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.     

The distribution of C12E8-solubilized oligomers of the (Na+ + K+)-ATPase

Gel filtration of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonother ( C12E8 ) has been performed under conditions where active (alpha beta)2 dimers (Mr 265000) are obtained, and under conditions where dissociation into alpha beta monomers occurs without appreciable loss of activity. It is shown that the alpha beta monomers aggregate with time to form (alpha beta)2 dimers at low detergent concentrations with no change in enzymatic activity. At high detergent concentrations the aggregation is much slower, but the enzymatic activity is lost rapidly. Polyacrylamide gel electrophoresis in the presence of C12E8 also suggest that high concentrations of detergent dissociate the (alpha beta)2 dimer into smaller particles, and conditions for gel electrophoresis are described. The inactivating effect of C12E8 at high C12E8 /protein ratios can be related to a delipidation of the enzyme, with about 0.19 mg phospholipid required per mg protein for optimal activity. The experiments suggest that the solubilized (Na+ + K+)-ATPase can be disrupted into particles containing only one alpha-chain and one or two beta-chains without irreversible loss of activity, and that the stable form of the enzyme is an (alpha beta)2 dimer.     

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.     

Stability of [3H]ouabain binding to the (Na+ + K+)-ATPase solubilized with C12E8

The (Na+ + K+)-ATPase from dog kidney and partially purified membranes from HK dog erythrocytes were labeled with [3H]ouabain, solubilized with C12E8 and analyzed by HPLC through a TSK-GEL G3000SW column in the presence of C12E8, Mg2+, HPO4(2-) and glycerol at 20-23 degrees C. The peaks of [3H]ouabain bound to the enzyme from dog kidney and HK dog erythrocyte membranes corresponded to each other with apparent molecular weights of 470 000-490 000. In addition, these bindings of [3H]ouabain to the (Na+ + K+)-ATPase were observed to be stable at 20-23 degrees C for at least 18 h after the solubilization.     

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.     

ATP binding to solubilized (Na+ + K+)-ATPase. The abolition of subunit-subunit interaction and the maximum weight of the nucleotide-binding unit

Membrane-bound (Na+ + K+)-ATPase from pig kidney outer medulla shows apparent heterogeneity in its ATP-binding site population when assays are carried out in the presence of K+. This finding has been interpreted as being due to interaction between (at least) two subunits, each containing an ATP-binding site. Treating the membrane-bound enzyme with the detergent, C12E8, has been shown to solubilize enzymatically active alpha beta-protomers. We show that in the dissolved enzyme all ATP-binding sites in the population are identical both in the absence and in the presence of K+, which would be consistent with an abolition of identical both in the absence and in the presence of K+, which would be consistent with an abolition of subunit-subunit interaction. This supports previous suggestions that enzyme solubilized by C12E8 is monomeric and that the membrane-bound enzyme is not. Differential extraction of enzyme-containing membranes with C12E8 yielded preparations with an ATP-binding capacity of up to 5.8 nmol per mg protein, measured by the method of Lowry et al. (Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275), with bovine serum albumin as standard. Evidence is presented that makes it likely that preparations with an ATP-binding capacity of 7.5 nmol per mg protein (as determined by the above-mentioned assay) will be obtainable. This corresponds to an alpha beta-protomer molecular weight of 133 000 which approximates closely to the minimum value found in the literature for an alpha beta-protomer (i.e., 126 000).     

