the gastric magnesium-dependent, hydrogen ion transporting, and potassium-stimulated adenosinetriphosphatase (EC 3.6.1.3) (H,K-ATPase) has been studied. The principal experiment observations are the following: (1) Inhibition of adenosine 5'-triphosphate (ATP) hydrolysis is biphasic. Vanadate binding with a stoichiometry of 1.5 nmol mg-1 approximately halves K+-stimulated ATPase activity at physiological temperature. The remaining activity is inhibited by binding an additional 1.5 nmol mg-1 vanadate with lower apparent ions bind specifically to gastric vesicles with two affinities. Vanadate binding in the presence of nucleotide is compatible with competition for the kinetically defined high-affinity and low-affinity ATP sites. (3) Vanadate inhibits phosphoenzyme formation and the K+-stimulated p-nitrophenyl phosphatase activity of the enzyme monophasically. A maximum of 1.5 nmol mg-1 acid-stable phosphoenzyme is formed. The half-time for vanadate dissociation from the site that inhibits p-nitrophenyl phosphate hydrolysis is 5 min (4) At most, 3 nmol mg-1 vanadate is required to inhibit proton transport. The simplest interpretation of the data is that vanadate inhibits the H,K-ATPase by binding competitively with ATP at two catalytic sites. Different catalytic mechanisms at the high-affinity and low-affinity sites are suggested by the different stoichiometries found for vanadate binding and phosphoenzyme formation.     

Mechanisms of urinary K+ and H+ excretion: primary structure and functional expression of a novel H,K-ATPase

The kidney plays an essential role in regulating potassium and acid balance. A major site for these regulations is in the collecting tubule. In the present study, we report the primary sequence of a novel alpha subunit of the P-ATPase gene family, which we isolated from the urinary bladder epithelium of the toad Bufo marinus, the amphibian equivalent of the mammalian collecting tubule. The cDNA encodes a protein of 1,042 amino acids which shares approximately 67% identity with the alpha 1 subunit of the ouabain-inhibitable Na,K-ATPase and approximately 69% identity with the alpha subunit of the SCH28080- inhibitable gastric H,K-ATPase. When coexpressed in Xenopus oocytes with a beta subunit isolated from the same cDNA library, the ATPase is able to transport rubidium (a potassium surrogate) inward, and hydrogen outward, leading to alkalization of the intracellular compartment and acidification of the external medium. The novel ATPase has a unique pharmacological profile showing intermediate sensitivity to both ouabain and SCH28080. Our findings indicate that the bladder ATPase is a member of a new ion motive P-ATPase subfamily. The bladder ATPase is expressed in the urinary tract but not in the stomach or the colon. This H,K-ATPase may be one of the molecules involved in H+ and K+ homeostasis, mediating the transport of these ions across urinary epithelia and therefore regulating their urinary excretion.     

Studies on (K+ + H+)-ATPase. VI. Determination on the molecular size by radiation inactivation analysis

(1) A (K+ + H+)-ATPase containing membrane fraction, isolated from pig gastric mucosa, has been further purified by means of zonal electrophoresis, leading to a 20% increase in specific activity and an increase in ratio of (K+ + H+)-ATPase to basal Mg2+-ATPase activity from 9 to 20. (2) The target size of (Na+ + K+)-ATPase, determined by radiation inactivation analysis, is 332 kDa, in excellent agreement with the earlier value of 327 kDa obtained from the subunit composition and subunit molecular weights. This shows that the Kepner-Macey factor of 6.4 X 10(11) is valid for membrane-bound ATPases. (3) The target size of (K+ + H+)-ATPase is 444 kDa, which, in connection with a subunit molecular weight of 110000, suggests a tetrameric assembly of the native enzyme. The ouabain-insensitive K+-stimulated p-nitrophenylphosphatase activity has a target size of 295 kDa. (4) In the presence of added Mg2+ the target sizes of the (K+ + H+)-ATPase and its phosphatase activity are decreased by about 15%, while that for the (Na+ + K+)-ATPase is not significantly changed. This observation is discussed in terms of a Mg2+-induced tightening of the subunits composing the (K+ + H+)-ATPase molecule.     

