Photoaffinity labeling of the ouabain binding site in Na, K-ATPase in developing brine shrimp

Analysis of purified Na,K-ATPase from brine shrimp nauplii by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two large (alpha) subunits [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The band with lower mobility in a neutral or alkaline gel is designated alpha 1 and the band with higher mobility alpha 2. Ouabain prevents dephosphorylation of both alpha 1 and alpha 2 as documented by gel analysis, but a higher concentration of ouabain is required to prevent dephosphorylation of alpha 2. The photoaffinity label, [3H]4'(2-ethyldiazomalonyl) digitoxigenin monodigitoxiside, specifically labels alpha in a ouabain-protectable manner without labeling other contaminating proteins in the preparation. Greater than 93% of the total ouabain-protectable labeling of the alpha subunits is associated with alpha 1. The photoaffinity label, [3H]4"' (2-ethyldiazomalonyl) digitoxin, specifically labels alpha 1 and beta in a ouabain-protectable manner without labeling other contaminating proteins. These data show that in the brine shrimp the third digitoxose residue of digitoxin binds in a region in which the alpha 1 and beta chains are in close proximity. Less than 5% of the specific ouabain-protectable labeling of total alpha is associated with alpha 2. These studies indicate that cardioactive steroids have higher affinity for the alpha 1 subunit.     

Structural and biosynthetic studies on the two molecular forms of the (Na+ + K+)-activated adenosine triphosphatase large subunit in Artemia salina Nauplii

The large subunit of (Na+ + K+)-activated ATPase from brine shrimp, Artemia salina, migrates as two bands in sodium dodecyl sulfate-polyacrylamide gels. The slower migrating band, as observed in neutral or alkaline gel systems, is designated alpha 1 and the faster, alpha 2. Structural and biosynthetic studies have been performed to determine if these two bands represent independent molecular forms or precursor products. Peptide mapping of partial proteolytic digests of alpha 1 and alpha 2 showed no distinguishable difference between them whereas this technique produced very distinct differences in the large subunit derived from three different species. The two large subunit bands also behaved identically when cross linked with cupric phenanthroline either in the presence or absence of digitonin, whereas other proteins in these preparations were unaffected. The peptide mapping and cross-linking experiments demonstrate that alpha 1 and alpha 2 have identical or nearly identical primary and probably higher order structure. Their different mobilities may be due to post-translational modification leading, for example, to different oligosaccharide composition. During development of the brine shrimp nauplius, alpha 1 increases in relative abundance while alpha 2 decreases. NaH14CO3 incorporation and pulse-chase experiments indicate that alpha 1 and alpha 2, as well as the small subunit of the brine shrimp (Na+ + K+)-activated ATPase, are synthesized at the same time during development and that all changes in the rates of synthesis of these subunits occur at the same time. The apparent rates of degradation of the subunits are also similar. These results are inconsistent with a precursor-product relationship between alpha 1 and alpha 2.     

Regulation of Na,K-ATPase biosynthesis in developing Artemia salina

Regulation of the biosynthesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase) (EC 3.6.1.3) was studied in the developing brine shrimp, Artemia salina. Measurement of levels of the subunits of the Na,K-ATPase by radioimmunoassay indicated the presence of both alpha and beta subunits in undeveloped cysts and developing embryos prior to the appearance of enzymatic activity. The quantity of each subunit increased dramatically between 8 and 24 h of development and then reached a plateau at about 32 h. The quantities of translationally active mRNA alpha and mRNA beta were also determined. Undeveloped cysts contained mRNA alpha and mRNA beta, and the amounts increased 9- and 3-fold, respectively, during the first 24 h of development. The data suggest that the increase in Na,K-ATPase activity was at least in part due to increases in protein synthesis related to changes in mRNA levels. The data also suggest involvement of additional regulatory mechanisms. The alpha-subunit has been detected as two molecular weight forms (alpha 1 and alpha 2) which demonstrate changes in relative amounts during development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We show here that this was not due to changes in mRNA alpha 1 and mRNA alpha 2.     

