Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases.

The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.     

Na K ATPase activity in mammalian erythrocytes

The effects of membranotropic substances--nonionic detergent Tween-20 and EDTA--on the activity and some properties of Na,K-ATPase from mammalian erythrocytes were studied. It was shown that pretreatment of whole erythrocytes with Tween-20 (5 mg/ml) allows a detection of the enzyme activity, which cannot be detected in intact cells. It was also found that erythrocyte ghosts with a high and stable activity of Na,K-ATPase can be obtained by injections of EDTA (1-2 mM) into the hemolysis medium. Although the enzyme activity in whole erythrocytes and their ghosts was detected by the use of various membranotropic agents, the type of the dependence of the Na,K-ATPase activity on MgCl2 and EDTA concentration in the incubation medium was essentially the same for both cell preparations, the optimal concentrations of MgCl2 and EDTA being 3 and 1 mM, respectively. A rise in MgCl2 concentration above 3 mM caused a decrease of the enzymatic activity. Simple techniques have been developed for the detection of the Na,K-ATPase activity in mammalian erythrocytes which allow the determination of a higher enzymatic activity than those described in literature.     

Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.     

Interactions between Na,K-ATPase alpha-subunit ATP-binding domains

The reaction mechanism of the Na,K-ATPase is thought to involve a number of ligand-induced conformational changes. The specific amino acid residues responsible for binding many of the important ligands have been identified; however, details of the specific conformational changes produced by ligand binding are largely undescribed. The experiments described in this paper begin to identify interactions between domains of the Na,K-ATPase alpha-subunit that depend on the presence of particular ligands. The major cytoplasmic loop (between TM4 and TM5), which we have previously shown contains the ATP-binding domain, was overexpressed in bacteria either with a His(6) tag or as a fusion protein with glutathione S-transferase. We have observed that these polypeptides associate in the presence of MgATP. Incubation with [gamma-(32)P]ATP under conditions that result in phosphorylation of the full-length Na,K-ATPase did not result in (32)P incorporation into either the His(6) tag or glutathione S-transferase fusion proteins. The MgATP-induced association was strongly inhibited by prior modification of the fusion proteins with fluorescein isothiocyanate or by simultaneous incubation with 10 microm eosin, indicating that the effect of MgATP is due to interactions within the nucleotide-binding domain. These data are consistent with Na,K-ATPase associating within cells via interactions in the nucleotide-binding domains. Although any functional significance of these associations for ion transport remains unresolved, they may play a role in cell function and in modulating interactions between the Na,K-ATPase and other proteins.     

Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[alpha-32P]ATP

Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[alpha-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 microM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 microM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A-Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-beta-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.     

Electrogenic sodium-sodium exchange carried out by Na,K-ATPase containing the amino acid substitution Glu779Ala

Na,K-ATPase containing the amino acid substitution glutamate to alanine at position 779 of the alpha subunit (Glu779Ala) supports a high level of Na-ATPase and electrogenic Na+-Na+ exchange activity in the absence of K+. In microsomal preparations of Glu779Ala enzyme, the Na+ concentration for half maximal activation of Na-ATPase activity was 161 +/- 14 mM (n = 3). Furthermore, enzyme activity with 800 mM Na+ was found to be similar in the presence and absence of 20 mM K+. These results showed that Na+, with low affinity, could stimulate enzyme turnover as effectively as K+. To gain further insight into the mechanism of this enzyme activity, HeLa cells expressing Glu779Ala enzyme were voltage clamped with patch electrodes containing 115 mM Na+ during superfusion in K+-free solutions. Electrogenic Na+-Na+ exchange was observed as an ouabain-inhibitable outward current whose amplitude was proportional to extracellular Na+ (Na+(o)) concentration. At all Na+(o) concentrations tested (3-148 mM), exchange current was maximal at negative membrane potentials (V(M)), but decreased as V(M) became more positive. Analyzing this current at each V(M) with a Hill equation showed that Na+-Na+ exchange had a high-affinity, low-capacity component with an apparent Na+(o) affinity at 0 mV (K0(0.5)) of 13.4 +/- 0.6 mM and a low-affinity, high-capacity component with a K0(0.5) of 120 +/- 13 mM (n = 17). Both high- and low-affinity exchange components were V(M) dependent, dissipating 30 +/- 3% and 82 +/- 6% (n = 17) of the membrane dielectric, respectively. The low-affinity, but not the high-affinity exchange component was inhibited with 2 mM free ADP in the patch electrode solution. These results suggest that the high-affinity component of electrogenic Na+-Na+ exchange could be explained by Na+(o) acting as a low-affinity K+ congener; however, the low-affinity component of electrogenic exchange appeared to be due to forward enzyme cycling activated by Na+(o) binding at a Na+-specific site deep in the membrane dielectric. A pseudo six-state model for the Na,K-ATPase was developed to simulate these data and the results of the accompanying paper (Peluffo, R.D., J.M. Argüello, and J.R. Berlin. 2000. J. Gen. Physiol. 116:47-59). This model showed that alterations in the kinetics of extracellular ion-dependent reactions alone could explain the effects of Glu779Ala substitution on the Na,K-ATPase.     