Some properties of the adenosine triphosphatase systems of two yeast species,saccharomyces cerevisiae and rhodotorula glutinis

1. Total ATPase levels were determined in homogenate fractions of baker's yeast, Saccharomyces cerevisiae K and Rhodotorula glutinis. The maximum ATPase activities in 8000 X g supernatant of the three yeast strains were 6.0, 1.9, and 2.2 mmol Pih-1 (gDS)-1, respectively; the activities in the sediment were somewhat higher. Exponential cells of S. cerevisiae K and R. glutinis exhibited higher ATPase levels than did the stationary cells. 2. The total ATPase activity in both yeast species showed a maximum at ph 6.8 a minimum at pH 7.2, and another broader masimum around pH 8.0. 3. No significant NaK-ATPase activity was detected in baker's yeast, in either the exponential or the stationary cells of R. glutinis, and in exponential S. cerevisiae K cells in the pH range of 6.0-9.3. 4. Stationary cells of S. cerevisiae K exhibited, at pH 7.0-8.5, A Na,K-ATPase activity attaining 9% of total ATPase level. 5.3 X 10(-3) M phenylmethyl sulphonyl fluoride had no effect on the total ATPase level in S. cerevisiae and inhibited the activity in R. glutinis by 25%; it did not bring forth any Na,K-ATPase activity apart from that found in its absence. 6. 1.5 M urea lowered the ATPase activity in R. glutinis by 68% but had no effect on S. cerevisiae cells. 10(-5) M dicyclohexylcarbodiimide suppressed the ATPase activity in S. cerevisiae and R. glutinis by 74 and 79%, respectively. Neither agent revealed and additional Na,K-ATPase activity. 7. The comparison of Na,K-ATPase activities with data on K+ fluxes across the yeast plasma membrane suggested that even with the lower flux values the Na,K-ATPase, even if present, would account for a mere 40% of transported ions. The results imply that the active ion transport in yeasts is energized by mechanisms other than the Na,K-ATPase.     

Different molecular sizes for Na(+)-dependent phosphonoformic acid binding and phosphate transport in renal brush border membrane vesicles

We compared several features of Na(+)-dependent phosphono[14C]formic acid (PFA) binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles. From kinetic analyses, we estimated an apparent Km for PFA binding of 0.86 mM, an order of magnitude greater than that for phosphate and the high-affinity phosphate transport system. A hyperbolic Na(+)-saturation curve for PFA binding and a sigmoidal Na(+)-saturation curve for phosphate transport were demonstrated; based on these data, we estimated stoichiometries of 1:1 for Na+/PFA and 2:1 for Na+/phosphate. By radiation inactivation analysis, target sizes for brush border membrane protein(s) mediating Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport corresponded to molecular masses of 555 +/- 32 kDa and 205 +/- 36 kDa, respectively. Similar analysis of the phosphate-inhibitable component of Na(+)-dependent PFA binding gave a target size of 130 +/- 28 kDa. We also demonstrated that phosphate deprivation, which elicits a 2.6-fold increase in brush border membrane Na(+)-dependent phosphate transport, had no effect on either Na(+)-dependent PFA binding or on the target size for PFA binding. However, phosphate deprivation appeared to increase the target size for phosphate transport (from 255 +/- 32 to 335 +/- 75 kDa (P less than 0.01]. In summary, we present evidence for several differences between Na(+)-dependent PFA binding and Na(+)-dependent phosphate transport in rat renal brush border membrane vesicles and suggest that PFA may not interact exclusively with the proteins mediating Na(+)-phosphate co-transport.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

The human non-gastric H,K-ATPase has a different cation specificity than the rat enzyme

