Protective effect of Na+ and K+ against inactivation of (Na+ + K+-ATPase by high concentrations of 2-mercaptoethanol at high temperatures

Purified dog kidney (Na⁺ + K⁺)-ATPase (EC 3.6.1.3) was inactivated with high concentrations of 2-mercaptoethanol at 50–55°C. The inactivation was prevented by NaCl or KCl, with KCl being more effective than NaCl (the former ion being about one order more efficient under a typical set of experimental conditions). A disulfide bond in the β-subunit of the enzyme protein was prevented from reductive cleavage by NaCl or KCl in accordance with protection of the enzyme activity. Choline chloride did not exert a significant protective effect over a similar concentration range. (Na⁺ + K⁺)-ATPase was also inactivated with high concentrations of 2-mercaptoethanol in the presence of low concentrations of dodecyl sulfate. This inactivation was also prevented by NaCl or KCl, with the latter being again more efficient than the former. These results indicate that Na⁺ and K⁺ bound to their respective ion-binding sites on the α-subunit exert a protective effect on a disulfide bond on the β-subunit. This suggests some sort of interaction between the α- and the β-subunits.     

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.     

Reversible in activation of purified (Na+ + K+)-ATPase from human renal tissue by cyclic AMP-dependent protein kinase.

Human renal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations which exhibited a non-linear reaction rate, contained high levels of membrane-bound cyclic AMP-dependent protein kinase, while this latter activity was much less or absent in purified preparations. A non-linear reaction rate was observed in a purified preparation of (Na+ + K+)-ATPase by reconstituting the enzyme into lipid vesicles with cyclic AMP-dependent protein kinase. The addition of cyclic AMP to the ATPase assay of these lipid vesicles inactivated the (Na+ + K+)-ATPase. The cytoplasmic fraction of the cell contained a nondialyzable factor, which prevented (or reversed) the cyclic AMP-mediated inactivation of the enzyme.     

Involvement of sulfhydryl groups in the inhibition of brain (Na+ + K+)-ATPase by pyrithiamin

Brain (Na+ + K+)-ATPase was protected by low concentrations of GSH from the inhibitory effect of pyrithiamin. The possible involvement of sulfhydryl groups in the inhibition was then studied by comparing the effect of pyrithiamin with that of N-ethylmaleimide on the enzyme. The treatment of rat brain (Na+ + K+)-ATPase with thesee inhibitors caused a significant decrease in reactivity of the enzyme to N-ethyl[3H]maleimide. N-Ethylmaleimide, like pyrithiamin, inhibited the partial reactions of (Na+ + K+)-ATPase system in parallel with the inhibition of the overall reaction. An SDS-polyacrylamide gel electrophoresis procedure indicated that pyrithiamin and N-ethylmaleimide inhibited Na+-dependent phosphorylation of the alpha(+) form of rat brain (Na+ + K+)-ATPase more than that of alpha, though the selectivity for the alpha(+) seemed to be higher with the former inhibitor than in the latter. The treatment also decreased sensitivity of the enzyme to ouabain inhibition. However, pyrithiamin- and N-ethylmaleimide-induced inactivations of the enzyme differed in the efficacy of GSH for protection and in the effect of the kind of ligands present during the reaction. Furthermore, pyrithiamin did not appear to interact directly with sulfhydryl groups, but caused the formation of disulfide in bovine brain (Na+ + K+)-ATPase. In contrast to N-ethylmaleimide, pyrithiamin did not affect the sulfhydryl-enzymes such as alcohol dehydrogenase and L-alanine dehydrogenase. It is concluded that pyrithiamin modifies the functional sulfhydryl groups of brain (Na+ + K+)-ATPase in a way different from N-ethylmaleimide and causes a structural change and inactivation of the enzyme.     

Solubilized (Na+ + K+)-ATPase from shark rectal gland and ox kidney--an inactivation study

The bi-exponential time-course of detergent inactivation at 37 degrees C of C12E8-solubilized (Na+ + K+)-ATPase from shark rectal glands and ox kidney was investigated. The data for shark enzyme, obtained at detergent/protein weight ratios between 2 and 16, are interpreted in terms of a simple model where the membrane bound enzyme is solubilized predominantly as (alpha-beta)2 diprotomers at low detergent concentrations and as alpha-beta protomers at high C12E8 (octaethyleneglycoldodecylmonoether) concentrations. It is observed that the protomers are inactivated 15-fold more rapidly than the diprotomers, and that the rate of inactivation of both oligomers is proportional to the detergent/protein ratio. Inactivation of kidney enzyme was biexponential with a very rapid inactivation of up to 40% of the enzyme activity. The observed rate of inactivation of the slower phase varied with the detergent/protein ratio, but the inactivation pattern for the kidney enzyme could not readily be accommodated within the model for inactivation of the shark enzyme. The rates of inactivation at 37 degrees C were about the same in KCl and NaCl, i.e., in the E2(K) and E1 X Na forms, for both enzymes.     