Interaction of sodium and potassium ions with Na+,K+-ATPase. II. General properties of ouabain-sensitive K+ binding

General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.     

Ratio of brain synaptosome Na, K-ATPase to dopamine receptors

Activating (0.3-3 microM) or inhibitory (0.03-0.3 mM) effects of dopamine (DA) in the absence of Ca2+, and its inhibitory effect in the presence of Ca2+ on Na,K-ATPase activity of synaptosomes from the caudate nucleus of the rat brain were confirmed. Na,K-ATPase was shown to be inhibited by 6 neuroleptics, with the degree of inhibition stronger in the presence of Ca2+. It was found that: 1) the biphasic or monophasic nature of DA action on Na,K-ATPase activity was preserved in the presence of neuroleptics, 2) DA enhances the inhibitory effects of neuroleptics on the enzyme, 3) the inhibitory effects of DA on Na,K-ATPase are enhanced by Ca2+ ions. The mechanisms of the modifying action of DA on synaptosomal Na,K-ATPase are discussed.     

Kinetic cooperativity change after H2O2 modification of (Na,K)-ATPase

The kinetics of hydrolysis of ATP and p-nitrophenylphosphate and the action of the allosteric effectors, Na+ and K+, upon the hydrolysis of these substrates were used to study the H2O2-modified, uncoupled (Na,K)-ATPase isolated from cultured bovine lenses ( Garner , W. H., Garner , M. H., and Spector , A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2044-2048). Pure bovine renal (Na,K)-ATPase was modified by H2O2 in 150 mM KCl and 20 mM MgCl2 to yield an enzyme with kinetic properties similar to the enzyme isolated from the H2O2-treated, cultured bovine lens. H2O2 modification changes the interaction of the ATP hydrolysis site from negative to positive kinetic cooperativity. H2O2 modification dramatically alters Na+ stimulation of ATP hydrolysis and Na+ inhibition of p-nitrophenylphosphate hydrolysis while having little effect upon K+ control of the hydrolysis of these two substrates.     

A K(+)-competitive fluorescent inhibitor of the H,K-ATPase.

The interactions of a novel fluorescent compound, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ) with the gastric H,K-ATPase were determined. MDPQ was shown to inhibit the H,K-ATPase and its associated K(+)-phosphatase competitively with K+, with Ki values of 0.22 and 0.65 microM, respectively. It also inhibited H+ transport with an IC50 of 0.29 microM, but at a concentration of 3.5 microM, reduced the steady-state level of phosphoenzyme by only 28%. The fluorescence of the inhibitor increased upon binding to the enzyme. 70% of this increment was quenched by K+, independently of Mg2+. The binding of MgATP to a high affinity site (K0.5(ATP) less than 1 microM) markedly increased the fluorescence due to the formation of an inhibitor-phosphoenzyme complex saturating with a K0.5(MDPQ) of 0.94 microM. The K(+)-dependent fluorescent quench (K0.5(K+) = 1.8 mM) required the ionophore, nigericin, indicating that K+ and MDPQ were competing at an extracytosolic site on the enzyme. Formation also of an enzyme-vanadyl-inhibitor complex was shown by the fact that Mg2+ plus vanadate enhanced MDPQ fluorescence in the absence of MgATP and decreased fluorescence in the presence of MgATP. The minimal stoichiometry of bound MDPQ determined by fluorescence titrations in the presence of MgATP was 1.4 mol/mol phosphoenzyme. The data suggest that this compound can serve as a probe of conformation at an extracytosolic site of the H,K-ATPase.     