The primary sequence of non-gastric H,K-ATPase differs much more between species than that of Na,K-ATPase or gastric H,K-ATPase. To investigate whether this causes species-dependent differences in enzymatic properties, we co-expressed the catalytic subunit of human non-gastric H,K-ATPase in Sf9 cells with the beta(1) subunit of rat Na,K-ATPase and compared its properties with those of the rat enzyme (Swarts et al., J. Biol. Chem. 280, 33115-33122, 2005). Maximal ATPase activity was obtained with NH(4)(+) as activating cation. The enzyme was also stimulated by Na(+), but in contrast to the rat enzyme, hardly by K(+). SCH 28080 inhibited the NH(4)(+)-stimulated activity of the human enzyme much more potently than that of the rat enzyme. The steady-state phosphorylation level of the human enzyme decreased with increasing pH, [K(+)], and [Na(+)] and nearly doubled in the presence of oligomycin. Oligomycin increased the sensitivity of the phosphorylated intermediate to ADP, demonstrating that it inhibited the conversion of E(1)P to E(2)P. All three cations stimulated the dephosphorylation rate dose-dependently. Our studies support a role of the human enzyme in H(+)/Na(+) and/or H(+)/NH(4)(+) transport but not in Na(+)/K(+) transport.     

Solubilization and reconstitution of the gastric H,K-ATPase

Proteoliposomes containing the hog gastric H+,K+-ATPase were prepared from cholate and n-octyl glucoside extracts of native microsomes. Experiments were presented which show reconstitution-dependent selective purification of a 94-kDa peptide capable of Rb+/Rb+ exchange and active H+ transport. The absence of selective enrichment of residual protein contamination in this material suggests but does not prove that those transport reactions are attributable only to the 94-kDa peptide. Transport demonstrated inhibitor sensitivity and cation specificity comparable to the microsomal gastric ATPase. In K2SO4 media the H+ transport reaction was protonophore insensitive and correlated with MgATP-dependent 86Rb+ extrusion. This and other evidence suggested that active transport occurs via electroneutral H+in for K+out exchange. 86Rb+ exchange (uptake) in the proteoliposomes demonstrated both saturable and nonsaturable components. At a K0.5 = 1.5 mM, saturable 86Rb+ uptake accounted for about 90% of Rb+ influx. The vanadate-sensitive cation exchange indicated that the ATPase was reconstituted asymmetrically into the proteoliposomes (70% cis-/30% trans-vanadate site). 86Rb+ exchange was inhibited by ATP and stimulated about 2-fold by low Mg2+ and 5 mM phosphate. These ligand effects and the demonstration of comparable rates of passive exchange and active Rb+ efflux suggest that passive K+ exchange is not severely limited by a K+-occluded enzyme form in the H,K-ATPase. A model compatible with this hypothesis is suggested.     

Thiophosphorylation of hog gastric (H+ + K+)-ATPase membranes by endogenous protein kinases

(H+ + K+)-ATPase-enriched membranes from hog stomachs were tested for their capacity to autophosphorylate using [gamma-32P]ATP or [gamma-35S]ATP[S] as phosphate donors. The radioactive polypeptides were characterized by SDS-PAGE. In the presence of Mg2+ and 5 microM [gamma-32P]ATP, rapid and transient incorporation of 32P occurred at 0 degrees C. Radioactivity was essentially found in the major polypeptide of the material, the 95 kDa subunit of (H+ + K+)-ATPase. Under the same experimental conditions, thiophosphorylation was slower and reached a plateau within 1 h. Incorporation levels were higher with manganese than with magnesium. After one hour at 0 degrees C, and in the presence of 10 mM manganese and 5 microM ATP[S], 0.58 +/- 0.06 nmoles of thiophosphate were incorporated per mg of protein. Twenty seven percent of the thiophosphorylated amino acids were acylphosphates i.e. likely to be the ATPase thiophosphointermediate. The remaining thiophosphorylated amino acids (73%) were thought to be produced by protein kinases. This was supported by the autoradiographies of membrane SDS-PAGE which indicated that, in addition to the 95 kDa ATPase subunit, other polypeptides were thiophosphorylated especially at 108, 58, 47, 45 and 36-40 kDa. A previous study had provided strong evidence that chloride transport in gastric microsomes, is modulated by a protein kinase-dependent phosphorylation (Soumarmon, A., Abastado, M., Bonfils, S. and Lewin M.J.M. (1980) J. Biol. Chem. 255, 11682-11687). In the present work, we demonstrate that the peptidic inhibitor of cAMP-dependent protein kinases decreased thiophosphorylation of a 45 kDa polypeptide. We suggest that this polypeptide could be regarded as a candidate for the role of chloride transporter or chloride transport regulator.     

