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.     

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+.     

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.     

Activation of the Dunaliella acidophila plasma membrane H(+)-ATPase by trypsin cleavage of a fragment that contains a phosphorylation site.

Trypsin treatment of purified H(+)-ATPase from plasma membranes of the extreme acidophilic alga Dunaliella acidophila enhances ATP hydrolysis and H+ pumping activities. The activation is associated with an alkaline pH shift, an increase in Vmax, and a decrease in Km(ATP). The activation is correlated with cleavage of the 100-kD ATPase polypeptide to a fragment of approximately 85 kD and the appearance of three minor hydrophobic fragments of 7 to 8 kD, which remain associated with the major 85-kD polypeptide. The N-terminal sequence of the small fragments has partial homology to residues 713 to 741 of Arabidopsis thaliana plasma membrane H(+)-ATPases. Incubation of cells with 32P-labeled orthophosphate (32Pi) results in incorporation of 32P into the ATPase 100-kD polypeptide. Trypsin treatment of the 32Pi-labeled ATPase leads to complete elimination of label from the approximately 85-kD polypeptide. Cleavage of the phosphorylated enzyme with endoproteinase Glu-C (V-8) yields a phosphorylated 12-kD fragment. Peptide mapping comparison between the 100-kD and the trypsinized 85-kD polypeptides shows that the 12-kD fragment is derived from the trypsin-cleaved part of the enzyme. The N-terminal sequence of the 12-kD fragment closely resembles a C-terminal stretch of an ATPase from another Dunaliella species. It is suggested that trypsin activation of the D. acidophila plasma membrane H(+)-ATPase results from elimination of an autoinhibitory domain at the C-terminal end of the enzyme that carries a vicinal phosphorylation site.     

A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).     

Phosphorylation of the beta subunit of Na+K+-ATPase in Ehrlich ascites tumor by a membrane-bound protein kinase

We have shown previously that proteoliposomes reconstituted with purified Na+K+-ATPase from Ehrlich ascites tumor cells, transport Na+ with low efficiency (Spector, M., O'Neal, S. and Racker, E. (1980) J. Biol. Chem., 255, 5504-5507). We now present evidence that this low efficiency (expressed in the ratio of Na+-transported/ATP-hydrolyzed) is caused by the phosphorylation of the beta subunit of the Na+K+-ATPase by an endogenous protein kinase. On addition of [gamma-32P]ATP, crude tumor plasma membrane preparations phosphorylated the beta subunit of the ATPase, whereas crude mouse brain plasma membranes did not. However, solubilized Na+K+-ATPase from either tumor or brain wre phosphorylated by purified protein kinase from the tumor plasma membrane and dephosphorylated by a phosphatase. In both cases, the phosphorylated enzyme was inefficient; the dephosphorylated enzyme was efficient after reconstitution into liposomes. During isolation of the Na+K+-ATPase from Ehrlich ascites tumor or mouse brain, an endogenous protease partially cleaved from the beta subunit a polypeptide of 29,000 daltons that contained the phosphorylation site. The proteolytic cleavage of the beta subunit was partially inhibited by phenylmethylsulfonyl fluoride and the major site of phosphorylation was then seen in the 53,000-dalton beta subunit of the enzyme. The isolated 29,000-dalton polypeptide from mouse brain ATPase was phosphorylated by tumor protein kinase with a stoichiometry of 1 mol of phosphate/mol of protein. When this 29,000-dalton polypeptide from mouse brain was incorporated into the tumor Na+K+-ATPase after mild proteolytic digestion, a marked increase in efficiency was observed after reconstitution of the Na+ pump.     

Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin

Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.     

Dimethyl sulfoxide-induced conformational state of Na(+)/K(+)-ATPase studied by proteolytic cleavage

