Purification and characterization of (Na+ + K+)-ATPase from toad kidney.

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], J?rgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.     

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.     

Studies on (Na+ + K+)-activated ATPase. XLII. Evidence for two classes of essential sulfhydryl groups.

1. Preincubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from rabbit kidney outer medulla with 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits the (Na+ + 5+)-ATPase and K+-stimulated 4-nitro-phenylphosphatase activities. Phosphorylation of the enzyme by ATP and the Na+-stimulated ATPase activity are inhibited to the same extent as the (Na+ + K+)-ATPase activity, whereas the K+-stimulated 4-nitrophenylphosphatase activity is inhibited much less. 2. Titration with 5,5'-dithiobis-(2-nitrobenzoic acid) in sodium dodecyl sulphate shows the presence of 36 reactive sulfhydryl groups per molecule (Na+ + K+)-ATPase (Mr = 250 000). 3. Treatment with N-ethylmaleimide, resulting in complete inhibition of (Na+ + K+)-ATPase activity, leads to modification of 26 sulfhydryl groups, whereas treatment with 5,5'-dithiobis-(2-nitrobenzoic acid) results in modification of 12 sulfhydryl groups under the same conditions. 4. The reaction of N-ethylmaleimide with an essential SH-group is not prevented by previous blocking of sulfhydryl groups with 5,5'-dithiobis-(2-nitrobenzoic acid). 5. These findings indicate the existence of at least two classes of sulfhydryl groups on the enzyme, each containing at least one vital group. The difference between these classes consists in their different reactivity towards 5,5'-dithiobis-(2-nitrobenzoic acid) and N-ethylmaleimide.     

Phenytoin Dephosphorylates the Catalytic Subunit of the (Na+,K+)-ATPase in C57/BL Mice

The effects of phenytoin, a potent antiepileptic drug, on the active transport of cations within membranes remain controversial. To assess the direct effects of phenytoin on the Na+,K+ pump, we studied the drug's influence on the phosphorylation of partially purified (Na+,K+)-ATPase from mouse brain. (Na+,K+)-ATPase subunits were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phenytoin, in vitro, decreased net phosphorylation of the (Na+,K+)-ATPase catalytic subunit in a dose-dependent manner (approximately 50% at 10(-4) M). When the conversion of E1-P to E2-P, e.g., the two major phosphorylated conformational states of (Na+,K+)-ATPase, was blocked by oligomycin or N-ethylmaleimide, phenytoin had no effect. The results suggest that phenytoin acts on the phosphatasic component of the reaction cycle, decreasing the phosphorylation level of the enzyme.     

Separation of basolateral plasma membranes from smooth endoplasmic reticulum of the rat enterocyte by zonal electrophoresis on density gradients

Basolateral plasma membranes of rat small intestinal epithelium were purified by density gradient centrifugation followed by zonal electrophoresis on density gradients. Crude basolateral membranes were obtained by centrifugation in which the marker enzyme, (Na+ + K+)-ATPase, was enriched 10-fold with respect to the initial homogenate. The major contaminant was a membrane fraction derived from smooth endoplasmic reticulum, rich in NADPH-cytochrome c reductase activity. The crude basolateral membrane preparation could be resolved into the two major components by subjecting it to zonal electrophoresis on density gradients. The result was that (Na+ + K+)-ATPase was purified 22-fold with respect to the initial homogenate. Purification with respect to mitochondria and brush border membranes was 35- and 42-fold, respectively. Resolution of (Na+ + K+)-ATPase from NADPH-cytochrome c reductase by electrophoresis was best with membrane material from adult rats between 180 and 250 g. No resolution between the two marker enzymes occurred with material from young rats of 125 to 140 g. These results demonstrate that zonal electrophoresis on density gradients, a simple and inexpensive technique, has a similar potential to free-flow electrophoresis.     

Separation of vesicles of cardiac sarcolemma from vesicles of cardiac sarcoplasmic reticulum. Comparative biochemical analysis of component activities.