Modulation of Na, K-ATPase activity by prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin

Adenylyl cyclase is activated by prostaglandin E and inhibited by mu-opioids. Since cAMP-related events influence the activity of the Na Pump and its biochemical correlate Na,K-ATPase in many systems, we tested the hypothesis that prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid agonist, have opposing actions on Na,K-ATPase activity. Studies were conducted with alamethicin-permeabilized SH-SY5Y human neuroblastoma cells. Prostaglandin E1 (1 microM) transiently inhibited Na,K-ATPase activity for 10-15 min. A direct activator of protein kinase A, 8-Br-cAMP (150 and 500 microM), also inhibited, but more rapidly and for a shorter duration. Both DAMGO (1 microM) and Rp-adenosine 3',5'-cyclic monophosphorothioate (500 microM), a protein kinase A-inhibitor, reversed the inhibitory effect of prostaglandin E1. DAMGO alone (1 microM) stimulated Na,K-ATPase activity up to nearly three-fold control activity. The stimulatory action of DAMGO was blocked by cyclosporine A (2 microM), an inhibitor of calcineurin, and was dependent on Ca2+ entry through nifedipine-sensitive Ca2+ channels. In the presence of 1 mM EGTA, DAMGO inhibited Na,K-ATPase activity. DAMGO-induced inhibition was blocked by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C (1 microM). Na,K-ATPase is poised to modulate neuronal excitability through its roles in maintaining the membrane potential and transmembrane ion gradients. The differential effects of prostaglandin E1 and opioids on Na,K-ATPase activity may be related to their actions in hyperalgesia.     

Imidazole chloride and tris-chloride substitute for sodium chloride in inducing high-affinity AdoPP[NH]P binding to (Na+ + K+)-ATPase

Optimal binding of [2,8-3H]AdoPP[NH]P to (Na+ + K+)-ATPase requires 25 mM Na+ (Cl-), 50 mM imidazole+ (Cl-) or 50 mM Tris+ (Cl-). Chloride is essential as counterion. We conclude that imidazole+ and Tris+ are able to bind to the Na+ site, and recommend the use of dilute buffers for studying the partial reactions of (Na+ + K+)-ATPase. In NaCl or the substituting buffers the dissociation constant for the enzyme-AdoPP[NH]P complex at 0 degrees C and pH 7.25 is 0.4 microM, whereas in millimolar MgCl2 it is about 2 microM. These distinct levels in affinity with MgCl2 as compared to NaCl, together with the MgCl2-dependence of photolabelling of the enzyme with ATP analogues (Rempeters, G. and Schoner, W. (1981) Eur. J. Biochem. 121, 131-137), suggest significant changes within the substrate site of (Na+ + K+)-ATPase upon binding of Mg2+ (Cl-)2.     

Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization

Adenosine 5'-phosphosulfate (APS) kinase, the second enzyme in the pathway of inorganic sulfate assimilation, was purified to near homogeneity from mycelium of the filamentous fungus, Penicillium chrysogenum. The enzyme has a native molecular weight of 59,000-60,000 and is composed of two 30,000-dalton subunits. At 30 degrees C, pH 8.0 (0.1 M Tris-chloride buffer), 5.5 microM APS, 5 mM MgATP, 5 mM excess MgCl2, and "high" salt (70-150 mM (NH4)2SO4), the most highly purified preparation has a specific activity of 24.7 units X mg of protein-1 in the physiological direction of adenosine 3'-phosphate 5'-phosphosulfate (PAPS) formation. This activity is nearly 100-fold higher than that of any previously purified preparation of APS kinase. APS kinase is subject to potent substrate inhibition by APS. In the absence of added salt, the initial velocity at 5 mM MgATP plus 5 mM Mg2+ is maximal at about 1 microM APS and half-maximal at 0.2 and 4.4 microM APS. In the presence of 200 mM NaCl or 70-150 mM (NH4)2SO4, the optimum APS concentration shifts to 4-6 microM APS; the half-maximal values shift to 1-1.3 and 21-27 microM APS. The steady state kinetics of the reaction were investigated using a continuous spectrophotometric assay. The families of reciprocal plots in the range 0.25-5 mM MgATP and 0.8-5.1 microM APS are linear and intersect on the horizontal axis. Appropriate replots yield KmMgATP = 1.5 mM, KmAPS = 1.4 microM, and Vmax, = 38.7 units X mg of protein-1. Excess APS is an uncompetitive inhibitor with respect to MgATP (K1APS = 23 microM). PAPS, the product of the forward reaction, is also uncompetitive with MgATP. PAPS is not competitive with APS. In the reverse direction, the plots have the characteristics of a rapid equilibrium ordered sequence with MgADP adding before PAPS. The kinetic constants are KmPAPS = 8 microM, KiMgADP = 560 microM, and Vmaxr = 0.16 units X mg of protein-1. Iso-PAPS (the 2'-phosphate isomer of PAPS) is competitive with PAPS and uncompetitive with respect to MgADP (Ki = 6 microM). APS kinase is inactivated by phenylglyoxal, suggesting the involvement of an essential argininyl residue. MgATP or MgADP at 10 Ki protect against inactivation. APS or PAPS at 600 and 80 Km, respectively, are ineffective alone, but provide nearly complete protection in the presence of 0.1 Ki of MgADP or MgATP.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

