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.     

Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+)ATPase

A microsomal fraction rich in (Na+ + K+)ATPase activity has been isolated from the outer medulla of pig kidney. The ability of this preparation to form phosphoenzyme on incubation with [gamma-32P]ATP and to bind [3H]ouabain was studied when its sulfatide was hydrolyzed by arylsulfatase treatment. The K+-dependent hydrolysis of the Na+-dependent phosphorylated intermediate as well as the ouabain binding were inactivated in direct relation to the breakdown of sulfatide. Both characteristics of the (Na+ + K+)ATPase preparation, lost by arylsulfatase treatment, were partially restored by the sole addition of sulfatide. These experiments indicate that sulfatide may play a role in sodium ion transport either in the conformational transition of the K+-insensitive phosphointermediate, E1P, to the K+-sensitive intermediate, E2P, or in the configuration of the high-affinity binding site for K+ of the E2P form. In addition, this glycolipid may have a specific role in the proteolipidic subunit that binds ouabain.     

Molecular properties of purified (sodium + potassium)-activated adenosine triphosphatases and their subunits from the rectal gland of Squalus acanthias and the electric organ of Electrophorus electricus.

The chemical properties of two highly purified preparations of (sodium + potassium)-activated adenosine triphosphatase (NaK ATPase) and their subunits have been compared. One preparation is derived from the rectal gland of the spiny dogfish shark, Squalus acanthias and the other preparation is derived from the electric organ of the electric eel, Electrophorus electricus. Ouabain binding and phosphorylation from [gamma-32-P]ATP for both enzymes ranged from 4000 to 4300 pmol per mg of protein. This gives a stoichiometry for ouabain binding and phosphorylation of 1:1 for both enzymes. The molar ratios of catalytic subunit to glycoprotein was 2:1 for both enzymes, suggesting a minimum molecular weight of 250, 000, which agrees with the molecular weight obtained by radiation inactivation. Assuming that only one of the two catalytic subunits is phosphorylated and binds ouabain per (sodium + potassium)-activated adenosine triphosphatase molecule the data on phosphorylation and ouabain binding also give a molecular weight of 250, 000. The data on phosphorylatiion, ouabain binding, subunit composition, and molecular weight based on radiaion inactivation are thus all internally consistent. A technique has been developed for isolation of pure catalytic subunit and glycoprotein in good yields by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A variety of chemical studies have been carried out with the purified subunits. The amino acid composition of the catalytic subunit was different from that of the glycoprotein, but the amino acid composition of each of the two subunits was essentially the same for both species. However, the NH2-terminal amino acid for the catalytic subunit was alanine for the rectal gland enzyme and serine for the electric organ enzyme, suggesting some differencesin amino acid sequences for the two species. The NH2-terminal amino acid for the glycoprotein was alanine for the two species. The glycoproteins from both species contained the same carbohydrates but in quite differing amounts. The carbohydrates were glucosamine, sialic acid, fucose, galactose, mannose, and glucose. The release of all the sialic acid from the electric organ enzyme and the release of 40% of the sialic acid from the rectal gland enzyme did not affect (sodium + potassium)-activated adenosine triphosphatase activity. Both enzymes contained the following phospholipids, which accounted for 98 to 100% of the total phospholipid phosphorus: sphingomyelin, lecithin, phosphatidylserine, phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylethanolamine, and phosphatidylinositol. With the exception of phosphatidylserine, the amount of any phospholipid per mg of enzyme as well as the total phospholipid content were quite different for the two enzymes.     

Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.     

Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

Origin of the gamma polypeptide of the Na+/K+-ATPase

The Na+/K+-ATPase purified from lamb kidney contains a gamma polypeptide fraction which is a collection of fragments derived from the alpha and beta polypeptides of the enzyme. This fraction has the solubility characteristics of a proteolipid and was isolated either by high performance liquid chromatography (size exclusion chromatography) in 1% sodium dodecyl sulfate or by sequential organic extraction of purified lamb kidney Na+/K+-ATPase. Formation of gamma polypeptide(s) from detergent solubilized holoenzyme was accelerated by sulfhydryl containing reagents and was unaffected by addition of inhibitors of proteolytic enzymes. Treatment of the holoenzyme with the photoaffinity reagent N-(2-nitro-4-azidophenyl)[3H]ouabain ([3H]NAP-ouabain) labeled the alpha polypeptide and the gamma polypeptide fraction but not the beta polypeptide. Amino acid sequence analysis of one gamma polypeptide preparation revealed homology of one component of this fraction with the N-terminus of the beta subunit of the Na+/K+-ATPase. Amino acid analysis of two preparations of proteolipid showed similar amino acid compositions with a peptide derived from the alpha subunit. The insolubility and complexity of the gamma polypeptide(s)/proteolipid fraction appears to preclude a conclusive sequence analysis of all components of this fraction.     