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.     

Myometrial (Na+ + K+)-activated ATPase and its Ca2+ sensitivity

Ouabain-sensitive (Na+ + K+)-ATPase activity in the rat myometrial microsome fraction could only be determined following detergent treatment. The (Na+ + K+)-ATPase activity manifested by detergent treatment proved very stable even to high concentrations of NaN3, in contrast Mg+-ATPase activity was reduced to about 30 percent of the control. The major part of the Mg2+-ATPase in the myometrial membrane preparation was found to be identical with the NaN3-sensitive ATP diphosphohydrolase capable of ATP and ADP hydrolysis. This monovalent-cation-insensitive ATP hydrolysis could be extensively reduced by DMSO. Furthermore DMSO prevented the inactivation of the (Na+ + K+)-ATPase activity. 10-100 microM Ca2+ inhibited the (Na+ + K+)-ATPase activity obtained in the presence of SDS by 15-50 percent. The Ca2+ sensitivity of the enzyme was considerably decreased if the proteins solubilized by the detergent had been separated from the membrane fragments by ultracentrifugation. The inhibitory effect could be regained by combining the supernatant with the pellet. Ca2+ sensitivity of the (Na+ + K+)-ATPase activity was preserved even after removal of the solubilized proteins provided that DMSO had been applied. It appears that a factor in the plasma membrane solubilized by SDS may be responsible for the loss of Ca2+ sensitivity of the (Na+ + K+)-ATPase activity, the solubilization of which can be prevented by DMSO.     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.     

Activation by lithium ions of the inside sodium sites in (Na+ + K+)-ATPase.

1. Purified pig kidney ATPase was incubated in 30--160 mM Tris-HCl with various monovalent cations. 130 mM LiCl stimulated a ouabain-sensitive ATP hydrolysis (about 5% of the maximal (Na+ + K) activity), whereas 160 mM Tris-HCl did not stimulate hydrolysis. Similar results were obtained with human red blood cell broken membranes. 2. In the absence of Na+ and with 130 mM LiCl, the ATPase activity as a function of KCl concentration showed an initial slight inhibition (50 micrometer KCl) followed by an activation (maximal at 0.2 mM KCl) and a further inhibition, which was total at mM KCl. In the absence of LiCl, the rate of hydrolysis was not affected by any of the KCl concentrations investigated. 3. The lithium-activation curve for ATPase activity in the absence of both Na+ and K+ had sigmoid characteristics. It also showed a marked dependence on the total LiCl + Tris-HCl concentration, being inhibited at high concentrations. This inhibition was more noticeable at low LiCl concentrations. 4. In the absence of Na+, 130 mM Li+ showed promoted phosphorylation of ATPase from 1 to 3 mM ATP in the presence of Mg2+. In enzyme treated with N-ethylmaleimide, the levels of phosphorylation in Li+-containing solutions, amounted to 40% of those in Na+- and up to 7 times of those in K+-containing solutions. 5. The total (Na+ + K+)-ATPase activity was markedly inhibited at high buffer concentrations (Tris-HCl, Imidazole-HCl and tetramethylammonium-HEPES gave similar results) in cases when either the concentration of Na+ or K+ (or both) was below saturation. On the other hand, the maximal (Na+ + K+)-ATPase activity was not affected (or very slightly) by the buffer concentration. 6. Under standard conditions (Tris-HCl + NaCl = 160 mM) the Na+-activation curve of Na+-ATPase had a steep rise between 0 and 2.5 mM, a fall between 2.5 and 20 mM and a further increase between 20 and 130 mM. With 30 mM Tris-HCl, the curve rose more steeply, inhibition was noticeable at 2.5 mM Na+ and was completed at 5 mM Na+. With Tris-HCl + NaCl = 280 mM, the amount of activation decreased and inhibition at intermediate Na+ concentrations was not detected.     