Effects of dimethyl sulfoxide (Me(2)SO) on substrate affinity for phosphorylation by inorganic phosphate, on phosphorylation by ATP in the absence of Na(+), and on ouabain binding to the free form of the Na(+)/K(+)-ATPase have been attributed to changes in solvation of the active site or Me(2)SO-induced changes in the structure of the enzyme. Here we used selective trypsin cleavage as a procedure to determine the conformations that the Na(+)/K(+)-ATPase acquires in Me(2)SO medium. In water or in Me(2)SO medium, Na(+)/K(+)-ATPase exhibited after partial proteolysis two distinct groups of fragments: (1) in the presence of 0.1 M Na(+) or 0.1 M Na(+) + 3 mM ADP (enzyme in the E1 state) cleavage produced a main fragment of about 76 kDa; and (2) in the presence of 20 mM K(+) (E2 state) a 58-kDa fragment plus two or three fragments of 39-41 kDa were obtained. Cleavage in Me(2)SO medium in the absence of Na(+) and K(+) exhibited the same breakdown pattern as that obtained in the presence of K(+), but a 43-kDa fragment was also observed. An increase in the K(+) concentration to 0.5 mM eliminated the 43-kDa fragment, while a 39- to 41-kDa doublet was accumulated. Both in water and in Me(2)SO medium, a strong enhancement of the 43-kDa band was observed in the presence of either P(i) + ouabain or vanadate, suggesting that the 43-kDa fragment is closely related to the conformation of the phosphorylated enzyme. These results indicate that Me(2)SO acts not only by promoting the release of water from the ATP site, but also by inducing a conformation closely related to the phosphorylated state, even when the enzyme is not phosphorylated.     

Immunochemical studies on the large polypeptide of electrophorus electroplax (Na+ + K+)-ATPase.

Antibodies against Lubrol-solubilized Electrophorus electroplax (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and its 96 000-dalton polypeptide (P96) were raised in rabbits. The P96 antibody does not cross react with the (Na+ + K+)-ATPase from mammalian species and tissues, but it cross reacts with the (Na+ + K+)-ATPase from both Electrophorus electroplax and brain. The combination of enzyme with anti-P96 is found to inhibit phosphoryl enzyme formation to the same extent that it inhibits enzyme activity. The rate of K+-sensitive dephosphorylation of phosphoryl enzyme appears to be unchanged. These are also found to be true with the antibody against the whole enzyme. Upon tryptic digestion of the enzyme-anti-P96 complex only the large polypeptide of the enzyme is protected. In the case of enzyme-anti-Lubrol-solubilized enzyme complex, both the large and small polypeptides are protected, whereas preimmune sera are without any protecting effect. The data indicate that the phosphoryl acceptor polypeptide and the Lubrol-solubilized electroplax (Na+ + K+)-ATPase from which the polypeptide is derived are phylogenetically distinct from those of the mammalian (Na+ + K+)-ATPases. The selective tryptic resistance of the enzyme-anti-P96 complex indicates that the two polypeptides are spatially well separated, possibly on opposite sides of the membrane.     

Characterization of Bacillus subtilis glutamine synthetase by limited proteolysis.

The inactivation of native glutamine synthetase (GS) from Bacillus subtilis by trypsin, chymotrypsin, or subtilisin followed pseudo-fast order kinetics. Trypsin cleaved the polypeptide chain of GS into two principal fragments, one of about 43,000 (Mr) and the other of smaller than 10,000. Chymotrypsin and subtilisin caused similar cleavage of GS. A large fragment (Mr 35,000) and one smaller than 10,000 were detected on SDS-PAGE. The nicked protein remained dodecameric, as observed on gel filtration, electrophoresis, and electron micrography. In the presence of glutamate, ATP, and Mn2+, the digestion of GS by each of the three proteases was retarded completely; however, the presence of one substrate, L-glutamate, ATP+Mn2+, or ATP+Mg2+ led to partial protection. The product, L-glutamine, did not retard but altered the susceptibility of the protease sensitive sites. Amino acid sequence analysis of the two smaller polypeptide fragments showed that the nicked region was around serine 375 and serine 311, respectively, and that both large fragments (43,000 and 35,000) were N-terminal polypeptides of GS. The serine 311 region was involved in the formation of the enzyme-substrate complex. Tyrosine 372 near serine 375 corresponded to tyrosine 397 which was adenylylated by adenyltransferase in Escherichia coli GS.     

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.     