The role of Na,K-ATPase alpha subunit serine 775 and glutamate 779 in determining the extracellular K+ and membrane potential-dependent properties of the Na,K-pump

The roles of Ser775 and Glu779, two amino acids in the putative fifth transmembrane segment of the Na,K-ATPase alpha subunit, in determining the voltage and extracellular K+ (K+(o)) dependence of enzyme-mediated ion transport, were examined in this study. HeLa cells expressing the alpha1 subunit of sheep Na,K-ATPase were voltage clamped via patch electrodes containing solutions with 115 mM Na+ (37 degrees C). Na,K-pump current produced by the ouabain-resistant control enzyme (RD), containing amino acid substitutions Gln111Arg and Asn122Asp, displayed a membrane potential and K+(o) dependence similar to wild-type Na,K-ATPase during superfusion with 0 and 148 mM Na+-containing salt solutions. Additional substitution of alanine at Ser775 or Glu779 produced 155- and 15-fold increases, respectively, in the K+(o) concentration that half-maximally activated Na,K-pump current at 0 mV in extracellular Na+-free solutions. However, the voltage dependence of Na,K-pump current was unchanged in RD and alanine-substituted enzymes. Thus, large changes in apparent K+(o) affinity could be produced by mutations in the fifth transmembrane segment of the Na,K-ATPase with little effect on voltage-dependent properties of K+ transport. One interpretation of these results is that protein structures responsible for the kinetics of K+(o) binding and/or occlusion may be distinct, at least in part, from those that are responsible for the voltage dependence of K+(o) binding to the Na,K-ATPase.     

Properties and subcellular localization of adenosine diphosphatase in rat heart

Some properties and subcellular localization of adenosine diphosphatase (ADPase) activity from rat heart have been investigated. The pH optimum was 7.4, maximal activity was found with 5 mM MgCl2, and the apparent Km was 20 microM. ADPase activity was strongly inhibited by NaF and AppNHp, and to a lesser extent by AMP and GppNHp. The enzyme was not inhibited by p-nitrophenylphosphate, beta-glycerophosphate, or pyridoxal phosphate. The distribution of ADPase activity in subcellular fractions obtained by differential centrifugation parallel ouabain-sensitive (Na+-K+)ATPase and 5'-nucleotidase activities, suggesting a plasma membrane-bound localization. The functional significance of ADPase in adenosine production and hemostasis is discussed.     

Affinity labelling with MgATP analogues reveals coexisting Na+ and K+ forms of the alpha-subunits of Na+/K+-ATPase.

To test the hypothesis that Na+/K+-ATPase works as an (alpha beta)2-diprotomer with interacting catalytic alpha-subunits, tryptic digestion of pig kidney enzyme, that had been inactivated with substitution-inert MgATP complex analogues, was performed. This led to the demonstration of coexisting C-terminal Na+-like 80-kDa as well as K+-like 60-kDa peptides and N-terminal 40-kDa peptides of the alpha-subunit. To localize the ATP binding sites on tryptic peptides, studies with radioactive MgATP complex analogues were performed: Co(NH3)4-8-N3-ATP specifically modified the E2ATP (low affinity) binding site of Na+/K+-ATPase with an inactivation rate constant (k2) of 12 x 10-3.min-1 at 37 degrees C and a dissociation constant (Kd) of 207 +/- 28 microm. Tryptic digestion of the [gamma32P]Co(NH3)4-8-N3-ATP-inactivated and photolabelled alpha-subunit (Mr = 100 kDa) led, in the absence of univalent cations, to a K+-like C-terminal 60-kDa fragment which was labelled in addition to an unlabelled Na+-like C-terminal 80-kDa fragment. Tryptic digestion of [alpha32P]-or [gamma32P]Cr(H2O)4ATP - bound to the E1ATP (high affinity) site - led to the labelling of a Na+-like 80-kDa fragment besides the immediate formation of an unlabelled K+-like N-terminal 40-kDa fragment and a C-terminal 60-kDa fragment. Because a labelled Na+-like 80-kDa fragment cannot result from an unlabelled K+-like 60-kDa fragment, and because unlabelled alpha-subunits did not show any catalytic activity, the findings are consistent with a situation in which Na+- and K+-like conformations are stabilized by tight binding of substitution-inert MgATP complex analogues to the E1ATP and E2ATP sites. Hence, all data are consistent with the hypothesis that ATP binding induces coexisting Na+ and K+ conformations within an (alphabeta)2-diprotomeric Na+/K+-ATPase.     