Sarcolemmal and sarcoplasmic reticulum membrane vesicle fractions were isolated from cardiac microsomes. Separation of sarcolemmal and sarcoplasmic reticulum membrane markers was documented by a combination of correlative assay and centrifugation techniques. To facilitate the separation, the crude microsomes were incubated in the presence of ATP, Ca2+, and oxalate to increase the density of the sarcoplasmic reticulum vesicles. After sucrose gradient centrifugation, the densest subfraction (sarcoplasmic reticulum) contained the highest (K+,Ca2+)-ATPase activity and virtually no (Na2+,K+)-ATPase activity, even when latent (Na+,K+)-ATPase activity was unmasked. In addition, the sarcoplasmic reticulum fraction contained no significant sialic acid, beta receptor binding activity, or adenylate cyclase activity. Sarcolemmal membrane fractions were of low buoyant density. Preparations most enriched in sarcolemmal vesicles contained the highest level of all the other parameters and only about 10% of the (K+,Ca2+)-ATPase activity of the sarcoplasmic reticulum fraction. The results suggest that (Na+,K+)-ATPase, sialic acid, beta-adrenergic receptors, and adenylate cyclase can be entirely accounted for by the sarcolemmal content of cardiac microsomes. Gel electrophoresis of the sarcolemmal and sarcoplasmic reticulum membrane fractions showed distinct bands. Membrane proteins exclusive to each of the fractions were also demonstrated by phosphorylation. Cyclic AMP stimulated phosphorylation by [gamma-32P]ATP of two proteins of apparent Mr = 20,000 and 7,000 that were concentrated in sarcoplasmic reticulum, but the stimulation was markedly dependent on the presence of added soluble cyclic AMP-dependent protein kinase. Cyclic AMP also stimulated phosphorylation of membrane proteins in sarcolemma, but this phosphorylation was mediated by an endogenous protein kinase activity. The apparent molecular weights of these phosphorylated proteins were 165,000, 90,000, 56,000, 24,000, and 11,000. The results suggest that sarcolemma may contain an integral enzyme complex, not present in sarcoplasmic reticulum, that contains beta-adrenergic receptors, adenylate cyclase, cyclic AMP-dependent protein kinase, and several substrates of the protein kinase.     

Mass isolation of cell surface membrane fragments from pigeon heart.

Cell surface membrane fragments were isolated and purified by successive rate zonal and isopycnic centrifugation of calcium oxalate-loaded pigeon heart microsomes in sucrose density gradients. The most highly purified cell membrane fraction sediments at a buoyant density of 1.105 g/ml. Some of the membrane pieces are present as open fragments and leaky vesicles, while others form tightly sealed vesicles of both inside-in and inside-out membrane orientation. The pigeon heart cell membrane preparation exhibits high (Na+ + K+ + Mg2+)-ATPase and adenylate cyclase activities. Additional activity of these enzymes is uncovered by sodium dodecyl sulfate and alamethicin, respectively. Electron microscopic inspection of the cell surface membrane preparation revealed (a) a predominance of thick-walled vesicles with smooth surfaces on negative staining and (b) binding of concanavalin A to the bulk of isolated membrane pieces following their incubation with the lectin.     

Inhibition by lead ion of Electrophorus electroplax (Na+ + K+)-adenosine triphosphatase and K+-p-nitrophenylphosphatase.

Inorganic lead ion in micromolar concentrations inhibits Electrophorus electroplax microsomal (Na+ + K+)-adenosine triphosphatase ((Na+ + K+)-ATPase) and K+-p-nitrophenylphosphatase (NPPase). Under the same conditions, the same concentrations of PbCl2 that inhibit ATPase activity also stimulate the phosphorylation of electroplax microsomes in the absence of added Na+. Enzyme activity is protected from inhibition by increasing concentrations of microsomes, ATP, and other metal ion chelators. The kinetics follow the pattern of a reversible noncompetitive inhibitor. No kinetic evidence is elicited for interactions of Pb2+ with Na+, K+, Mg2+, ATP, or p-nitrophenylphosphate. Na+- ATPase, in the absence of K+, and (Na+ + K+)-NPPase activity at low [K+] are also inhibited. ATP inhibition of NPPase is not reversed by Pb2+. The calculated concentrations of free [Pb2+] that produce 50% inhibition are similar for ATPase and NPPase activities. Pb2+ may act at a single independent binding site to produce both stimulation of the kinase and inhibition of the phosphatase activities.     

A reconstituted Na+ + K+ pump in liposomes containing purified (Na+ + K+)-ATPase from kidney medulla.

