Inhibition of neuronal sodium and potassium ion activated adenosinetriphosphatase by pyrithiamin

Since one of the electrophysiological effects of pyrithiamin, an antimetabolite of thiamin, suggested an interference with sodium pump mechanisms, the effect of pyrithiamin on Na+,K+-ATPase was investigated. We found that whereas preincubation of the antimetabolite with nonneuronal preparations of Na+,K+-ATPase produced only minimal inhibition, the enzyme derived from brain preparations was markedly inhibited. This inhibition could be prevented by thiamin but not reversed. The kinetic study showed that pyrithiamin acts in a noncompetitive manner with respect to the activation of the enzyme by ATP, Na+, and K+. Pyrithiamin inhibited Na+-dependent phosphorylation and K+-stimulated phosphatase as well as ouabain binding, and these inhibitions were parallel with that of the overall Na+,K+-ATPase reaction. In addition, the antimetabolite caused a significant change in the turbidity of the enzyme suspension. The results suggest that pyrithiamin may induce a structural change of the enzyme complex.     

Any of several lysines can react with 5'-isothiocyanatofluorescein to inactivate sodium and potassium ion activated adenosinetriphosphatase

Determinations of reaction stoichiometry demonstrate that the covalent incorporation of one molecule of 5'-isothiocyanatofluorescein can inactivate one molecule of sodium and potassium ion activated adenosinetriphosphatase in agreement with earlier determination of this stoichiometry. Several different modified peptides are produced, however, when the modified enzyme is digested with trypsin. One of these peptides has been identified as HLLVMK (thioureidylfluorescein)GAPER by use of a specific immunoadsorbent. The modified lysine is lysine 501 in the amino acid sequence of the alpha polypeptide of (Na+ + K+)-ATPase. This peptide has been previously isolated from such digests [Farley, R. A., Tran, C. M., Carilli, C. T., Hawke, D., & Shively, J. E. (1984) J. Biol. Chem. 259, 9532-9535]. The other specifically modified peptides have been purified and identified by amino acid sequencing. Their sequences identify lysine 480 and lysine 766 from the alpha polypeptide as amino acids modified by 5'-isothiocyanatofluorescein in reactions sensitive to the addition of ATP and responsible for inactivation of the enzyme.     

Topological localization of proteolytic sites of sodium and potassium ion stimulated adenosinetriphosphatase

The (Na+ and K+)-stimulated adenosinetriphosphatase [(Na+,K+)-ATPase] consists of two different polypeptides, alpha and beta, both of which are embedded in the plasma membrane. The alpha chain from dog kidney (Na+,K+)-ATPase can be hydrolyzed at specific sites by trypsin and chymotrypsin [Castro, J., & Farley, R. A. (1979) J. Biol. Chem. 254, 2221-2228]. In order to position these sites with respect to the lipid bilayer, we have treated sealed, inside out vesicles from human red cells and unsealed kidney enzyme membranes with trypsin and chymotrypsin and have used ouabain-stimulated phosphorylation to identify the (Na+,K+)-ATPase and its fragments. All of the proteolytic sites observed in the kidney membranes are accessible in the inside out vesicles. The ouabain-inhibitable uptake of 86Rb+ in human red blood cells is resistant to externally added chymotrypsin. These results indicate that the proteolytic sites of the (Na+,K+)-ATPase are exposed on the cytoplasmic side of the membrane.     

Acid dissociation constant and apparent nucleophilicity of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase

A combination of competitive labeling with [3H]acetic anhydride [Kaplan, H., Stevenson, K. J., & Hartley, B. S. (1971) Biochem. J. 124, 289-299] and immunoaffinity chromatography is described that permits the assignment of the acid dissociation constant and the absolute nucleophilicity of individual lysines in a native enzyme. The acid dissociation constant of lysine-501 of the alpha-polypeptide in native (Na+ + K+)-ATPase was determined. This lysine had a normal pKa of 10.4. The rate constant for the reaction of the free base of lysine-501 with acetic anhydride at 10 degrees C is 400 M-1 s-1. This value is only 30% that for a fully accessible lysine in a protein. The lower than normal apparent nucleophilicity suggests that lysine-501 is hindered from reacting with its intrinsic nucleophilicity by the tertiary structure of the enzyme and is consistent with its location within a pocket that forms the active site upon the surface of the native protein.     