Gel electrophoretic identity of the (Na+ + Mg-2+)- and (Na+ + Ca-2+)-stimulated phosphorylations of rat brain ATPase.

The classical E2-P intermediate of (Na+ + K+)-ATPase dephosphorylates readily in the presence of K+ and is not affected by the addition of ADP. To determine the significance in the reaction cycle of (Na+ + K+)-ATPase of kinetically atypical phosphorylations of rat brain (Na+ + K+)-ATPase we compared these phosphorylated components with the classical E2-P intermediate of this enzyme by gel electrophoresis. When rat brain (Na+ + K+)-ATPase was phosphorylated in the presence of high concentrations of Na+ a proportion of the phosphorylated material formed was sensitive to ADP but resistant to K+. Similarly, if phosphorylation was carried out in the presence of Na+ and Ca-2+ up to 300 pmol/mg protein of a K+ -resistant, ADP-sensitive material were formed. If phosphorylation was from [gamma-32-P]CTP up to 800 pmol-32-P/mg protein of an ADP-resistant, K+ -sensitive phosphorylated material were formed. On gel electrophoresis these phosphorylated materials co-migrated with authentic Na+ -stimulated, K+ -sensitive, E2-P-phosphorylated intermediate of (Na+ + K+)-ATPase, supporting suggestions that they represent phosphorylated intermediates in the reaction sequence of this enzyme.     

Determination of monoclonal antibody-induced alterations in Na+/K+-ATPase conformations using fluorescein-labeled enzyme

The fluorescein 5'-isothiocyanate (FITC)-labeled lamb kidney Na+/K+-ATPase has been used to investigate enzyme function and ligand-induced conformational changes. In these studies, we have determined the effects of two monoclonal antibodies, which inhibit Na+/K+-ATPase activity, on the conformational changes undergone by the FITC-labeled enzyme. Monitoring fluorescence intensity changes of FITC-labeled enzyme shows that antibody M10-P5-C11, which inhibits E1 approximately P intermediate formation (Ball, W.J. (1986) Biochemistry 25, 7155-7162), has little effect on the E1 in equilibrium E2 transitions induced by Na+, K+, Mg2+ Pi or Mg2+. ouabain. The M10-P5-C11 epitope, which appears to reside near the ATP-binding site, does not significantly participate in these ligand interactions. In contrast, we find that antibody 9-A5 (Schenk, D.B., Hubert, J.J. and Leffert, H.L. (1984) J. Biol. Chem. 259, 14941-14951) inhibits both the Na+/K+-ATPase and p-nitrophenylphosphatase activity. Its binding produces a 'Na+-like' enhancement in FITC fluorescence, reduces the ability of K+ to induce the E1 in equilibrium E2 transition and converts E2.K+ to an E1 conformation. Mg2+ binding to the enzyme alters both the conformation of this epitope region and its coupling of ligand interactions. In the presence of Mg2+, 9-A5 binding stabilizes an E1.Mg2+ conformation such that K+-, Pi- and ouabain-induced E1----E2 or E1----E2-Pi transitions are inhibited. Oubain and Pi added together overcome this stabilization. These studies indicate that the 9-A5 epitope participates in the E1 in equilibrium E2 conformational transitions, links Na+-K+ interactions and ouabain extracellular binding site effects to both the phosphorylation site and the FITC-binding region. Antibody-binding studies and direct demonstration of 9-A5 inhibition of enzyme phosphorylation by [32P]Pi confirm the results obtained from the fluorescence studies. Antibody 9-A5 has also proven useful in demonstrating the independence of Mg2+ ATP and Mg2+Pi regulation of ouabain binding. In addition, [3H]ouabain and antibody-binding studies demonstrate that FITC-labeling alters the enzyme's responses to Mg2+ as well as ATP regulation.     

Inhibition of (Na+, K+)adenosine triphosphatase and its partial reactions by quercetin.