Substituting manganese for magnesium alters certain reaction properties of the (Na+ + K+)-ATPase

MnCl2 was partially effective as a substitute for MgCl2 in activating the K+- dependent phosphatase reaction catalyzed by a purified (Na+ + K+)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl2. Estimates of the concentration of free Mn2+ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187--3197). MnCl2 competed with MgCl2 as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl2 appreciable ouabain-inhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K+ as activator of the reaction and for Na+ as inhibitor were both decreased. For the (Na+ + K+)-ATPase reaction substituting MnCl2 for MgCl2 was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K+ was increased by the substitution, although that for Na+ was decreased as in the phosphatase reaction. Substituting MnCl2 also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [48V] -vanadate to the enzyme; furthermore, binding in the presence of MnCl2 was, unlike that with MgCl2, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na+ + K+)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution. These findings are considered in terms of Mn2+ at the divalent cation site being a better selector than Mg2+ of the E2 conformational states of the enzyme, states also selected by K+ and by dimethylsulfoxide and reactive with ouabain and vanadate; the E1 conformational states, by contrast, are those selected by Na+ and ATP, and also by Triton X-100.     

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.     

Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.     

Inhibition of gill (Na+ + K+)-ATPase in rainbow trout (Salmo gairdneri) by a purinedisulfide analog of adenosine triphosphate.

The effect of the adenosine triphosphate analog, 6,6'-dithiobis(inosinyl imidodiphosphate), (sIMP-PNP)2, was tested on the ouabain-sensitive (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and the ouabain-insensitive Mg2+ - ATPase in microsomes prepared from gill tissue of sea water-adapted rainbow trout, Salmo gairdneri. The (Na+ + K+)-ATPase was completely inhibited by low concentrations of (sIMP-PNP)2 (6 micrometer) but the Mg2+ - ATPase was unaffected by the inhibitor at concentrations as high as 28 micrometer, supporting the suggestion that the two activities represent separate enzymes. The specificity of inactivation could be demonstrated both at a physiological temperature (13 degrees C) and at 37 degrees C. The rates of inactivation were similar at both temperatures. Inactivation of the (Na+ + K+)-ATPase by (sIMP-PNP)2 was reversed by dithiothreitol, suggesting that the inhibitor forms a mixed disulfide with sulfhydryl groups on the enzyme. The inability of substrate (either ATP or its analog, adenyl-5'-yl imidodiphosphate) to protect against inactivation suggests that (sIMP-PNP)2 is reacting with sulfhydryl groups which are not associated with the active site.     

All three potential N-glycosylation sites of the dog kidney (Na+ + K+)-ATPase beta-subunit contain oligosaccharide

The beta-subunit of dog kidney (Na+ + K+)-ATPase is a sialoglycoprotein and contains three potential N-glycosylation sites. In this study, the oligosaccharide chains of purified dog kidney beta-subunit were labeled with tritium by oxidation with sodium periodate or galactose oxidase followed by NaB3H4 reduction. The beta-subunit was extensively digested by trypsin and the radioactive peptides were purified by HPLC. The enzyme, glycopeptidase A, which catalyzes the removal of N-linked oligosaccharide chains and the conversion of the glycosylated Asn residue to Asp, was used to demonstrate that a number of purified beta-subunit tryptic peptides were glycosylated. Amino-acid analysis of these beta-subunit peptides following glycopeptidase-A treatment revealed the expected Asn to Asp conversion for Asn-157, Asn-192 and Asn-264, demonstrating that all three potential N-glycosylation sites of the dog kidney beta-subunit are glycosylated. In addition, amino-acid sequence data suggest that a disulfide bond exists between Cys-158 and Cys-174.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

Kinetic properties of C12E8-solubilized (Na+ + K+)-ATPase

The properties of the rectal gland (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether ( C12E8 ) have been investigated. The kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that Km for ATP for the (Na+ + K+)-ATPase is about 30 microM for the solubilized enzyme and about 100 microM for the membrane-bound enzyme. The Na+-form (E1) and the K+-form (E2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and eosin fluorescence responses to Na+ and K+. The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95-100% pure supernatant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C12E8 /protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C12E8 is required both for solubilization and inactivation. It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C12E8 , and its advantages in the assay of detergent-solubilized (Na+ + K+)-ATPase are discussed. The results show that (Na+ + K+)-ATPase can be solubilized in C12E8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C12E8 -solubilized (Na+ + K+)-ATPase is therefore a good model system for a solubilized membrane protein.     