Specific photoaffinity labeling of the digitalis binding site of the sodium and potassium ion activated adenosinetriphosphatase induced by energy transfer

A ouabain p-aminobenzenediazonium derivative with a high specific radioactivity has been synthesized from ouabain and used as a photolabel for the (sodium plus potassium)-activated adenosinetriphosphatase from Electrophorus electricus electric organ and from dog kidney. In the dark it binds reversibly to the digitalis receptor site, with binding characteristics comparable to those of ouabain. The photoactivation of the ouabain derivative to produced covalent labeling of the receptor was obtained by energy transfer from a tryptophan residue in the (Na+,K+)ATPase to the ouabain p-aminobenzenediazonium molecule bound at the active site. The great advantage of this procedure compared to previous methods is that free molecules of the photoactivatable derivative are not photodecomposed. Analysis of the photolabeled polypeptides on sodium dodecyl sulfate gel electrophoresis showed that over 90% of the total radioactivity incorporated was found in the large molecular weight alpha-chain of the kidney enzyme (Mr 93 000). The same specific labeling of the alpha-subunit was obtained with a crude microsomal fraction from Electrophorus electricus. A mild tryptic fragmentation of the subunit into two peptide fragments of Mr 58 000 and 41 000, respectively, shows that the digitalis receptor is located in the N-terminal 41 000 fragment.     

Saturation-transfer electron spin resonance studies on the mobility of spin-labeled sodium and potassium ion activated adenosinetriphosphatase in membranes from Squalus acanthias

The sodium and potassium ion activated adenosinetriphosphatase [(Na+,K+)-ATPase] in membranous preparations from Squalus acanthias has been spin-labeled on sulfhydryl groups after prelabeling with N-ethylmaleimide. Saturation-transfer electron spin resonance spectroscopy has been used to study the rotational motions of the labeled protein on the microsecond time scale. Effective rotational correlation times deduced from the diagnostic line-height ratios in the second-harmonic, 90 degrees out-of-phase (V2') spectra are much larger than those deduced from the spectral integrals, indicating the presence of large-scale segmental motions, in addition to rotation of the protein as a whole. Experiments involving controlled cross-linking of the protein by glutaraldehyde, as well as measurements of the line broadening of the conventional electron spin resonance spectra, support this interpretation. Both the spectral integrals and diagnostic line-height ratios are found to increase irreversibly with time on incubation at temperatures greater than 20 degrees C, corresponding to a decrease in the segmental motion of the protein and probably also in the overall protein rotation. The native enzyme displays a marked nonlinearity in the Arrhenius temperature dependence of the activity at temperatures above 20 degrees C, and the activity decreases with a half-life of ca. 70 min on incubation at 37 degrees C (but not on incubation at low temperature), paralleling the time- and temperature-dependent changes in the saturation-transfer spectra of the labeled protein. Both of these observations suggest that the changes observed in the molecular dynamics could correspond to functional properties of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Demonstration that lysine-501 of the alpha polypeptide of native sodium and potassium ion activated adenosinetriphosphatase is located on its cytoplasmic surface

Evidence that the peptide HLLVMKGAPER, which can be released from intact sodium and potassium ion activated adenosinetriphosphatase by tryptic digestion, is located on the cytoplasmic surface of the native enzyme has been obtained. An immunoadsorbent directed against the carboxy-terminal sequence of this tryptic peptide has been constructed. The peptide KGAPER was synthesized by solid-phase techniques. Antibodies against the sequence -GAPER were purified by immunoadsorption, using the synthetic peptide attached to agarose beads. These antibodies, in turn, were coupled to agarose beads to produce an immunoadsorbent. Sealed, right-side-out vesicles, prepared from canine kidneys, were labeled with pyridoxal phosphate and sodium [3H]borohydride in the absence or presence of saponin, respectively. A tryptic digest of these labeled vesicles was passed over the immunoadsorbent. Large increases in the incorporation of radioactivity into the peptides bound by the immunoadsorbent were observed in the digests obtained from the vesicles exposed to saponin. From the results of several control experiments examining the labeling reaction as applied to these vesicles, it could be concluded that this increase in incorporation resulted only from the access that the reagents gained to the inside of the vesicles in the presence of saponin and that the increase in the extent of modification was due to the cytoplasmic disposition of this segment in the native enzyme.     