Molecular weights of alpha beta-protomeric and oligomeric units of soluble (Na+, K+)-ATPase determined by low-angle laser light scattering after high-performance gel chromatography

The (Na+, K+)-ATPase of canine renal outer medulla was solubilized with a nonionic surfactant, octaethylene glycol n-dodecyl ether (C12E8), in the presence of 0.2 M sodium ion. The solubilized ATPase retained 74% of the enzymatic activity expressed before solubilization. Molecular species of the solubilized ATPase were analyzed by high-performance chromatography through a TSK-GEL G3000SW column in the presence of 1 mg/ml C12E8 at 23 degrees C. The eluate was monitored by one or two monitors chosen from the following: an ultraviolet absorption monitor, a precision differential refractometer and a low-angle laser light scattering photometer. The three kinds of elution pattern thus obtained can best be interpreted by assuming the presence of at least four kinds of protein component with molecular weights 1 740 000 +/- 230 000, 836 000 +/- 82 000, 286 000 +/- 30 000 and 123 000 +/- 8 000, respectively. Among them, those with the last two molecular weight were the major components. The amounts of the first three components were found to increase with time during the incubation before application to the column at the expense of that of the last one. The amounts of the last two were 18 and 73%, respectively, when measured immediately after the solubilization. A stoichiometric composition of 1:1 molar ratio for the alpha and beta polypeptide chains was obtained for the two major components as well as for the intact ATPase by high-performance gel chromatography in the presence of sodium dodecyl sulfate using the same column as above. The (Na+, K+)-ATPase was, thus, indicated to be solubilized with C12E8 to give the alpha beta-protomer and its dimer as the main components.     

Properties of oligomycin-induced occlusion of Na+ by detergent-solubilized Na,K-ATPase from pig kidney or shark rectal gland.

Oligomycin induces occlusion of Na+ in membrane-bound Na,K-ATPase. Here it is shown that Na,K-ATPase from pig kidney or shark rectal gland solubilized in the nonionic detergent C12E8 is capable of occluding Na+ in the presence of oligomycin. The apparent affinity for Na+ is reduced for both enzymes upon solubilization, and there is an increase in the sigmoidicity of binding curves, which indicates a change in the cooperativity between the occluded ions. A high detergent/protein ratio leads to a decreased occlusion capacity. De-occlusion of Na+ by addition of K+ is slow for solubilized Na,K-ATPase, with a rate constant of about 0.1 s-1 at 6 degrees C. Stopped-flow fluorescence experiments with 6-carboxyeosin, which can be used to monitor the E1Na-form in detergent solution, show that the K(+)-induced de-occlusion of Na+ correlates well with the fluorescence decrease which follows the transition from the E1Na-form to the E2-form. There is a marked increase in the rate of fluorescence change at high detergent/protein ratios, indicating that the properties of solubilized enzyme are subject to modification by detergent in other respects than mere solubilization of the membrane-bound enzyme. The temperature dependence of the rate of de-occlusion in the range 2 degrees C to 12 degrees C is changed slightly upon solubilization, with activation energies in the range 20-23 kcal/mol for membrane-bound enzyme, increasing to 26-30 kcal/mol for solubilized enzyme. Titrations of the rate of transition from E1Na to E2K with oligomycin can be interpreted in a model with oligomycin having an apparent dissociation constant of about 2.5 microM for C12E8-solubilized shark Na,K-ATPase and 0.2 microM for solubilized pig kidney Na,K-ATPase.     

Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na+ + K+)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C12E8). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K+-phosphatase or (Na+ + K+)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the alpha- and beta-subunits. The (Na+ + K+)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis alpha-toxin-Sepharose columns. The data suggest that the alpha-subunit of (Na+ + K+)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.     

Purification from brain of an intrinsic membrane protein fraction enriched in (Na+ + K+)-ATPase.

A microsomal fraction from canine brain gray matter has been extracted with the detergent sodium dodecyl sulfate to partially purify the membrane-bound (Na+ + K+)-stimulated adenosine triphosphatase. Phospholipid, glycolipid, and a family of other glycoproteins are also enriched by the procedure; it is proposed that the product is an intrinsic membrane protein fraction. 6--8-fold purification of (Na+ + K+)-ATPase is obtained without solubilizing the enzyme and without irreversibly altering its turnover number. Final specific activities are 350--400 mumol of ATP hydrolyzed/h per mg protein. The stimulation and reversible inactivation of the (Na+ + K+)-ATPase by dodecyl sulfate were examined for information relevant to the mechanism of action of the detergent.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified (Na+ + K+)-ATPase accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to beta-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of (Na+ + K+)-ATPase and p-nitrophenylphosphate activity by digitonin (which occurs during the rapid phase of inactivation) is unlikey to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either (Na+ + K+)-ATPase or p-nitrophenylphosphatase activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Thermoinactivation and aggregation of alpha beta units in soluble and membrane-bound (Na,K)-ATPase