Liposomes containing either purified or microsomal (Na+,K+)-ATPase preparations from lamb kidney medulla catalyzed ATP-dependent transport of Na+ and K+ with a ratio of approximately 3Na+ to 2K+, which was inhibited by ouabain. Similar results were obtained with liposomes containing a partially purified (Na+,K+)-ATPase from cardiac muscle. This contrasts with an earlier report by Goldin and Tong (J. Biol. Chem. 249, 5907-5915, 1974), in which liposomes containing purified dog kidney (Na+,K+)-ATPase did not transport K+ but catalyzed ATP-dependent symport of Na+ and Cl-. When purified by our procedure, dog kidney (Na+,K+)-ATPase showed some ability to transport K+ but the ratio of Na+ : K+ was 5 : 1.     

Kinetics of the Mechanism of Action of Monoamine Oxidase in the Regulation of Na+, K+-ATPase Activity in Rat Brain

Kinetic studies on the action of monoamine oxidase (MAO) in the regulation of Na+,K+-ATPase were performed using 3-methoxy-4-hydroxybenzaldehyde (MHB), which is an analogue of 3-methoxy-4-hydroxy-phenylacetylaldehyde (product of MAO-catalysed reaction with dopamine as substrate). It was observed that at 2.6 microM MHB, the activation of Na+,K+-ATPase may be the result of the removal of the inhibitory Ca2+, thereby increasing the Vmax. Double-reciprocal plots of Pi versus MHB showed that Ca2+ counteracted the effect of the aldehyde not by changing the Km, but be decreasing the Vmax of the Na+,K+-ATPase stimulation. The removal of 3',5'-cyclic AMP-dependent protein kinase from the microsomes by sodium dodecyl sulphate treatment abolished the activation and/or inhibition of the Na+,K+-ATPase by aldehyde; it can therefore be inferred that 3',5'-cyclic AMP-dependent protein kinase is involved in the regulation of Na+,K+-ATPase.     

Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo

A Na+ -ATPase was partially purified from plasma membranes of the marine alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were collected by differential centrifugation with subsequent discontinuous gradient centrifugation. Na+ -ATPase activity was associated with the resultant plasma membrane fraction and was stimulated to the greatest extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions, pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in the same fraction in the presence of both Mg2+ and Na+ ions, and this protein was dephosphorylated upon the addition of K+ ions. We could partially purify the 140-kD protein after solubilization by Suc monolaurate and fractionation by sequential column chromatography on Sephacryl S-300, DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide could also be phosphorylated and be detected after acid sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+ ions.     

Membrane ATPase of Bacillus subtilis. I. Purification and properties.

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s20,w of 13,4 and an amino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (alpha) and 57 000 (beta) with different charges. Kinetic studies showed that Ca2+ or Zn2+ are required for ATPase activity, although Mg2+ was uneffective. At optimal Ca2+ concentration, the Mg2+ has an inhibitory effect. The Km for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na+ + K+)-ATPase are studied.     

A tyrosine-specific protein kinase from Ehrlich ascites tumor cells

A protein tyrosine kinase that phosphorylates both alpha and beta subunits of inactivated (Na+,K+)-ATPase from dog kidney was purified about 500-fold from Ehrlich ascites tumor cell membranes. The enzyme required divalent cations Mn2+, Mg2+, or Fe2+ but was inhibited by Cu2+ or Zn2+. The purified enzyme phosphorylated the beta subunit about five times faster than the alpha subunit of the (Na+,K+)-ATPase. The random polymer poly(Glu80Tyr20) was an excellent substrate while casein was only marginally phosphorylated. In contrast, the purified transforming gene product of Rous sarcoma virus phosphorylated all three substrates and the (Na+,K+)-ATPase was preferentially phosphorylated on the alpha subunit. The transforming gene product of Fujinami sarcoma visue and EGF receptor kinase from A431 cells phosphorylated (Na+,K+)-ATPase poorly whereas casein was an excellent substrate. The molecular weight of the partially purified protein tyrosine kinase from Ehrlich ascites tumor cells determined by gel filtration was about 60,000. One of two major phosphorylated phosphopeptides resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis had an Mr of 60 kDa, thus suggesting that it might be the autophosphorylated protein tyrosine kinase. A phosphatase that hydrolyzes phosphorylated histones or poly(Glu80Tyr20) was partially purified from the same membrane.     