Assembly of the (Na+ + K+)-adenosine triphosphatase. Post-translational membrane integration of the alpha subunit

Guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates and wheat germ extracts. Antibodies to the holoenzyme (Na/K-ATPase) immunoprecipitated only a 96,000-dalton product which was identified as the alpha subunit with a molecular weight that was indistinguishable from that of mature alpha subunit. To explore the possibility that the primary translational product is integrated as such into membranes, guinea pig kidney poly(A+) RNA was translated in reticulocyte lysates in the presence of dog pancreas microsomes; two immunoprecipitated products were detected, the 96,000-dalton alpha subunit and a 135,000-dalton new component that was integrated into the microsomal membrane since it was completely resistant to extraction with alkali. Addition of purified alpha subunit inhibited the binding of antibody to the 135,000-dalton product and extraction with urea-sodium dodecyl sulfate recovered the 96,000-dalton product, implying that the 135,000-dalton product was an alpha-chi dimer. Translation of size-fractionated poly(A+) RNA yielded evidence that the 135,000-dalton product is encoded in two separate mRNAs. The integration in vitro of the alpha subunit is, therefore, dependent on the co-translational integration into the membranes of a smaller peptide (35,000 to 40,000 daltons) which is presumably the beta subunit. Evidence was also obtained that this mechanism is present in vivo by isolation of mRNA alpha from free polysomes, as well as detection of the cytosolic form of the alpha subunit in pulse-chase experiments in MDCK cells.     

Differences in phosphorylation of the two large subunits of brine shrimp Na,K-ATPase

Analysis of purified Na,K-ATPase from brine shrimp nauplii revealed two molecular forms of the alpha subunit separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The molecular form with lower mobility is designated alpha 1 and the one with higher mobility, alpha 2, in a neutral or alkaline gel system. Differences in Na+-dependent, K+-sensitive phosphorylation of these two molecular forms have been investigated by directly measuring the radioactivity present in each phosphoprotein after separation of the two forms by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of Na+,Mg2+, and ATP, when the ATP concentration is above 1 microM, both alpha subunits are phosphorylated, although the phosphoprotein content of alpha 1 is considerably greater than that of alpha 2. Below 1 microM ATP, the phosphoprotein content of alpha 2 is even further reduced. These striking differences in phosphorylation at low ATP concentrations are not due to a greater instability of the alpha 2 phosphoprotein during the long electrophoresis times or during fixation, staining, and destaining. The proportion of total phosphoprotein content in alpha 2, as well as the relationship between phosphoprotein content and ATP concentration, is unchanged when the radioactive analysis is performed on frozen gels that have been electrophoresed for shorter times, even though the actual amount of phosphorylation is 15 times greater than with fixed gels. Since the concentration of alpha 1 and alpha 2 vary during development [G.L. Peterson, L. Churchill, J.A. Fisher, and L.E. Hokin (1982) J. Exp. Zool. 221:295], the differences in phosphorylation may be relevant to differences in Na,K-ATPase activity during different development stages.     

Identification of two isoforms of the catalytic subunit of Na,K-ATPase in myocytes from adult rat heart

The present study demonstrates that two forms of the alpha catalytic subunit of the Na,K-ATPase are present in rat heart and originate from cardiomyocytes. They were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis after reduction and alkylation of the sulfhydryl groups. The two forms were identified on immunoblots using two specific antisera against either the alpha subunit from Bufo marinus kidney and the alpha and beta subunits from lamb kidney. Comparison of the two forms to the alkylated Na,K-ATPase from rat kidney (containing one catalytic subunit) and from rat brain (containing alpha and alpha + subunits) suggested that, in rat cardiac myocytes, the form with a fast migration rate (alpha F) corresponds to the alpha subunit of low ouabain affinity and the one with a slow migration rate (alpha S), to a subunit of high ouabain affinity. Thus, the existence of two isoforms of the catalytic subunit in cardiac myocytes accounts well for the biphasic ouabain inhibition of the Na,K-ATPase activity and for the biphasic inotropic responsiveness to cardiac glycosides of the rat heart.     

Structure of extracellular hemoglobin from the brine shrimp Artemia salina.

1. Hemoglobin from the brine shrimp Artemia salina, purified by ultracentrifugation and preparative gel electrophoresis in non-denaturing medium, gave in sodium dodecyl sulfate-polyacrylamide gel electrophoresis a single band corresponding to a polypeptide chain with Mr 150,000. 2. Crosslinking by glutardialdehyde resulted in the appearance of a band corresponding to a molecular mass twice that of a polypeptide chain. 3. Limited trypsinolysis gave eight proteolytic bands corresponding to submultiples 8/9-1/9 of a polypeptide chain. 4. We conclude that a molecule of Artemia hemoglobin is composed of two single polypeptide chain subunits and that each subunit consists of nine structural units roughly equal in size.     