The bioflavonoid, quercetin, inhibited the (Na+, K+)adenosine triphosphatase purified from the electric organ of electric eel (Electrophorus electricus) or from lamb kidney. An analysis of its mode of action revealed that the formation of phosphoenzyme from Pi but not from ATP was inhibited. Quercetin increased the amount of ADP-sensitive phosphoenzyme (E1--P), indicating an inhibition of the conversion of E1--P to the ADP-insensitive form (E2--P). The rate of dephosphorylation of the phosphoenzyme formed from ATP was slowed by quercetin. These results suggest that quercetin inhibits the formation of E2--P from either Pi or E1-P as well as the hydrolysis of the phosphoenzyme. Its mode of action is therefore different from that of ouabain and other inhibitors of the Na+, K+)adenosine triphosphatase.     

Phosphorylated intermediates of (Ca2+ + Mg2+)-ATPase and alkaline phosphatase in renal plasma membranes

Renal basal-lateral and brush border membrane preparations were phosphorylated in the presence of [gamma-32P]ATP. The 32P-labeled membrane proteins were analysed on SDS-polyacrylamide gels. The phosphorylated intermediates formed in different conditions are compared with the intermediates formed in well defined membrane preparations such as erythrocyte plasma membranes and sarcoplasmic reticulum from skeletal muscle, and with the intermediates of purified renal enzymes such as (Na+ + K+)-ATPase and alkaline phosphatase. Two Ca2+-induced, hydroxylamine-sensitive phosphoproteins are formed in the basal-lateral membrane preparations. They migrate with a molecular radius Mr of about 130 000 and 100 000. The phosphorylation of the 130 kDa protein was stimulated by La3+-ions (20 microM) in a similar way as the (Ca2+ + Mg2+)-ATPase from erythrocytes. The 130 kDa phosphoprotein also comigrated with the erythrocyte (Ca2+ + Mg2+)-ATPase. In addition in the same preparation, another hydroxylamine-sensitive 100 kDa phosphoprotein was formed in the presence of Na+. This phosphoprotein comigrates with a preparation of renal (Na+ + K+)-ATPase. In brush border membrane preparations the Ca2+-induced and the Na+-induced phosphorylation bands are absent. This is consistent with the basal-lateral localization of the renal Ca2+-pump and Na+-pump. The predominant phosphoprotein in brush border membrane preparations is a 85 kDa protein that could be identified as the phosphorylated intermediate of renal alkaline phosphatase. This phosphoprotein is also present in basal-lateral membrane preparations, but it can be accounted for by contamination of those membranes with brush border membranes.     

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

The MgATP complex analogue cobalt-tetrammine-ATP [Co(NH3)4ATP] inactivates (Na+ + K+)-ATPase at 37 degrees C slowly in the absence of univalent cations. This inactivation occurs concomitantly with incorporation of radioactivity from [alpha-32P]Co(NH3)4ATP and from [gamma-32P]Co(NH3)4ATP into the alpha subunit. The kinetics of inactivation are consistent with the formation of a dissociable complex of Co(NH3)4ATP with the enzyme (E) followed by the phosphorylation of the enzyme: (Formula: see text). The dissociation constant of the enzyme-MgATP analogue complex at 37 degrees C is Kd = 500 microM, the inactivation rate constant k2 = 0.05 min-1. ATP protects the enzyme against the inactivation by Co(NH3)4ATP due to binding at a site from which it dissociates with a Kd of 360 microM. It is concluded, therefore, that Co(NH3)4ATP binds to the low-affinity ATP binding site of the E2 conformational state. K+, Na+ and Mg2+ protect the enzyme against the inactivation by Co(NH3)4ATP. Whilst Na+ or Mg2+ decrease the inactivation rate constant k2, K+ exerts its protective effect by increasing the dissociation constant of the enzyme.Co(NH3)4ATP complex. The Co(NH3)4ATP-inactivated (Na+ + K+)-ATPase, in contrast to the non-inactivated enzyme, incorporates [3H]ouabain. This indicates that the Co(NH3)4ATP-inactivated enzyme is stabilized in the E2 conformational state. Despite the inactivation of (Na+ + K+)-ATPase by Co(NH3)4ATP from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 microM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na+ + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the E1 and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na+ + K+)-transport.     

The adenosine-triphosphatase system responsible for cation transport in electric organ: exclusion of phospholipids as intermediates

1. Subcellular fractions were prepared from the electric organs of Electrophorus and Torpedo and assayed for adenosine-triphosphatase activity. 2. Treatment of the `low-speed' fraction from Torpedo with m-urea gave an adenosine-triphosphatase preparation that was almost completely (98%) inhibited by ouabain (0·1mg./ml.) and dependent on the simultaneous presence of Na<sup>+</sup> and K<sup>+</sup>. 3. The adenosine-triphosphatase preparations were exposed to [γ-<sup>32</sup>P]ATP for 30sec. in the presence of (i) Na<sup>+</sup>, (ii) K<sup>+</sup>, (iii) Na<sup>+</sup>+K<sup>+</sup> and (iv) Na<sup>+</sup>+K<sup>+</sup>+ouabain. No significant labelling of phosphatidic acid, triphosphoinositide or any other phospholipid was observed. 4. The results suggest that phospholipids do not act as phosphorylated intermediates in the `transport adenosine-triphosphatase' system of electric organ.     