The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport

The (Na+ + K+)-ATPase of cultured chick sensory neurons was studied with the aid of antibodies specific for this enzyme. Immunofluorescent labeling indicated the (Na+ + K+)-ATPase is evenly distributed on the neuronal cell surface; cell bodies, neurites, and growth cones were labeled with comparable intensity. Pulse-chase experiments with [35S]methionine, followed by immunoprecipitation, indicated concurrent synthesis and rapid association of the alpha (Mr = 105,000) and beta (Mr = 47,000) subunits. The alpha subunit is oligosaccharide-free while the beta subunit contains three Asn-linked oligosaccharide chains attached to a core peptide of 32,000 molecular weight. The time required for oligosaccharide processing of the newly synthesized beta subunit to endoglycosidase H-resistance suggests the (Na+ + K+)-ATPase takes 45-60 min to move from the site of polypeptide synthesis to the Golgi apparatus. Significantly less time was required for transport through the Golgi apparatus and insertion in the plasma membrane. From 30% to 55% of the newly synthesized (Na+ + K+)-ATPase did not appear on the cell surface but accumulated intracellularly. When tunicamycin was used to inhibit glycosylation of the beta subunit, there was no effect upon subunit assembly, intracellular transport, or degradation rate (t1/2 = 40 h).     

Effect of hemolysate on calcium inhibition of the (Na+ + K+)-ATPase of human red blood cells

The sensitivity of the (Na+ + K+)-ATPase in human red cell membranes to inhibition by Ca2+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca2+ causing 50% inhibition of the (Na+ + K+)-ATPase is shifted from greater than 50 microM free Ca2+ in the absence of hemolysate to less than 10 microM free Ca2+ when hemolysate diluted 1:60 compared to in vivo concentrations is added to the assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Na+ + K+)-ATPase to Ca2+ in a dose-dependent fashion, causing over 80% inhibition of the (Na+ + K+)-ATPase at 10 microM free Ca2+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca2+-dependent, because it had no effect on the (Na+ + K+)-ATPase in the absence of Ca2+. Trypsin digestion prior to the assay destroyed the ability of this protein extract to increase the sensitivity of the (Na+ + K+)-ATPase to Ca2+.     

Mechanism of ethonium effect on membrane preparations of brain Na+, K+-ATPase

The effect of ethonium, a local anesthetic, on the membrane preparations of brain N+, K+-ATPase was studied by fluorescent, radioisotopic, electron-microscopic and electrophysiological methods. Ethonium is established to affect the formation of an intermediate phosphorylated product and has no pronounced destructive effect on the membrane. It changes the fluorescence intensity of 2-toluidinonaphthalen-6-sulphonate (TNS), astrafloxin (AF) and fluorene probes in a suspension of the Na+, K+-ATPase preparations, which evidences for ethonium-induced changes in the structure of membrane fragments.     

Conformational states of the (Na+ + K+)-transporting ATPase. Formation of 240 000-Mr and 116 000-Mr polypeptides in the presence of a bifunctional thiol probe.

Interpeptide cross-linking of alpha-subunits with concomitant loss of Na+ + K+-transporting ATPase (Na+, K+-ATPase) activity was found when the purified lamb kidney enzyme was treated with the bifunctional thiol reagent 4,4'-difluoro-3,3'-dinitrodiphenyl sulphone (F2DNS). Several forms of the enzyme could be clearly distinguished: one binding ATP (non-phosphorylated enzyme, E1 X ATP), a phosphorylated form (E2-P) and a phosphoenzyme-ouabain complex (E2P X ouabain). A polypeptide of approx. Mr 240 000 and probable alpha 2 composition comprised up to 5-20% of the total polypeptides after reaction of the lamb kidney Na+, K+-ATPase with F2DNS. The amount of this polypeptide formed was related to the conformational state of the enzyme. The presence of adenine nucleotide greatly diminished the amount of 240 000-Mr polypeptide formed and provides evidence for an enzyme-adenine-nucleotide complex under conditions where the enzyme is not phosphorylated. F2DNS reacted with the enzyme in the presence of Mg2+, Pi and ouabain to form a new polypeptide with an approx. Mr of 116 000, and comprised 23% of the total, whereas the 240 000-Mr polypeptide comprised 9% of the total. This suggests that the 116 000-Mr polypeptide is a characteristic marker of the E2P X ouabain complex. By using specific antibodies it was established that both the 240 000- and 116 000-Mr polypeptides contained alpha-, but not beta-, subunits of the Na+, K+-ATPase.     

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.     

Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla

When purified on a sucrose gradient, basolateral membranes from dog kidney outer medulla are found to be very rich in (Na,K)-ATPase; about 50% of the membrane protein is comprised of this enzyme. (Na,K)-ATPase activity is activated 3- to 5-fold by detergent treatment, and this has been previously attributed to the impermeable vesicular nature of the membranes. Porcine trypsin inactivates only that fraction of (Na,K)-ATPase activity seen without detergent, consistent with a right-side-out orientation of membrane vesicles; the trypsin sensitivity and detergent activation of [3H]ouabain binding in the presence of Na+ + Mg2+ + ATP or Mg2+ + Pi are also consistent with this hypothesis. Using nearly isosmotic Hypaque density gradient centrifugation a population of impermeable right-side-out membrane vesicles (H1) is separated from a leaky population (H2). (Na,K)-ATPase activity in the H1 population is 20-fold activated by detergent and insensitive to porcine trypsin. The vesicle volume is 2.4 microliters/mg, and monovalent cations passively equilibrate with the intravesicular volume on a time scale of 5-30 min. Very rapid ouabain sensitive 22Na efflux from the vesicles is observed when ATP is photolytically released from intravesicular caged ATP.     

Captopril inhibits ouabain-sensitive Na+/K+-ATPase

Captopril has been reported to inhibit ouabain-sensitive Na+/K+-ATPase activity in erythrocyte membrane fragments. We investigated the effect of captopril on two physiological measures of Na+/K+ pump activity: 22Na+ efflux from human erythrocytes and K+-induced relaxation of rat tail artery segments. Captopril inhibited 22Na+ efflux from erythrocytes in a concentration-dependent fashion, with 50% inhibition of total 22Na+ efflux at a concentration of 4.8 X 10(-3) M. The inhibition produced by captopril (5 X 10(-3) M) and ouabain (10(-4) M) was not greater than that produced by ouabain alone (65.3 vs. 66.9%, respectively), and captopril inhibited 50% of ouabain-sensitive 22Na+ efflux at a concentration of 2.0 X 10(-3) M. Inhibition by captopril of ouabain-sensitive 22Na efflux was not explained by changes in intracellular sodium concentration, inhibition of angiotensin-converting enzyme or a sulfhydryl effect. Utilizing rat tail arteries pre-contracted with norepinephrine (NE) or serotonin (5HT) in K+-free solutions, we demonstrated dose-related inhibition of K+-induced relaxation by captopril (10(-6) to 10(-4) M). Concentrations above 10(-4) M did not significantly inhibit K+-induced relaxation but did decrease contractile responses to NE, although not to 5HT. Inhibition of K+-induced relaxation by captopril was not affected by saralasin, teprotide or indomethacin. We conclude that captopril can inhibit membrane Na+/K+-ATPase in intact red blood cells and vascular smooth muscle cells. The mechanism of pump suppression is uncertain, but inhibition of ATPase should be considered when high concentrations of captopril are employed in physiological studies.     

Structural organization of alpha-subunit from purified and microsomal toad kidney (Na+ + K+)-ATPase as assessed by controlled trypsinolysis

The membrane organization of the alpha-subunit of purified (Na+ + K+)-ATPase ((Na+ + K+)-dependent adenosine triphosphate phosphorylase, EC 3.6.1.3) and of the microsomal enzyme of the kidney of the toad Bufo marinus was compared by using controlled trypsinolysis. With both enzyme preparations, digestions performed in the presence of Na+ yielded a 73 kDa fragment and in the presence of K+ a 56 kDa, a 40 kDa and small amounts of a 83 kDa fragment from the 96 kDa alpha-subunit. In contrast to mammalian preparations (J?rgensen, P.L. (1975) Biochim. Biophys. Acta 401, 399-415), trypsinolysis of the purified amphibian enzyme led to a biphasic loss of (Na+ + K+)-ATPase activity in the presence of both Na+ and K+. These data could be correlated with an early rapid cleavage of 3 kDa from the alpha-subunit in both ionic conditions and a slower degradation of the remaining 93 kDa polypeptide. On the other hand, in the microsomal enzyme, a 3 kDa shift of the alpha-subunit could only be produced in the presence of Na+. Our data indicate that (1) purification of the amphibian enzyme with detergent does not influence the overall topology of the alpha-subunit but produces a distinct structural alteration of its N-terminus and (2) the amphibian kidney enzyme responds to cations with similar conformational transitions as the mammalian kidney enzyme. In addition, anti alpha-serum used on digested enzyme samples revealed on immunoblots that the 40 kDa fragment was better recognized than the 56 kDa fragment. It is concluded that the NH2-terminal of the alpha-subunit contains more antigenic sites than the COOH-terminal domain in agreement with the results of Farley et al. (Farley, R.A., Ochoa, G.T. and Kudrow, A. (1986) Am. J. Physiol. 250, C896-C906).