Stability and conformational transitions of soluble and fully active alpha beta units of (Na,K)-ATPase in n-dodecyl octaethylene glycol monoether (C12E8) are examined. Sedimentation equilibrium centrifugation gave a molecular weight of 143 000 for the alpha beta unit eluting from TSK 3000 SW gel chromatography columns. Fluorescence analysis and phosphorylation experiments show that E1-E2 transitions between both dephospho and phospho forms of soluble (Na,K)-ATPase are similar to those previously observed in the membrane-bound state. The two conformations can also be identified by their different susceptibilities to irreversible temperature-dependent inactivation. E1 forms of both soluble and membrane-bound (Na,K)-ATPase are more thermolabile than E2 forms. Gel chromatography on TSK 3000 SW and 4000 SW columns shows that thermal inactivation of soluble (Na,K)-ATPase at 40 degrees C is accompanied by aggregation of alpha beta units to (alpha beta)2 units and higher oligomers. The aggregates are stable in C12E8 but dissolve in sodium dodecyl sulfate. Similar aggregation accompanies inactivation of membrane-bound (Na,K)-ATPase at 55-60 degrees C. These data suggest that inactivation both in the soluble and in the membrane-bound state involves exposure of hydrophobic residues to solvent. The instability of the soluble E1 form may be related to inadequate length of the dodecyl alkyl chain of C12E8 for stabilization of hydrophobic protein domains that normally associate with alkyl chains of phospholipids in the membrane. Interaction between alpha beta units-does not seem to be required for the E1-E2 conformational change, but irreversible aggregation appears to be a consequence of denaturation of (Na,K)-ATPase in both soluble and membranous states.     

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.     

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.     

Mechanisms of detergent effects on membrane-bound (Na+ + K+)-ATPase

Because the nonionic detergent octaethylene glycol dodecyl ether has been used extensively for studies on active solubilized preparations of (Na+ + K+)-ATPase, we tried to see if the detergent alters the properties of the membrane-bound enzyme prior to solubilization. Addition of the detergent, at concentrations below its critical micellar concentration, to reaction mixtures containing the highly purified membrane-bound enzyme reduced the K0.5 of ATP for (Na+ + K+)-dependent ATPase activity without affecting the maximal velocity or abolishing the negative cooperativity of the substrate-velocity curve. Under these conditions, however, the enzyme was not solubilized as evidenced by complete sedimentation of the membrane fragments containing the enzyme upon centrifugation at 100,000 X g for 30 min. Other nonsolubilizing effects of the detergent included an increase in K0.5 of K+, inhibition of Na+-dependent ATPase with no effect on K0.5 of ATP for this activity, and reductions in the spontaneous decomposition rates of the K+-sensitive phosphoenzyme obtained from ATP and the phosphoenzyme obtained from Pi. The nonsolubilizing effects of the detergent on the purified enzyme were obtained with no detectable lag, were readily reversible, and could be distinguished from its vesicle-opening effects on crude membrane preparations. Several other nonionic and ionic detergents had similar effects on the enzyme. The findings indicate (a) detergent binding to hydrophobic sites on extramembranous segments of enzyme subunits; (b) that occupation of these sites mimics the effects of ATP at a low-affinity regulatory site with no effect on high-affinity ATP binding to the catalytic site; and (c) that in studies on detergent-solubilized preparations, it is necessary to distinguish between the effects of solubilization per se and detergent effects at the regulatory site.     