A reversal in relative mobility of the two large subunits of brine shrimp (Na+ + K+)-adenosinetriphosphatase

The two large subunits of brine shrimp Na,K-ATPase can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at neutral pH and at acidic pH. These subunits appear to reverse their positions on the gel relative to each other when resolved at acidic pH relative to neutral pH. The migration of both subunits is apparently affected by charge, even in the presence of 2.5% sodium dodecyl sulfate.     

The alpha and beta subunits of the Na,K-ATPase can assemble at the plasma membrane into functional enzyme

Synthesis and assembly of most oligomeric plasma membrane proteins occurs in the ER. However, the role the ER plays in oligomerization is unknown. We have previously demonstrated that unassociated alpha and beta subunits of the Na,K-ATPase are targeted to the plasma membrane when individually expressed in baculovirus-infected Sf-9 cells. This unique property allows us to determine if assembly of these two polypeptides is restricted to the ER, or if it can also occur at the plasma membrane. To investigate the assembly of the Na,K-ATPase we have taken advantage of the ability of baculovirus-infected cells to fuse. Lowering the extracellular pH of the infected cells triggers an endogenously expressed viral protein to initiate plasma membrane fusion. When individual Sf-9 cells expressing either the Na,K-ATPase alpha or beta subunits are plated together and subjected to a mild acidic shock, they form large syncytia. In the newly continuous plasma membrane the separate alpha and beta polypeptides associate and assemble into functional Na,K-ATPase molecules. However, a hybrid ATPase molecule consisting of a Na,K-ATPase alpha subunit and a H,K- ATPase beta subunit, which efficiently assembles in the ER of coinfected cells, does not assemble at the plasma membrane of fused cells. When cells expressing the Na,K-ATPase alpha subunit are fused to cells coexpressing the Na,K-ATPase beta subunit and the H,K-ATPase beta subunit, the Na,K-ATPase alpha subunit selectively assembles with the Na,K-ATPase beta subunit. However, when cells are coinfected and expressing all three polypeptides, the Na,K-ATPase alpha subunit assembles with both beta subunits in the ER, in what appears to be a random fashion. These experiments demonstrate that assembly between some polypeptides is restricted to the ER, and suggests that the ability of the Na,K-ATPase alpha and beta subunits to leave the ER and assemble at the plasma membrane may represent a novel mechanism of regulation of activity.     

Solubilization and purification of Artemia salina (Na,K)-activated ATPase and NH2-terminal amino acid sequence of its larger subunit

Membrane bound (Na,K)-ATPase partially purified from the nauplius larva of the brine shrimp, Artemia salina, was solubilized with the non-ionic detergent C12E8 in the presence of KCl. The addition of KCl was essential for protecting the enzyme against inactivation. With solubilization the enzyme could then be purified to apparent homogeneity. Electron microscopic observation of the purified enzyme revealed a homogeneous population of particles with a diameter of approximately 4 nm. The larger (alpha) subunit of the enzyme formed double bands on sodium dodecyl sulfate-polyacrylamide gels. NH2-terminal sequence analysis of the alpha subunit revealed the possible presence of two isoforms of (Na,K)-ATPase. At the third position a small but distinct amount of lysine was found in addition to glycine, suggesting that the two forms are different from each other at least at the third residue. The NH2-terminal sequence determined is as follows. NH2-Ala-Lys-Gly (Lys)-Lys-Gln-Lys-Lys-Gly-Lys-Asp-Leu-Asn-Glu-Leu-Lys-Lys-Glu-Leu-Asp-Il e-Asp -Phe-His-Lys-Ile-Pro- The sequence is abundant in hydrophilic amino acids, especially lysine, and is quite different from those of vertebrate enzymes reported so far.     