Inhibition of (Na+,K+)-ATPase by dicyclohexylcarbodiimide. Evidence for two carboxyl groups that are essential for enzymatic activity

N,N'-Dicyclohexylcarbodiimide (DCCD), a reagent that reacts with carboxyl groups under mild conditions, irreversibly inhibits (Na+,K+)-ATPase activity (measured by using 1 mM ATP) with a pseudo-first-order rate constant of 0.084 min-1 (0.25 mM DCCD and 37 degrees C). The partial activities of the enzyme, including (Na+,K+)-ATPase at 1 microM ATP, Na+-ATPase, and the formation of enzyme-acyl phosphate (E-P), decayed at about one-third the rate at which (Na+,K+)-ATPase at 1 mM ATP was lost. The formation of E-P from inorganic phosphate was unaffected by DCCD while K+-phosphatase activity decayed at the same rate as (Na+,K+)-ATPase measured at 1 mM ATP. The enzyme's substrates (i.e., sodium, potassium, magnesium, and ATP) all decreased the rate of DCCD inactivation of (Na+,K+)-ATPase activity measured at either 1 mM or 1 microM ATP. The concentration dependence of the protection afforded by each substrate is consistent with its binding at a catalytically relevant site. DCCD also causes cross-linking of the enzyme into species of very high molecular weight. This process occurs at about one-tenth the rate at which (Na+,K+)-ATPase activity measured at 1 mM ATP is lost, too slowly to be related to the loss of enzymatic activity. Labeling of the enzyme with [14C]DCCD shows the incorporation of approximately 1 mol of DCCD per mole of large subunit; however, the incorporation is independent of the loss of enzymatic activity. The results presented here suggest that (Na+,K+)-ATPase contains two carboxyl groups that are essential for catalytic activity, in addition to the previously known aspartate residue which is involved in formation of E-P.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Ion-gated channel induced in planar bilayers by incorporation of (Na+,K+)-ATPase

An ion-gated channel was conferred on a planar lipid bilayer membrane upon incorporation of (Na+,K+)-ATPase. The channel exhibited two conductance states. The high conductance state was only observed when an ion gradient was present across the planar membrane. This state corresponded to an enzyme conformation which was ouabain and vanadate sensitive (i.e. conductance was inhibited by these compounds), while the low conductance state showed no sensitivity to either inhibitor. Single channel conductance behavior was observed when minimal amounts of enzyme were incorporated into the planar bilayer. The observed single channel conductance was 270 +/- 14 picosiemens. Similar transport behavior was observed for enzyme purified from ovine kidney using sodium dodecyl sulfate (anionic), eel electroplax using Lubrol-WX (nonionic), and kidney microsomes. In addition, the data strongly suggest that enzyme from the kidney microsomes was asymmetrically incorporated into the planar bilayer.     

Isolation of plasma membrane vesicles from rabbit skeletal muscle and their use in ion transport studies

A method has been developed for the isolation of sealed plasma membrane vesicles from rabbit white skeletal muscle. The final preparation was highly purified as indicated by enrichment of plasma membrane marker enzymes (i.e. ouabain-sensitive (Na+,K+)-ATPase, adenylate cyclase, and acetylcholinesterase). The absence of sarcoplasmic reticulum and mitochondria as contaminants was indicated by the low specific activity of marker enzymes, i.e. Ca2+-ATPase, succinate-cytochrome c reductase, and monoamine oxidase. Thin section and negative staining electron microscopy confirmed the absence of sarcoplasmic reticulum and mitochondrial contamination. The plasma membrane preparation consisted largely of sealed vesicles as observed by electron microscopy and as also demonstrated by latency of enzymic activities, which were unmasked by preincubation with detergent (sodium dodecyl sulfate). Membrane sidedness was estimated from latency of ouabain-sensitive (Na+,K+)-ATPase activity and acetylcholinesterase activity. The latency studies suggest that most of the vesicles are oriented inside out with respect to the orientation of the sarcolemma membrane in the muscle fiber. The inside-out plasma membrane vesicles actively accumulated sodium ions upon addition of ATP. The sodium ions were concentrated greater than 8-fold inside the vesicles and were released upon addition of the ionophore monensin. The sodium ions were taken up in the presence of K+ or NH4+ but not of choline. Uptake was inhibited by low concentrations of vanadate or digitoxin. The Na+ uptake was concomitant with Rb+ efflux. Therefore, the sodium ion transport and the resulting gradients formed appear to have been generated by the ouabain-sensitive (Na+,K+)-ATPase. Batrachotoxin, which opens Na+ channels in excitable tissues, prevents most of the Na+ uptake, suggesting the presence of toxin-activated Na+ channels in these plasma membrane vesicles.     

The measurement of ouabain binding and some related properties of digitonin-treated (Na+,K+)-ATPase.

