Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A.

(Na+ + K+)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na+ + K+)-ATPase activity, while their ouabain-insensitive Mg2+-ATPase is markedly lowered. A solubilized (Na+ +K+)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21 mumol Pi/h per mg protein). Concanavalin A has no effect on these partially purified (Na+ + K+)-ATPase, while inhibits (40%) this activity in less purified fractions which still contain Mg2+-ATPase activity.     

Variations in (Na+ + K+)-ATPase and Mg2+-ATPase activity of the ground squirrel brain during hibernation.

1. The specific activity of brain (Na+ + K+)-ATPase and Mg2+ -ATPase of the ground squirrel (Spermophilus richardsonii) is significantly increased after long-term hibernation. 2. The markedly non-linear thermal dependence of (Na+ + K+)-ATPase is unchanged during hibernation whereas the near linear thermal dependence of Mg2+-ATPase undergoes minor alteration after prolonged hibernation. 3. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is significantly decreased after 100 days of hibernation as is both the rate and amount of [3H]-ouabain binding. 4. These changes may be related to alteration in the phospholipid matrix of the membrane rather than alteration in the protein structure of the enzyme.     

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

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

Subcellular fractionation of pig stomach smooth muscle. A study of the distribution of the (Ca2+ + Mg2+)-ATPase activity in plasmalemma and endoplasmic reticulum

Isolated membrane vesicles from pig stomach smooth muscle (antral part) were subfractionated by a density gradient procedure modified in order to obtain an efficient extraction of extrinsic proteins. By using this method in combination with digitonin-treatment, an endoplasmic reticulum fraction contaminated with maximally 10 to 20% of plasma membranes was isolated, together with a plasma membrane fraction containing at most 30% endoplasmic reticulum. The endoplasmic reticulum and plasma membrane fractions differed in protein composition, reaction to digitonin, binding of wheat germ agglutinin, activities of marker enzymes and in the characteristics of the Ca2+ uptake. The Ca2+ uptake by the endoplasmic reticulum was much more stimulated by oxalate than the uptake by plasma membranes. Both fractions showed a (Ca2+ + Mg2+)-ATPase activity, but the largest amount of this enzyme was present in the plasma membranes. The study of the phosphorylated intermediates of the (Ca2+ + Mg2+)-ATPase by polyacrylamide gel electrophoresis revealed two phosphoproteins one of 130 kDa and one of 100 kDa (Wuytack, F., Raeymaekers, L., De Schutter, G. and Casteels, R. (1982) Biochim. Biophys. Acta 693, 45-52). The 130 kDa enzyme was predominant in the fraction enriched in plasma membrane whereas the distribution of the 100 kDa polypeptide correlated with the endoplasmic reticulum markers. The 130 kDa ATPase was the main 125I-calmodulin binding protein detected on nitrocellulose blots of proteins separated by gel electrophoresis. The (Ca2+ + Mg2+)-ATPase activity of the plasma membranes was higher than the (Na+ + K+)-ATPase activity, suggesting that the Ca2+ extrusion from these cells depends much more on the activity of the (Ca2+ + Mg2+)-ATPase than on Na+-Ca2+ exchange.     

Correlation between microsomal (Na+ + K+)-ATPase activity and [3H]ouabain binding to heart tissue homogenates.

ATP plus Mg2+ plus Na+ supported [3H]ouabain binding to canine left ventricular tissue homogenates and microsomal (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity from the same tissue were measured. A linear relationship was found between the initial velocity of [3H]ouabain binding to tissue homogenates and microsomal (Na+ + K+)-ATPase activity from the same tissue in the presence and absence of in vivo bound digoxin. In vivo bound digoxin reduced both measurements. With tissue from digoxin-free hearts, a linear relationship was also obtained between the initial velocity and the maximum level of [3H]ouabain binding to tissue homogenate. Binding of [3H]ouabain to whole tissue homogenate is a convenient method for estimating (Na+ + K+)-ATPase activity in small left ventricular biopsy samples.     

Radiation inactivation of (Na+ + K+)-ATPase. A small target size for the K+-occluding mechanism

Radiation inactivation of partially purified (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from pig kidney outer medulla shows that the target size for Rb+ occlusion by the enzyme (in the absence of phosphorylation) is much smaller than the target size for p-nitrophenyl phosphatase activity, which is itself smaller than the reported target size for (Na+ + K+)-ATPase activity.     