The adhesion molecule on glia (AMOG) is a homologue of the beta subunit of the Na,K-ATPase

AMOG (adhesion molecule on glia) is a Ca2(+)-independent adhesion molecule which mediates selective neuron-astrocyte interaction in vitro (Antonicek, H., E. Persohn, and M. Schachner. 1987. J. Cell Biol. 104:1587-1595). Here we report the structure of AMOG and its association with the Na,K-ATPase. The complete cDNA sequence of mouse AMOG revealed 40% amino acid identity with the previously cloned beta subunit of rat brain Na,K-ATPase. Immunoaffinity-purified AMOG and the beta subunit of detergent-purified brain Na,K-ATPase had identical apparent molecular weights, and were immunologically cross-reactive. Immunoaffinity-purified AMOG was associated with a protein of 100,000 Mr. Monoclonal antibodies revealed that this associated protein comprised the alpha 2 (and possibly alpha 3) isoforms of the Na,K-ATPase catalytic subunit, but not alpha 1. The monoclonal AMOG antibody that blocks adhesion was shown to interact with Na,K-ATPase in intact cultured astrocytes by its ability to increase ouabain-inhibitable 86Rb+ uptake. AMOG-mediated adhesion occurred, however, both at 4 degrees C and in the presence of ouabain, an inhibitor of the Na,K-ATPase. Both AMOG and the beta subunit are predicted to be extracellularly exposed glycoproteins with single transmembrane segments, quite different in structure from the Na,K-ATPase alpha subunit or any other ion pump. We hypothesize that AMOG or variants of the beta subunit of the Na,K-ATPase, tightly associated with an alpha subunit, are recognition elements for adhesion that subsequently link cell adhesion with ion transport.     

Na,K-ATPase in dog red cells. Immunological identification and maturation-associated degradation by the proteolytic system

The Na,K-ATPase of red cells from high K+ and low K+ dogs was studied immunologically by using antibodies raised against dog kidney enzyme. Anti-alpha subunit IgGs, which also recognized alpha (+) from brain enzyme, identified the larger subunit of erythrocyte Na,K-ATPase as a homogeneous polypeptide with Mr = 96,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by immunoblotting. In addition, erythrocyte Na,K-ATPase, purified by immunoaffinity chromatography on a monoclonal antibody-coupled column, showed the identity of its polypeptide composition to that of the renal enzyme. Furthermore, it was shown that reticulocyte lysates from high K+ and low K+ dogs substantially degraded 125I-Bolton-Hunter reagent-labeled Na,K-ATPase. This degradation of the enzyme protein was significantly enhanced by the addition of ATP and Mg2+. These results indicate that dog reticulocytes possess some mechanism for protein breakdown involving an ATP-dependent proteolytic system, resulting in the dramatic breakdown of Na,K-ATPase activity during dog reticulocyte maturation into erythrocytes (Maede, Y., and Inaba, M. (1985) J. Biol. Chem. 260, 3337-3343).     

Modulation of the Na,K-pump function by beta subunit isoforms

To study the role of the Na,K-ATPase beta subunit in the ion transport activity, we have coexpressed the Bufo alpha 1 subunit (alpha 1) with three different isotypes of beta subunits, the Bufo Na,K-ATPase beta 1 (beta 1NaK) or beta 3 (beta 3NaK) subunit or the beta subunit of the rabbit gastric H,K-ATPase (beta HK), by cRNA injection in Xenopus oocyte. We studied the K+ activation kinetics by measuring the Na,K- pump current induced by external K+ under voltage clamp conditions. The endogenous oocyte Na,K-ATPase was selectively inhibited, taking advantage of the large difference in ouabain sensitivity between Xenopus and Bufo Na,K pumps. The K+ half-activation constant (K1/2) was higher in the alpha 1 beta 3NaK than in the alpha 1 beta 1NaK groups in the presence of external Na+, but there was no significant difference in the absence of external Na+. Association of alpha 1 and beta HK subunits produced active Na,K pumps with a much lower apparent affinity for K+ both in the presence and in the absence of external Na+. The voltage dependence of the K1/2 for external K+ was similar with the three beta subunits. Our results indicate that the beta subunit has a significant influence on the ion transport activity of the Na,K pump. The small structural differences between the beta 1NaK and beta 3NaK subunits results in a difference of the apparent affinity for K+ that is measurable only in the presence of external Na+, and thus appears not to be directly related to the K+ binding site. In contrast, association of an alpha 1 subunit with a beta HK subunit results in a Na,K pump in which the K+ binding or translocating mechanisms are altered since the apparent affinity for external K+ is affected even in the absence of external Na+.     