The conformation of H,K-ATPase determines the nucleoside triphosphate (NTP) selectivity for active proton transport

The gastric H,K-ATPase is related to other cation transport ATPases, for example, Na,K-ATPase and Ca-ATPase, which are called E1-E2 ATPases in recognition of conformational transitions during their respective transport and catalytic cycles. Generally, these ATPases cannot utilize NTPs other than ATP for net ion transport activity. For example, under standard assay conditions, rates of NTP hydrolysis and H+ pumping by the H,K-ATPase for CTP are about 10% of those for ATP and undetectable with GTP, ITP, and UTP. However, we observed that H,K-ATPase will catalyze NTP/ADP phosphate exchange at similar rates for all of these NTPs, suggesting that a common phosphoenzyme intermediate is formed. The present study was undertaken to evaluate the specificity of nucleotides to power the H,K-ATPase and several of its partial reactions, including NTP/ADP exchange, K+-catalyzed phosphatase activity, and proton pumping. Results demonstrate that under conditions that promote the conformational change of the K+ bound form of the enzyme, K.E2, to E1, all NTPs tested support K+-stimulated NTPase activity and H+ pumping up to 30-50% of that with ATP. These conditions include (1) the presence of ADP as well as the NTP energy source and (2) reduced K+ concentration on the cytoplasmic side to approximately 0. These data conform to structural models for E1-E2 ATPases whereby adenosine binding promotes the K.E2 to E1 conformational change and K+ deocclusion.     

Thallium binding to native and radiation-inactivated Na+/K+-ATPase

The number of high-affinity K+-binding sites on purified Na+/K+-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl+, under conditions (low ionic strength, absence of Na+ and Tris+) where the enzyme is in the E2-form. Na+/K+-ATPase has two identical Tl+ sites per ADP site, and the dissociation constant varies between 2 and 9 microM. These values are identical to those for Tl+ occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl+ to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl+-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl+ binding is 71 kDa, and the RIS for Tl+ binding with original affinity is approx. 190 kDa, equal to that of Na+/K+-ATPase activity and to that for Tl+ occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na+/K+-ATPase are closely associated. The 71 kDa value obtained for total Tl+ sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl+-binding experiments reported thus supports the notion that radiation inactivation of Na+/K+-ATPase is a stepwise rather than an all or none process.     

Effect of subunit interactions on enzymatic activity of glutathione S-transferases: a radiation inactivation study.

The glutathione S-transferases are a family of dimeric enzymes. Three isozymes from the alpha family, termed YaYa, YaYc, and YcYc, and three from the mu family, termed Yb1Yb1, Yb1Yb2, and Yb2Yb2, were purified from rat liver. Binding studies were performed by equilibrium dialysis using a radiolabeled product, S(-)[14C](dinitrophenyl)glutathione. Each isozyme contained two independent binding sites which had equal affinity for the ligand. The presence of two independent active sites per enzyme dimer suggests that each subunit contains a complete active site. This conclusion was examined further using radiation inactivation which also allowed for assessment of the importance of subunit interactions in catalytic activity. The activity target size of YaYa (47 kDa) was significantly larger than the protein monomer target size (31 kDa); similarly the activity target size of YaYc was that of the dimer (54 kDa). In contrast, the activity target sizes of Yb1Yb1 and Yb2Yb2 were the same, being 35 and 29 kDa, respectively, and the protein monomer target size of Yb1Yb1 also was similar, being 32 kDa. These data indicate that interactions between subunits are critical for the maintenance of enzymatic activity of alpha class enzymes whereas each subunit of the two mu class proteins is capable of independent catalytic activity.     