Calcium inhibition of the ATPase and phosphatase activities of (Na+ + K+)-ATPase

In experiments performed at 37 degrees C, Ca2+ reversibly inhibits the Na+-and (Na+ + K+)-ATPase activities and the K+-dependent phosphatase activity of (Na+ + K+)-ATPase. With 3 mM ATP, the Na+-ATPase was less sensitive to CaCl2 than the (Na+ + K+)-ATPase activity. With 0.02 mM ATP, the Na+-ATPase and the (Na+ + K+)-ATPase activities were similarly inhibited by CaCl2. The K0.5 for Ca2+ as (Na+ + K+)-ATPase inhibitor depended on the total MgCl2 and ATP concentrations. This Ca2+ inhibition could be a consequence of Ca2+-Mg2+ competition, Ca . ATP-Mg . ATP competition or a combination of both mechanisms. In the presence of Na+ and Mg2+, Ca2+ inhibited the K+-dependent dephosphorylation of the phosphoenzyme formed from ATP, had no effect on the dephosphorylation in the absence of K+ and inhibited the rephosphorylation of the enzyme. In addition, the steady-state levels of phosphoenzyme were reduced in the presence both of NaCl and of NaCl plus KCl. With 3 mM ATP, Ca2+ alone sustained no more than 2% of the (Na+ + K+)-ATPase activity and about 23% of the Na+-ATPase activity observed with Mg2+ and no Ca2+. With 0.003 mM ATP, Ca2+ was able to maintain about 40% of the (Na+ + K+)-ATPase activity and 27% of the Na+-ATPase activity seen in the presence of Mg2+ alone. However, the E2(K)-E1K conformational change did not seem to be affected. Ca2+ inhibition of the K+-dependent rho-nitrophenylphosphatase activity of the (Na+ + K+)-ATPase followed competition kinetics between Ca2+ and Mg2+. In the presence of 10 mM NaCl and 0.75 mM KCl, the fractional inhibition of the K+-dependent rho-nitrophenylphosphatase activity as a function of Ca2+ concentration was the same with and without ATP, suggesting that Ca2+ indeed plays the important role in this process. In the absence of Mg2+, Ca2+ was unable to sustain any detectable ouabain-sensitive phosphatase activity, either with rho-nitrophenylphosphate or with acetyl phosphate as substrate.     

Target sizes of human erythrocyte membrane Ca2+-ATPase and Mg2+-ATPase activities in the presence and absence of calmodulin

We have investigated the subunit structure of Ca2+-transport ATPase in human erythrocyte membranes using radiation inactivation analysis. All inactivation data were linear on a semilog plot down to at least 20% of the control activity. We found a target size for the calmodulin-dependent Ca2+-ATPase activity of 331 kDa, consistent with the presence of this enzyme as a dimer in calmodulin-depleted ghosts. Membranes which had been saturated with calmodulin before irradiation yield a a similar size of 317 kDa, implying that activation of Ca2+-transport ATPase by calmodulin does not involve significant change in oligomeric structure. Basal (calmodulin-independent) Ca2+-ATPase activity corresponded to a size of 290 kDa, suggesting that this activity resides in the same, or similar-sized, complex as the calmodulin-dependent activity. Mg2+-ATPase activity, however, was found to reside in a smaller complex of 224 kDa, which proved to be statistically distinct from the target size of Ca2+-ATPase activity. It would appear that Mg2+-ATPase is a distinct entity whose function is likely unrelated to the Ca2+-transport ATPase.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Effect of concanavalin A on membrane-bound enzymes from mouse lymphocytes.

The ionic influence and ouabain sensitivity of lymphocyte mg-2+-atpase and Mg-2+-(Na+ +K+)-activated ATPase were studied in intact cells, microsomal fraction and isolated plasma membranes. The active site of 5'-nucleotidase and Mg2+-ATPase seemed to be localized on the external side of the plasma membrane whereas the ATP binding site of (Na+ +K+)-ATPase was located inside the membrane. Concanavalin A induced an early stimulation of Mg2+-APTase and (Na+ +K+)-ATPase both on intact cells and purified plasma membranes. In contrast, 5'-nucleotidase activity was not affected by the mitogen. Although the thymocyte Mg2+-ATPase activity was 3-5 times lower than in spleen lymphocytes, it was much more stimulated in the former cells (about 40 versus 20%). (Na+ +K+)-ATPase activity was undectectable in thymocytes. However, in spleen lymphocytes (Na+ +K+)-ATPase activity can be detected and was 30% increased by concanavalin A. Several aspects of this enzymic stimulation had also characteristic features of blast transformation induced by concanavalin A, suggesting a possible role of these enzymes, especially Mg2+-ATPase, in lymphocyte stimulation.     