Interaction with the Na,K-ATPase and tissue distribution of FXYD5 (related to ion channel)

FXYD5 (related to ion channel, dysadherin) is a member of the FXYD family of single span type I membrane proteins. Five members of this group have been shown to interact with the Na,K-ATPase and to modulate its properties. However, FXYD5 is structurally different from other family members and has been suggested to play a role in regulating E-cadherin and promoting metastasis (Ino, Y., Gotoh, M., Sakamoto, M., Tsukagoshi, K., and Hirohashi, S. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 365-370). The goal of this study was to determine whether FXYD5 can modulate the Na,K-ATPase activity, establish its cellular and tissue distribution, and characterize its biochemical properties. Anti-FXYD5 antibodies detected a 24-kDa polypeptide that was preferentially expressed in kidney, intestine, spleen, and lung. In kidney, FXYD5 resides in the basolateral membrane of the connecting tubule, the collecting tubule, and the intercalated cells of the collecting duct. However, there is also labeling of the apical membrane in long thin limb of Henle's loop. FXYD5 was effectively immunoprecipitated by antibodies to the alpha subunit of Na,K-ATPase and the anti-FXYD5 antibody immunoprecipitates alpha. Co-expressing FXYD5 with the alpha1 and beta1 subunits of the Na,K-ATPase in Xenopus oocytes elicited a more than 2-fold increase in pump activity, measured either as ouabain-blockable outward current or as ouabain-sensitive (86)Rb(+) uptake. Thus, as found with other FXYD proteins, FXYD5 interacts with the Na,K-ATPase and modulates its properties.     

Antisera specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the Na,K-ATPase: differential expression of alpha and beta subunits in rat tissue membranes

We have developed a panel of antibodies specific for the alpha 1, alpha 2, alpha 3, and beta subunits of the rat Na,K-ATPase. TrpE-alpha subunit isoform fusion proteins were used to generate three antisera, each of which reacted specifically with a distinct alpha subunit isotype. Western blot analysis of rat tissue microsomes revealed that alpha 1 subunits were expressed in all tissues while alpha 2 subunits were expressed in brain, heart, and lung. The alpha 3 subunit, a protein whose existence had been inferred from cDNA cloning, was expressed primarily in brain and copurified with ouabain-inhibitable Na,K-ATPase activity. An antiserum specific for the rat Na,K-ATPase beta subunit was generated from a TrpE-beta subunit fusion protein. Western blot analysis showed that beta subunits were present in kidney, brain, and heart. However, no beta subunits were detected in liver, lung, spleen, thymus, or lactating mammary gland. The distinct tissue distributions of alpha and beta subunits suggest that different members of the Na,K-ATPase family may have specialized functions.     

Synthesis and assembly of functional mammalian Na,K-ATPase in yeast.

The yeast Saccharomyces cerevisiae was investigated as an in vivo protein expression system for mammalian Na,K-ATPase. Unlike animal cells, yeast cells lack endogenous Na,K-ATPase. Expression of high affinity ouabain binding sites, ouabain-sensitive ATPase activity, or ouabain-sensitive p-nitrophenylphosphatase activity in membrane fractions of yeast cells was observed to require the expression of both alpha subunit and beta subunit polypeptides of Na,K-ATPase in the same cell. High affinity ouabain binding sites are also expressed at the cell surface of intact yeast cells containing both the alpha subunit and the beta subunit of Na,K-ATPase. These observations demonstrate that both the alpha subunit and the beta subunit of Na,K-ATPase are required for the expression of functional Na,K-ATPase activity and that yeast cells can correctly assemble this oligomeric membrane protein and transport it to the cell surface.     

Synthesis of a possible precursor of alpha-amylase in wheat aleurone cells

α-Amylase from wheat aleurone (Triticum aestivum) was synthesized in a S-150 wheat germ readout system using polysomes, and a messenger RNA-dependent reticulocyte lysate system using polyadenylic acid [poly(A)]-enriched RNA. The product was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, precipitation by specific λ-globulin for α-amylase, and proteolysis. Two immunoprecipitated products were synthesized from the readout system, the predominant species migrating coincidentally with authentic α-amylase on sodium dodecyl sulfate-polyacrylamide gels. A putative precursor, 1,500 daltons larger, was evident but was less abundant. The relationship between the two polypeptides was established by proteolytic analysis using Staphylococcus aureus V8 protease. At least nine fragments were generated and were identical in both species. The poly(A)-enriched RNA synthesized only the putative precursor in the reticulocyte lysate system. Attempts to process the precursor to the mature size of α-amylase failed. These findings are discussed in connection with the signal hypothesis (proposed for the transport of proteins across membranes) and the mode of secretion of α-amylase in aleurone cells.