Factors affecting the inhibition of yeast plasma membrane ATPase by vanadate

Inhibition of yeast plasma membrane ATPase by vanadate occurs only if either Mg2+ or MgATP2- is bound to the enzyme. The dissociation constant of the complex of vanadate and inhibitory sites is 0.14-0.20 microM in the presence of optimal concentrations of Mg2+ and of the order of 1 microM if the enzyme is saturated with MgATP2-. The dissociation constants of Mg2+ and MgATP2- for the sites involved are 0.4 and 0.62-0.73 mM, respectively, at pH 7. KCl does not increase the affinity of vanadate to the inhibitory sites as was found with (Na+ + K+)-ATPase. On the other hand, the effect of Mg2+ upon vanadate binding is similar to that upon (Na+ + K+)-ATPase, and the corresponding affinity constants of Mg2+ and vanadate for the two enzymes are of the same order of magnitude.     

Properties of rat heart adenosine kinase.

Adenosine kinase was purified 870-fold from rat heart by a combination of gel filtration and affinity chromatography. The preparation was free of purine-metabolizing enzymes that could interfere in the assay of the kinase. A study of the properties of the purified enzyme showed that it is activated by Na+ and K+, it possesses a broad pH optimum between 6 and 8, MgATP is the nucleotide substrate, free Mg2+ is an inhibitor with respect to both MgATP and adenosine, and the enzyme is subject to substrate inhibition by adenosine. The severity of this inhibition increases as the concentration of free Mg2+ increase. The Km for MgATP was calculated to be 0.8 mM and that for adenosine, at likely physiological concentrations of MgATP and free MgCl2, was about 0.2 microM. In vivo the enzyme is likely to be saturated with both MgATP and adenosine. Indeed, the adenosine concentration in rat heart in vivo is probably sufficient to cause substrate inhibition, and this would be increased by an increase in free Mg2+ concentration. Changes in the concentrations of adenosine and free Mg2+ may play a role in modifying the activity of the enzyme in vivo.     

Calmodulin increases Ca-dependent inhibition of the Na,K-ATPase in human red blood cells.

Proteins in human red cell hemolysate were purified to determine which of them increase inhibition of the Na,K-ATPase in the presence of 2 microM free Ca. Samples purified 600,000-fold inhibited the Na,K-ATPase of human red cells in a Ca-dependent manner and stimulated the (Ca+Mg)-ATPase. These samples contained two proteins as analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE): calmodulin (18,000 Mr), which comprised most (greater than 90%) of the total protein, and an unidentified protein of approximately 13,000 Mr. Both proteins were a distinctive light yellow when stained with silver. Calmodulin from bovine testes also inhibited the Na,K-ATPase and stimulated the (Ca+Mg)-ATPase. This preparation also contained two proteins as analyzed by SDS-PAGE: calmodulin (95 to 99% of the total protein) and another protein of approximately 13,000 Mr (1 to 5% of the total protein). Both were light yellow when stained with silver. Since the amount of red cell protein was limited, the remainder of the study was carried out with the bovine testes preparation. Heating the testes preparation decreased, but did not abolish, inhibition of the Na,K-ATPase and reduced stimulation of the (Ca+Mg)-ATPase. When corrected for denatured calmodulin, both heated and unheated proteins increased inhibition of the Na,K-ATPase to the same extent. The Na,K-ATPase was inhibited at 2 microM free Ca in a dose-dependent manner over a range of 15 to 100 nM calmodulin. To establish if the inhibition was due to the calmodulin or the 13,000 Mr protein, both were electroeluted after SDS-PAGE. Electroeluted calmodulin stimulated the (Ca+Mg)-ATPase and increased Ca inhibition of the Na,K-ATPase. Electroeluted amounts of the smaller Mr protein slightly stimulated the (Ca+Mg)-ATPase, but had no effect on the Na,K-ATPase. This protein was digested with cyanogen bromide, partially sequenced, and thereby identified as a fragment of calmodulin. We conclude that intact calmodulin increases inhibition of the Na,K-ATPase at 2 microM free Ca. We suggest that calmodulin is part of a mechanism mediating the effects of physiological free Ca on the Na,K-ATPase.