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.     

Thallium binding to native and radiation-inactivated Na+/K+-ATPase

The number of high-affinity K+-binding sites on purified Na+/K+-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl+, under conditions (low ionic strength, absence of Na+ and Tris+) where the enzyme is in the E2-form. Na+/K+-ATPase has two identical Tl+ sites per ADP site, and the dissociation constant varies between 2 and 9 microM. These values are identical to those for Tl+ occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl+ to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl+-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl+ binding is 71 kDa, and the RIS for Tl+ binding with original affinity is approx. 190 kDa, equal to that of Na+/K+-ATPase activity and to that for Tl+ occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na+/K+-ATPase are closely associated. The 71 kDa value obtained for total Tl+ sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl+-binding experiments reported thus supports the notion that radiation inactivation of Na+/K+-ATPase is a stepwise rather than an all or none process.     

An essential arginine residue in the ATP-binding centre of (Na+ + K+)-ATPase.

1. Incubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rabbit kidney outer medulla with butanedione in borate buffer leads to reversible inactivation of the (Na+ + K+)-ATPase activity. 2. The reaction shows second-outer kinetics, suggesting that modification of a single amino acid residue is involved in the inactivation of the enzyme. 3. The pH dependence of the reaction and the effect of borate ions strongly suggest that modification of an arginine residue is involved. 4. Replacement of Na+ by K+ in the butanedione medium decreases inactivation. 5. ATP, ADP and adenylyl imido diphosphate, particularly in the presence of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid to complex Mg2+, protect the enzyme very efficiently against inactivation by butanedione. 6. The (Na+ + Mg2+)-dependent phosphorylation capacity of the enzyme is inhibited in the same degree as the (Na+ + K+)-ATPase activity by butanedione. 7. The K+-stimulated p-nitrophenylphosphatase activity is much less inhibited than the (Na+ + K+)ATPase activity. 8. The ATP stimulation of the K+-stimulated p-nitrophenylphosphatase activity is inhibited by butanedione to the same extent as the (Na+ + K+)-ATPase activity. 9. Modification of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) protects partially against the inactivating effect of butanedione. 10. The results suggest that an arginine residue is present in the nucleotide binding centre of the enzyme.     

The presence of two (Na+ + K+)-ATPase inhibitors in equine muscle ATP: vanadate nad a dithioerythritol-dependent inhibitor.

A potent inhibitor of (Na+ + K+)-ATPase activity was purified from Sigma equine muscle ATP by cation- and anion-exchange chromatography. The isolated inhibitor was identified by atomic absorption spectroscopy and proton resonance spectroscopy to be an inorganic vanadate. The isolated vanadate and a solution of V2O5 inhibit sarcolemma (Na+ + K+)-ATPase with an I50 of 1 micrometer in the presence of 1 mM ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid (EGTA), 145 mM NaCl, 6mM MgCl2, 15 mM KCl and 2 mM synthetic ATP. The potency of the isolated vanadate is increased by free Mg2+. The inhibition is half maximally reversed by 250 micrometer epinephrine. Equine muscle ATP was also found to contain a second (Na+ + K+)-ATPase inhibitor which depends on the sulfhydryl-reducing agent dithioerythritol for inhibition. This unknown inhibitor does not depend on free Mg2+ and is half maximally reversed by 2 micrometer epinephrine. Prolonged storage or freeze-thawing of enzyme preparations decreases the susceptibility of the (Na+ + K+)-ATPase to this inhibitor. The adrenergic blocking agents, propranolol and phentolamine, do not block the catecholamine reactivation. The inhibitors in equine muscle ATP also inhibit highly purified (Na+ + K+)-ATPase from shark rectal gland and eel electroplax. The inhibitors in equine muscle ATP have no effect on the other sarcolemmal ATPases, Mg2+-ATPase, Ca2+-ATPase and (Ca2+ + Mg2+)-ATPase.     

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.     