Nucleophilic behavior of lysine-501 of the alpha-polypeptide of sodium and potassium ion activated adenosinetriphosphatase consistent with a role in binding adenosine triphosphate

An immunoadsorbent specific for the carboxy-terminal sequence -GAPER, which comprises residues 502-506 of the alpha-polypeptide of ovine sodium and potassium ion activated adenosinetriphosphatase [(Na+ + K+)-ATPase], was used to isolate the products of the reaction between the lysine immediately preceding this sequence in the intact protein and either [3H]acetic anhydride or fluorescein 5'-isothiocyanate. Changes in the apparent nucleophilicity of this lysine, Lys501, were observed with both reagents when ATP was bound by the intact, native enzyme poised in the E1 conformation or when the structure of the enzyme was changed from the E1 conformation into the E2-P conformation. With both reagents, a decrease of more than 4-fold in the yield of incorporation occurred during the former change, but a decrease of only 2-fold occurred during the latter. Because a much larger decrease occurred when ATP was bound in the absence of a conformational change than occurred when a major conformational change took place in the absence of the occupation of the active site, these changes in the incorporation of [3H]acetyl suggest that Lys501 from the alpha polypeptide is directly involved in binding ATP within the active site of (Na+ + K+)-ATPase. The immunochemical reactions between the specific polyclonal antibodies raised against the sequence-GAPER and denatured or enzymically active (Na+ + K+)-ATPase were also investigated. Western blots and the inhibition of enzymic activity caused by the antibody have shown that it can bind to both the denatured and the native form of the alpha-polypeptide, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

The sodium ion translocating adenosinetriphosphatase of Propionigenium modestum pumps protons at low sodium ion concentrations

The purified ATPase (F1F0) of Propionigenium modestum has its pH optimum at pH 7.0 or at pH 6.0 in the presence or absence of 5 mM NaCl, respectively. The activation by 5 mM NaCl was 12-fold at pH 7.0, 3.5-fold at pH 6.0, and 1.5-fold at pH 5.0. In addition to its function as a primary Na+ pump, the ATPase was capable of pumping protons. This activity was demonstrated with reconstituted proteoliposomes by the ATP-dependent quenching of the fluorescence of 9-amino-6-chloro-2-methoxyacridine. No delta pH was formed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone or by blocking the ATPase with dicyclohexylcarbodiimide. In the presence of valinomycin and K+, the delta pH increased, in accord with the operation of an electrogenic proton pump. The proton pump was only operative at low Na+ concentrations (less than 1 mM), and its activity increased as the Na+ concentration decreased. Parallel to the decrease of H+ pumping, the velocity of the Na+ transport increased about 6-fold from 0.1 to 4 mM NaCl, indicating a switch from H+ to Na+ pumping, as the Na+ concentration increases. Due to proton leaks in the proteoliposomal membranes, fluorescence quenching was released after blocking the ATPase with dicyclohexylcarbodiimide, by trapping residual ATP with glucose and hexokinase, or by the Na+-induced conversion of the proton pump onto a Na+ pump. Amiloride, an inhibitor of various Na+-coupled transport systems, was without effect on the kinetics of Na+ transport by the P. modestum ATPase.     

Spatial relationship and conformational changes between the cardiac glycoside site and beta-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

(Na,K)-ATPase, the enzyme responsible for active transport of Na and K across the plasma membranes of animal cells, consists of a catalytic subunit (alpha) and a glycoprotein subunit (beta) with unknown function. We have determined the distance between fluorescent probes directed to specific sites on the alpha- and beta-subunits and ligand-induced changes in the fluorescence of a probe specifically attached to the beta-subunit. The cardiac glycoside site on the alpha-subunit was labeled with anthroylouabain [Fortes, P. A. G. (1977) Biochemistry 16, 531-540]. The oligosaccharides on the beta-subunit were labeled with lucifer yellow carbohydrazide [Lee, J. A., & Fortes, P. A. G. (1985) Biochemistry 24, 322-330]. Resonance energy transfer from anthroylouabain to lucifer yellow was measured by steady-state and time-resolved fluorescence spectroscopy. The distance between these probes was determined from the efficiency of energy transfer. The average distance between anthroylouabain and lucifer yellow was 47 A and was independent of the number of acceptor molecules attached to the beta-subunit. The measured distance corresponds to the distance between the cardiac glycoside site and the center of the labeled oligosaccharides on the beta-subunit within one alpha beta dimer. The distance was the same (47 A) when anthroylouabain was bound with ATP or Pi as phosphorylating ligands but increased to 49 A in the presence of vanadate. The change in average distance provides quantitative evidence of a conformational difference between the complexes of cardiac glycosides with (Na,K)-ATPase induced by phosphorylating ligands or by vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modification of the conformational equilibria in the sodium and potassium dependent adenosinetriphosphatase with glutaraldehyde