1. Digitonin treated membrane preparations purified from dog kidney lose their (Na+,K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, but the K+-phosphatase and Na+-dependent ADP-ATP exchange activities survive and remain ouabain-sensitive. Because the enzyme preparations consist largely of pure (Na+,K+)-ATPase, these effects of digitonin must be intrinsic to the Na+ pump. 2. Concomitant with these enzymatic changes, digitonin treatment alters the sensitivity of the phosphatase and exchange activities to ouabain. 3. Attempts to measure ouabain binding by the usual centrifugation or filtration methods proved unsuccessful. A filtration method involving a double 0.01 mum filter and omitting water washes is necessary to demonstrate ouabain binding. Under these conditions, ouabain binding capacity appears to be unchanged in the presence of digitonin, but the apparent dissociation constant is doubled. 4. Ouabain binding is rendered more reversible by digitonin treatment, since washing filters with water removes a large fraction of bound ouabain without affecting the retention of exchange activity. 5. The double filter method traps essentially all of the ADP-ATP exchange activity on the filter. However, a large and somewhat variable proportion of the K+-phosphatase activity passes through the filter. Sodium dodecyl sulfate polyacrylamide gel analysis of the filtrate shows that a small amount of filtrable protein catalyzed this phosphatase activity at greatly increased turnover rates. Both subunits of the (Na+, K+)-ATPase are present in this latter protein fraction.     

Purification and characterization of (Na+, K+)-ATPase. V. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool.

1. Purified (Na+, K+)-ATPase consisting of membrane fragments was digested with trypsin. The time course of enzyme inactivation was related to the electrophoretic pattern of native and cleaved proteins remaining in the membrane. 2. Differences in both the inactivation kinetics and the cleavage of the large chain (mol. wt 98 000) allow distinction of two patterns of tryptic digestion of (Na+, K+)-ATPase seen with Na+ or K+ in the medium. 3. With K+, the inactivation of (Na+, K+)-ATPase is linear with time in semilogarithmic plots and the activity is lost in parallel with cleavage of the large chain to fragments with molecular weights 58 000 and 48 000. 4. With Na+, the inactivation curves are biphasic. In the initial phase of rapid inactivation, 50% of the activity is lost with minor changes in the composition of the large chain. In the final phase, the large chain is cleaved at a low rate to a fragment with a molecular weight of 78 000. 5. It is concluded that the regions of the large chain exposed in the presence of K+ are distinct from the regions exposed in presence of Na+ and that two conformations of (Na+, K+)-ATPase can be sensed with trypsin, a (t)K-form and a (t)Na-form. 6. Reaction of the (t)K-form with ATP cause transition to the (t)Na-form. Relatively high concentrations of ATP are required and Mg2+ is not necessary. Phosphorylation of (Na+, K+)-ATPase is accompanied by transition from the (t)Na-form to the (t)K-form. Previous kinetic data suggest that these conformational changes are accompanied by shifts in the affinities of the enzyme for Na+ and K+.     

Activation of (Na+, K+)-ATPase by Nanomolar Concentrations of GM1 Ganglioside

GM1 ganglioside binding to the crude mitochondrial fraction of rat brain and its effect on (Na+, K+)-ATPase were studied, the following results being obtained: (a) the binding process followed a biphasic kinetics with a break at 50 nM-GM1; GM1 at concentrations below the break was stably associated, while over the break it was loosely associated; (b) stably bound GM1 activated (Na+, K+)-ATPase up to a maximum of 43%; (c) the activation was dependent upon the amount of bound GM1 and was highest at the critical concentration of 20 pmol bound GM1 X mg protein-1; (d) loosely bound GM1 suppressed the activating effect on (Na+, K+)-ATPase elicited by firmly bound GM1; (e) GM1-activated (Na+, K+)-ATPase had the same pH optimum and apparent Km (for ATP) as normal (Na+, K+)-ATPase but a greater apparent Vmax; (f) under identical binding conditions (2 h, 37 degrees C, with 40 nM substance) all tested gangliosides (GM1, GD1a, GD1b, GT1b) activated (Na+, K+)-ATPase (from 26-43%); NeuNAc, sodium dodecylsulphate, sulphatide and cerebroside had only a very slight effect. It is suggested that the ganglioside activation of (Na+-K+)-ATPase is a specific phenomenon not related to the amphiphilic and ionic properties of gangliosides, but due to modifications of the membrane lipid environment surrounding the enzyme.