Seasonal variations in the renal cortical (Na+ + K+)-ATPase and Mg2+-ATPase of a hibernator, the ground squirrel (Spermophilus richardsonii).

1. The specific activity of renal cortical (Na+ + K+)-ATPase of the Richardson ground squirrel is markedly reduced during hibernation, in contrast to the specific activity of the accompanying Mg2+-ATPase which is markedly increased. 2. The sensitivity of (Na+ + K+)-ATPase to inhibition by ouabain is unchanged by hibernation. 3. Both the non-linear thermal dependence of (Na+ + K+)-ATPase and the linear thermal dependence of Mg2+-ATPase are also unchanged by hibernation. 4. The energy of activation of both enzymes is unchanged during hibernation, or by comparison with that determined in awake controls. 5. There is no evidence for inherent "cold resistance" in these enzyme preparations compared to similar preparations from the non-hibernating rabbit. This parameter does not change during hibernation. 6. Both the rate and amount of specific [3H]-ouabain binding to the renal cortical preparations of (Na+ + K+)-ATPase decrease during hibernation. This decrease matches the fall in enzyme activity so that the ratio of pumping sites/unit of enzyme activity shows no seasonal variations. 7. These findings suggest that the amount of renal cortical (Na+ + K+)-ATPase enzyme falls during hibernation, but that the enzyme which remains functions with the same thermodynamic efficiency and identical biochemical characteristics of that found in the awake summer controls.     

Inhibition of gastric (H+ + K+)-ATPase by the substituted benzimidazole, picoprazole

The substituted benzimidazole, picoprazole, inhibited the gastric (H+ + K+)-ATPase in a concentration-and time-dependent manner. Half-maximal inhibition of the (H+ + K+)-ATPase activity was obtained at about 2 . 10(-6)M under standard conditions. In addition to the inhibition of ATPase activity, parallel inhibition of phosphoenzyme formation and the proton transport activity were achieved. Radiolabelled picoprazole was found to bind to 100 kDa peptide; this peptide was shown by phosphorylation experiments to contain the catalytic centre of the (H+ + K+)-ATPase. Studies on the (Na+ + K+)-ATPase indicated that this enzyme was unaffected by picoprazole. From the data presented and from other pharmacological studies, it is proposed that this compound inhibits acid secretion at the level of the parietal cell by its ability to inhibit the gastric proton pump, the (H+ + K+)-ATPase.     

Orientation of the beta subunit polypeptide of (Na+ + K+)ATPase in the cell membrane

Although the animal cell (Na+ + K+)-ATPase is composed of two polypeptide subunits, alpha and beta, very little is known about the beta subunit. In order to obtain information about the structure of this polypeptide, the beta subunit has been investigated using proteolytic fragmentation, chemical modification of carbohydrate residues, and immunoblot analysis. The sialic acid moieties on the oligosaccharide groups on the beta subunit of (Na+ + K+)-ATPase were labeled with NaB3H4 after oxidation by sodium periodate, or the penultimate galactose residues on the oligosaccharides were similarly labeled after removal of sialic acid with neuraminidase and oxidation by galactose oxidase. All of the carbohydrate residues of the protein are located on regions of the beta subunit that are found on the non-cytoplasmic surface of the membrane. Cleavage of the galactose oxidase-treated, NaB3H4-labeled beta subunit by chymotrypsin at an extracellular site produced labeled fragments of 40 and 18 kDa, indicating multiple glycosylation sites along the polypeptide. Neither the 40 kDa fragment nor the 18 kDa fragment was released from the membrane by chymotrypsin digestion alone, but after cleavage the 40 kDa fragment could be removed from the membrane by treatment with 0.1 M NaOH. This indicates that the 40 kDa fragment does not span the lipid bilayer. The 40 kDa fragment and the 18 kDa fragment are also linked by at least one disulfide bond. The 18 kDa fragment also contains all of the binding sites found on the (Na+ + K+)-ATPase for anti-beta subunit antibodies. Both the 40 kDa fragment and the 18 kDa fragment were also generated using papain or trypsin to cleave the beta subunit. These data indicate that the beta subunit of (Na+ + K+)-ATPase contains multiple sites of glycosylation, that it inserts into the cell membrane near only one end of the polypeptide, and that one region of the polypeptide is particularly sensitive to proteolytic cleavage relative to the rest of the polypeptide.