Some peculiarities of functioning of H+-ATPase from the membranes of the anaerobic bacterium Lactobacillus casei

Radiation inactivation analysis gave the target sizes of 176 +/- 5 kDa and 275 +/- 33 kDa for ATPase from anaerobic Lactobacillus casei and aerobic Micrococcus luteus bacteria respectively. The values are close to the known molecular masses of the enzymes. Thus, to function the L. casei ATPase, like the F1-ATPases, requires a complete structure composed of all the enzyme subunits. L. casei ATPase is inhibited by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole owing to modification of an amino acid residue(s) with pK greater than 8.5. L. casei ATPase consists of six identical subunits and differs from alpha 3 beta 3 gamma delta epsilon-type F1-ATPases in a number of catalytic properties. Namely, ATP hydrolysis under the 'unisite' conditions proceeds at a relatively high rate suggesting the absence of cooperative interactions between the catalytic sites. Contrary to mitochondrial F1-ATPase. L. casei ATPase does not form an inactive complex with ADP. These findings imply essential differences in the operating mechanism for L. casei ATPase and F1 ATPase.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Comparison of the substrate dependence properties of the rat Na,K-ATPase alpha 1, alpha 2, and alpha 3 isoforms expressed in HeLa cells.

The role of multiple isoforms for the alpha subunit of Na,K-ATPase is essentially unknown. To examine the functional properties of the three alpha subunit isoforms, we developed a system for the heterologous expression of Na,K-ATPase in which the enzymatic activity of each isoform can be independently analyzed. Ouabain-resistant forms of the rat alpha 2 and alpha 3 subunits were constructed by site-directed mutagenesis of amino acid residues at the extracellular borders of the first and second transmembrane domains (L111R and N122D for alpha 2 and Q108R and N119D for alpha 3). cDNAs encoding the rat alpha 1 subunit, which is naturally ouabain-resistant, and rat alpha 2 and alpha 3, which were mutated to ouabain resistance (designated rat alpha 2* and rat alpha 3*, respectively) were cloned into an expression vector and transfected into HeLa cells. Resistant clones were isolated and analyzed for ouabain-inhibitable ATPase activity in the presence of 1 microM ouabain, which inhibits the endogenous Na,K-ATPase present in HeLa cells (I50 approximately equal to 10 nM). The remaining activity corresponds to Na,K-ATPase molecules containing the transfected rat alpha 1, rat alpha 2*, or rat alpha 3* isoforms. Utilizing this system, we examined Na+, K+, and ATP dependence of enzyme activity. Na,K-ATPase molecules containing rat alpha 1 and rat alpha 2* exhibited a 2-3-fold higher apparent affinity for Na+ than those containing rat alpha 3* (apparent KNa+ (millimolar): rat alpha 1 = 1.15 +/- 0.13; rat alpha 2* = 1.05 +/- 0.11; rat alpha 3* = 3.08 +/- 0.06). Additionally, rat alpha 3* had a slightly higher apparent affinity for ATP (in the millimolar concentration range) compared with rat alpha 1 or rat alpha 2* (apparent K0.5 (millimolar): rat alpha 1 = 0.43 +/- 0.12; rat alpha 2* = 0.54 +/- 0.15; rat alpha 3* = 0.21 +/- 0.04) and all three isoforms has similar apparent affinities for K+ (apparent KK+: rat alpha 1 = 0.45 +/- 0.01; rat alpha 2* = 0.43 +/- 0.004; rat alpha 3* = 0.27 +/- 0.01). This study represents the first comparison of the functional properties of the three Na,K-ATPase alpha isoforms expressed in the same cell type.