Characterization of (Na+ + K+)-ATPase liposomes. II. Effect of alpha-subunit digestion on intramembrane particle formation and Na+,K+-transport

The effect of the protein structure of (Na+ + K+)-ATPase on its incorporation into liposome membranes was investigated as follows: the catalytic alpha-subunit of (Na+ + K+)-ATPase was split into low-molecular weight fragments by trypsin treatment and the digested enzyme was reconstituted at the same protein concentration as intact control enzyme. The reconstitution process was quantified by the average number of intramembrane particles appearing on concave and convex fracture faces after freeze-fracture of the (Na+ + K+)-ATPase liposomes. The number of intramembrane particles as well as their distribution on concave and convex fracture faces is not modified by the proteolysis. In contrast, the ATPase activity and the transport capacity of the (Na+ + K+)-ATPase decrease progressively with increasing incubation times in the presence of trypsin and are abolished when the original 100 000 molecular weight alpha-subunit is no longer visible by sodium dodecylsulfate gel electrophoresis. Apparently, functional (Na+ + K+)-ATPase with intact protein structure and digested, non functional enzyme consisting of fragments of the alpha-subunit reconstitute in the same manner and to the same extent as judged by freeze-fracture analysis. We conclude that, while trypsin treatment modifies the (Na+ + K+)-ATPase molecule in a functional sense, it appears not to modify its interaction with the bilayer in producing intramembrane particles. On the basis of our results, we propose a lipid-lipid interaction mechanism for reconstitution of (Na+ + K+)-ATPase.     

Enrichment of Na+-Ca2+ exchange in cardiac sarcolemmal vesicles by alkaline extraction

Exposure of canine cardiac sarcolemmal vesicles to alkaline media (greater than or equal to pH 12) results in the extraction of 33% of the protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that specific proteins are being solubilized. Most of the phospholipid and sialic acid remains with the pellet after centrifugation. Electron microscopy reveals that alkaline treatment does not cause gross morphological damage to the vesicles, although freeze-fracture demonstrates some aggregation of intramembrane particles. The data indicate that high pH probably removes peripheral proteins and leaves the integral proteins in place. We find complete recovery of Na+-Ca2+ exchange activity in alkaline-extracted membranes after solubilization and reconstitution. These vesicles contain only 50% of the protein of vesicles reconstituted from control sarcolemma. Thus, the specific activity of Na+-Ca2+ exchange is doubled. Alkaline extraction is a useful and reproducible procedure for enrichment of the Na+-Ca2+ exchange protein. (Na+ + K+)-ATPase is completely inactivated by exposure to pH 12 medium though immunodetection shows that the (Na+ + K+)-ATPase proteins are not extracted. We detect both alpha and alpha + forms of (Na+ + K+)-ATPase and deduce that the Na+ pump proteins do not comprise a major fraction of sarcolemmal protein.     

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.     

Difference in phospholipid dependence between two isozymes of brain (Na+ + K+)-ATPase

The effect of phospholipase C on two isozymes (alpha (+) and alpha forms) of rat brain (Na+ + K+)-ATPase and the temperature-dependence of their activities were investigated. Phospholipase C from Clostridium welchii inhibited the activities of the enzymes treated with and without pyrithiamin or N-ethylmaleimide, a preferential inhibitor of the alpha (+) form, but the extent of the inhibition was higher in the control enzyme than in the treated enzymes. The treatment of the (Na+ + K+)-ATPase with phospholipase C altered a ratio between high- and low-affinity components for ouabain inhibition. It also caused the similar change in a ratio between the alpha (+) and alpha forms of Na+-stimulated phosphorylation from [gamma-32P]ATP. These findings indicate that the alpha (+) form of rat brain (Na+ + K+)-ATPase is more sensitive to phospholipase C than the alpha form. Analysis of Arrhenius plots of the activities of the control and pyrithiamin-treated enzymes showed that there was a difference between the two enzymes in a break point. We suggest that two isozymes of rat brain (Na+ + K+)-ATPase differ in the interaction with phospholipids or in the lipid-environment.     

A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.