Glutaraldehyde treatment of electroplax membrane preparations of Na,K-ATPase leads to irreversible changes in the enzymic behavior of the protein, which are not due to modification of the active site. When the glutaraldehyde treatment is carried out in a medium containing K+ and without Na+, the "K+-modified enzyme" so produced shows the following changes in enzymic properties: The steady-state phosphorylation by ATP and the rate of ATP-ADP exchange are decreased to approximately 40% of control, while Na,K-ATPase activity decreases to approximately 15% of control. Phosphatase activity is decreased very little, but the potassium activation parameters of the reaction are changed, from K0.5 approximately equal to 5 mM and nH = 1.9 in control to K0.5 approximately equal to 0.5 mM and nH = 1 in K+-modified enzyme. KI(app) for nucleotide inhibition of phosphatase activity is increased significantly. Changes in the cation dependence of the ATPase reaction are also observed. All of these effects can be explained by assuming that the cross-linking of surface groups in protein subunits when they are in conformation E2 shifts the intrinsic conformational equilibrium of the enzyme toward E2. We considered the simplest mathematical model for the coupling between K+ binding and the conformational equilibrium, with equivalent potassium sites that must be simultaneously in the same state. If one assumes that the potassium activation of phosphatase activity in the K+-modified enzyme reflects the affinity for K+ of E2, the behavior of the phosphatase activity in the native enzyme can be fit if there are only two potassium sites, whose affinity is 80-fold higher in E2 than in E1, and the equilibrium constant for E2 in equilibrium E1 is about 250. The same sites can explain the activation of dephosphorylation during ATP hydrolysis. Independent of the model chosen, potassium ions must be required for the catalytic action of form E2 and cannot be merely "allosteric activators". The enzyme modified with glutaraldehyde in a medium containing Na+ also has interesting properties, but their rationalization is less straightforward. The Na,K-ATPase activity is inhibited more than the "partial reactions", as in the K+-modified enzyme. We suggest that this is a generally expected result of modifications of the enzyme.     

Functional characterization of a testes-specific alpha-subunit isoform of the sodium/potassium adenosinetriphosphatase.

Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) alpha and beta subunits have been identified in mammals. The association of the various alpha and beta polypeptides results in distinct Na,K-ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase alpha4 isoform in Sf-9 cells using recombinant baculoviruses. When alpha4 and the Na pump beta1 subunit are coexpressed in the cells, Na, K-ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na(+)-dependent, K(+)-sensitive, and ouabain-inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K(+). Furthermore, the activity of alpha4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase alpha4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the beta1 and beta3 subunits. In insect cells, the alpha4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the alpha4beta1 and alpha4beta3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na(+), K(+), ATP, and ouabain. The enzymatic properties of alpha4beta1 and alpha4beta3 are, however, distinct from the other Na pump isozymes. A Na, K-ATPase activity with similar properties as the alpha4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the alpha4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.     

An enzymatic ion exchange model for active sodium transport

An enzymatic ion exchange model for active sodium transport is described. Kinetic equations relating net flux to time, and to concentration difference across the actively transporting membrane are derived. The second of these equations is tested, using the isolated frog skin in the "short-circuit" apparatus of Ussing. Reasonable linearity, as predicted by this equation, is observed. The passive permeability coefficient for Na⁺, is calculated as 5.3 x 10^-4 ± 5.3 x 10^-4 cm./hr. If cholinesterase is assumed to be the enzyme responsible for transport, the activity required to account for the observations reported here is 17.7 x 10^-4 mmoles/cm.²/hr.     

Determination of polyhydroxyalkanoates in activated sludge by ion chromatographic and enzymatic methods

Two new detection methods for the determination of poly-beta-hydroxybutyrate (PHB) and -valerate (PHV) are described. Both methods are based on depolymerization of PHB/PHV to 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Depolymerization was achieved by either propanolic or hydrolytic digestion. Propanolic digestion transformed commercial PHB/PHV stoichiometrically into 3HB/3HV and yielded apparently complete recoveries of bacterial PHB/PHV from activated sludge. Hydrolytic digestion was suitable only for PHB determination. For quantification of 3HB and 3HV directly from digested sludge, a method based on ion-exchange chromatography and conductivity detection was developed (IC-method). Alternatively, the total of 3HB and 3HV was quantified using a commercial enzymatic test kit and colorimetric detection (enzyme method). Both detection methods are easier to perform than previous methods and are suitable for complex matrices such as activated sludge. The IC-method is recommended for high sample throughputs or if distinction between PHB and PHV is essential. Enzymatic detection is recommended if a few samples per day have to be measured immediately or if an ion chromatograph is unavailable.