Phosphorylation of two isozymes of (Na+ + K+)-ATPase by inorganic phosphate

The phosphorylation of two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase by 32Pi was studied under equilibrium conditions in various enzyme preparations from rat medulla oblongata, rat cerebral cortex, rat cerebellum, rat kidney, guinea pig kidney, and rabbit kidney. In ouabain-sensitive (Na+ + K+)-ATPases such as the brain, guinea pig kidney, and rabbit kidney enzymes, ouabain stimulated the Mg2+-dependent phosphorylation at lower concentrations, while a higher concentration was required for the stimulation of rat kidney (Na+ + K+)-ATPase, an ouabain-insensitive enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that two isozymes of the brain (Na+ + K+)-ATPase were also phosphorylated by 32Pi in the presence of ouabain. The properties of the phosphorylation were compared between the medullar oblongata (referred to as alpha(+] and the kidney (referred to as alpha) (Na+ + K+)-ATPases. The steady-state level of phosphorylation was achieved faster in the kidney enzymes than in the medulla oblongata enzyme. Phosphorylation without ouabain was greater in the kidney enzymes than in the brain enzymes. Furthermore, the former enzymes were inhibited by K+ much more than the latter. These findings suggest that the two isozymes of (Na+ + K+)-ATPase differ in their conformational changes during enzyme turnover.     

Difference between neuronal and nonneuronal (Na+ + K+)-ATPases in their conformational equilibrium

Several experiments were carried out to study the difference between two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase in the conformational equilibrium. Rat brain (Na+ + K+)-ATPase was much more thermolabile than the kidney enzyme. Both enzymes were protected from heat inactivation not only by Na+ and K+, but also by choline in varying degrees, though there was a difference between the two enzymes in the protection by the ligands. The brain enzyme was partially protected from N-ethylmaleimide (NEM) inactivation by both Na+ and K+, but the effects of the ligands on NEM inactivation of the kidney enzyme were more complex. Though ligands differentially affected the thermostability and NEM sensitivity of the two enzymes, the effects were not simply related to the conformational states. The sensitivity of phosphoenzyme (EP) formed in the presence of ATP, Na+, and Mg2+ to ADP or K+ and K+-p-nitrophenyl phosphatase (pNPPase) was then studied as a probe of the differences in the conformational equilibrium between the two isozymes. The EP of the brain enzyme was partially sensitive to ADP, while those of the heart and kidney enzymes were not. At physiological Na+ concentrations the percentages of E1P formed by the brain and kidney enzymes were determined to be about 40-50 and 10-20% of the total EP, respectively. The hydrolytic activity of pNPP in the presence of Li+, a selective activator at catalytic sites of the reaction, was much higher in the kidney enzyme than in the brain enzyme. The inhibition of K+-stimulated pNPPase by ATP and Na+ was greater in the latter enzyme than in the former. These results suggest that neuronal and nonneuronal (Na+ + K+)-ATPases differ in their conformational equilibrium: the E1 or E1P may be more stable in the alpha(+) than in the alpha during the turnover, and conversely the E2 or E2P may be more stable in the latter than in the former.     

Effects of Na+ and K+ on Artemia salina (Na,K)-ATPase solubilized with a zwitterionic detergent, CHAPS

The membrane bound (Na,K)-ATPase prepared from Artemia salina nauplii was solubilized with a zwitterionic detergent, 3[3(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and then purified on a Bio-Gel A-1.5 m column in the presence of the detergent. 1) Upon solubilization, both NaCl and KCl protected the enzyme against loss of activity, KCl being more effective than NaCl. 2) Gel filtration of the solubilized enzyme on a Bio-Gel A-1.5 m column in the presence of 5 mM CHAPS resulted in loss of the enzyme activity even when one of the cations was added. Most of the phospholipids in the solubilized enzyme preparation were removed during the gel filtration (delipidation) and 10-25 phospholipids were left on a protomer (alpha beta) of the enzyme irrespective of the cation present during the gel filtration. With the addition of exogenous phospholipids, the activity was restored. The activity of the enzyme delipidated in the presence of KCl was restored to 3-4 times higher than in the case of that delipidated in the presence of NaCl. 3) Relipidation experiments with a fluorescent phospholipid, dansyl phosphatidylethanolamine (Dans-PE), suggested that the enzyme delipidated in the presence of KCl reassociated with phospholipids more firmly than the enzyme delipidated in the presence of NaCl. From these results we concluded that K+ stabilized the (Na,K)-ATPase more effectively than Na+, even when the enzyme was delipidated.