Ca2+-dependent and cAMP-dependent control of nicotinic acetylcholine receptor phosphorylation in muscle cells

Mouse BC3H1 myocytes were incubated with 32Pi before acetylcholine receptors were solubilized, immunoprecipitated, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. More than 90% of the 32P found in the receptor was bound to the delta subunit. Two phosphorylation sites in this subunit were resolved by reverse phase high performance liquid chromatography after exhaustive proteolysis of the protein with trypsin. Sites 1 and 2 were phosphorylated to approximately the same level in control cells. The divalent cation ionophore, A23187, increased 32P in site 1 by 40%, but did not affect the 32P content of site 2. In contrast, isoproterenol increased 32P in site 2 by more than 60%, while increasing 32P in site 1 by only 20%. When dephosphorylated receptor was incubated with [gamma-32P]ATP and the catalytic subunit of cAMP-dependent protein kinase, the delta subunit was phosphorylated to a maximal level of 1.6 phosphates/subunit. Approximately half of the phosphate went into site 2, with the remainder going into a site not phosphorylated in cells. The alpha subunit was phosphorylated more slowly, but phosphorylation of both alpha and delta subunits was blocked by the heat-stable protein inhibitor of cAMP-dependent protein kinase. Phosphorylation of the receptor was also observed with preparations of phosphorylase kinase. In this case phosphorylation occurred in the beta subunit and site 1 of the delta subunit, neither of which were phosphorylated by cAMP-dependent protein kinase. The rate of receptor phosphorylation by phosphorylase kinase was slow relative to that catalyzed by cAMP-dependent protein kinase. Therefore, it can not yet be concluded that phosphorylase kinase phosphorylates the beta subunit and the delta subunit site 1 in cells. However, the results strongly support the hypothesis that phosphorylation by cAMP-dependent protein kinase accounts for phosphorylation of the alpha subunit and the delta subunit site 2 in response to elevations in cAMP.     

Direct evidence for an ADP-sensitive phosphointermediate of (K+ + H+)-ATPase

Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 microM [gamma-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7-15 microM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.     

A new simple preparation method for NaK-ATPase-rich membrane fragments

A method for the isolation of highly active membrane bound NaK-ATPase without detergents in quantity from the electric organ of the electric eel (Electrophorus electricus) is described. This method consists of the homogenation of electric organ with an isotonic solution containing sucrose, histidine, EDTA, and arginine, and of the separation of the higher active membrane fraction from the microsomal fraction by density gradient centrifugation. The enzyme has a specific activity of about 20 μmol Pi/min/mg at 37°C, and 13 μmol Pi/min/mg at 30°C. Although it is not as pure as the detergent-treated enzyme preparation based on the level of phosphorylated protein, ouabain binding, or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, its enzyme activity is comparable to that of the purified enzymes. This preparation is very stable and is able to change its medium by Sephadex chromatography without any loss of enzyme activity and protein content. This preparation is also expected to keep the original characteristics as well as the enzyme in the tissue.     

Phosphorylation of rat kidney pyruvate kinase type L by cyclic 3',5'-AMP-dependent protein kinase.

Pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) type L was partly purified from rat kidney. During the last two purification steps, the incorporation of [32P]phosphate into protein on incubation with [32P]ATP and cyclic 3',5'-AMP-dependent protein kinase was found to parallel the pyruvate kinase activity. After phosphorylation of the enzyme, a major radioactive band with a molecular weight of 57 000 was found on polyacrylamide gel electrophoresis [32P]Phosphorylserine was isolated from the kidney pyruvate kinase. Immunological identity was found between the liver and kidney pyruvate kinases type L. By autoradiography of high-voltage electropherograms after partial acid hydrolysis of the phosphorylated rat liver and kidney pyruvate kinases type L, identical results were obtained. The affinity for phosphoenolpyruvate was found to be decreased by phosphorylation of the enzyme with a change in the apparent Km from 0.15 mM to 0.35 mM. After incubation of the phosphorylated kidney pyruvate kinase with phosphatase the phosphoenolpyruvate saturation curve was found to be identical to that for the unphosphorylated enzyme. Thus, the activity of the rat kidney pyruvate kinase type L is with all probability regulated by a reversible phosphorylation-dephosphorylation reaction, thereby indicating that hormonal regulation of gluconeogenesis via cyclic AMP may be of importance in the renal cortex.     

Reversible change in light scattering following formation of ADP-sensitive phosphoenzyme in Na+,K+-ATPase modified with N-[p-(2-benzimidazolyl)phenyl]maleimide

An increase in light scattering (3.5 +/- 0.2%) was observed when pig kidney Na+,K+-ATPase preparations modified with N-[p-(2-benzimidazolyl)phenyl] maleimide were phosphorylated by ATP in the presence of 2 M Na+ with Mg2+ to form ADP-sensitive phosphoenzyme (E1P), which had a negative fluorescence intensity (-1.5 +/- 0.3%). Addition of K+ or ouabain to E1P reduced the light scattering to the original level observed in the absence of ATP. Stopped flow measurements showed that the fluorescence change accompanying the E1P formation (t1/2 = 0.1 s) occurred preceding the light-scattering change (t1/2 = 1 s). Oligomycin affected the rate of the scattering increase little, but it diminished the effect of K+ on E1P to reduce the light scattering and increase the fluorescence. The addition of 2 M Na+ to K+-sensitive phosphoenzyme (E2P) immediately decreased the fluorescence (t1/2 = 0.02 s) to form E1P which was followed by a slow increase in the light scattering (t1/2 = 0.25 s). Oligomycin reduced both rates of the above changes accompanying the transition of E2P to E1P. The data suggest the sequential appearance of species of E1P that precede E2P formation during the hydrolysis of ATP.     

Immunochemical comparison of cardiac glycoside-sensitive (lamb) and -insensitive (rat) kidney (Na+ + K+)-ATPase

The immunological cross-reactivity of the ouabain-sensitive lamb kidney and the ouabain-insensitive rat kidney (Na+ + K+)-ATPase (EC 3.6.1.37) was examined using polyclonal and monoclonal antibodies. Studies using rabbit antisera prepared against both the lamb kidney and rat kidney holoenzymes showed the existence of substantial antigenic differences as well as similarities between the holoenzymes and the respective denatured alpha and beta subunits of these two enzymes. Quantitation of the extent of cross-reactivity using holoenzyme-directed antibodies showed a 40-60% cross-reactivity. In addition, rabbit antisera monospecific to the purified, denatured alpha and beta subunits of the lamb kidney enzyme showed about a 50% cross-reactivity towards the respective subunit of the rat enzyme. In contrast to the cross-reactivity observed using the polyclonal antibodies, six monoclonal antibodies specific for the alpha subunit of the lamb holoenzyme exhibited no cross-reactivity with the rat holoenzyme. Four of these monoclonal antibodies, however, showed substantial cross-reactivity with rat alpha subunit as resolved by SDS-polyacrylamide gel electrophoresis. A fifth antibody did not bind to the denatured alpha subunit of either the lamb or the rat enzyme. Another monoclonal antibody (M7-PB-E9), which is specific for an epitope previously implicated in the regulation of both ATP and ouabain binding to (Na+ + K+)-ATPase (Ball, W.J., Jr. (1984) Biochemistry 2275-2281) was found to bind to the denatured lamb alpha but not to the rat alpha. This antibody has identified a region of the lamb alpha that has an altered amino acid sequence in the ouabain-insensitive rat enzyme. These immunological studies indicate that there are substantial antigenic differences between the lamb and rat kidney (Na+ + K+)-ATPases. The majority of these antigenic differences appear to be due to variations in the tertiary structures rather than to variations in the primary structures of the alpha subunits.     

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.     

Phosphorylation ofEscherichia coli NADP+-specific glutamate dehydrogenase

Glutamate dehydrogenase fromEscherichia coli is phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein, when exposed to acid conditions, releases the phosphate; this implies that the phosphorylation site is not on a serine, tyrosine, or threonine residue(s). Treatment of glutamate dehydrogenase with diethyl pyrocarbonate, a highly specific histidine-modifying reagent, blocks incorporation of³²P-phosphate from [-³²P]ATP into the enzyme, suggestive that the phosphorylation site is a histidine residue(s). The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogenase, isolated fromE. coli, on denaturing, nondenaturing, and isoelectric focusing polyacrylamide gels and by sequence analysis.