Beta-subunits Of Na Research Articles

Abstract 151: A Polymorphic T-Rich Element in ATP1B1 Is Associated with Blood Pressure and Regulates Alternative Polyadenylation

Genome-wide linkage and association studies of hypertension (HTN) aim to uncover loci that are involved in blood pressure (BP) regulation and the pathophysiology of HTN. However, even when such loci are successfully identified, the mechanism by which the implicated genes increase HTN susceptibility is not well understood. ATP1B1, which encodes the beta subunit of Na+, K+ ATPase, a co-transporter involved in multiple BP-regulating physiological processes, is located within a well-established BP linkage peak. Although a single nucleotide polymorphism (rs12079745) in the 3’UTR of ATP1B1 has previously been shown to be associated with BP levels, the molecular mechanism underlying this association is unknown. We identified a multi-allelic T-rich element (TRE) in the 3’UTR of ATP1B1 that varies in length and sequence composition (T22-27 and T12GT3GT6). The 3’UTR of ATP1B1 contains 2 functional polyadenylation signals and the TRE is downstream of the proximal site (A2). Because U-rich elements (UREs) in 3’UTRs are known to play a role in the cleavage and polyadenylation of mRNA, and ATP1B1 transcripts with shorter 3’UTRs are translationally more efficient, we hypothesized that alleles of this TRE might influence ATP1B1 expression by regulating alternative polyadenylation. Consistent with our hypothesis, the A2-polyadenylated mRNA isoform was more abundant in human kidneys with at least one copy of T12GT3GT6 than in those homozygous for the T22-27 alleles. Functionally, in vitro, we demonstrated that the T12GT3GT6 allele has greater luciferase activity than the T22 allele and that the T12GT3GT6 allele shows increased polyadenylation at the A2 site than the T22 allele. In addition, the TRE element is associated with systolic BP in European Americans of the GenNet network of the NHLBI Family Blood Pressure Program, with the T12GT3GT6 allele being associated with higher BP (effect size = 2.4 mmHg SBP, P = 0.003). Sharing strong linkage disequilibrium with rs12079745, this TRE is likely the functional site underlying the original association. In summary, we have identified a novel multi-allelic TRE in the 3’UTR of ATP1B1. Alleles at this site are associated with HTN and may mediate their effect on BP by regulating polyadenylation of the ATP1B1 mRNA.

Analysis of Alpha and Beta Subunits of Na+/K+-ATPase in the Uterine Endometrium During the Estrous Cycle and Pregnancy in Pigs.

Analysis of Alpha and Beta Subunits of Na+/K+-ATPase in the Uterine Endometrium During the Estrous Cycle and Pregnancy in Pigs.

T cell activation causes diarrhea by increasing intestinal permeability and inhibiting epithelial Na+/K+-ATPase.

Inflammatory bowel disease (IBD) is associated with mucosal T cell activation and diarrhea. We found that T cell activation with anti-CD3 mAb induces profound diarrhea in mice. Diarrhea was quantified by intestinal weight-to-length (wt/l) ratios, mucosal Na(+)/K(+)-ATPase activity was determined and ion transport changes were measured in Ussing chambers. Anti-CD3 mAb increased jejunal wt/l ratios by more than 50% at 3 hours, returning to base line after 6 hours. Fluid accumulation was significantly reduced in TNF receptor-1 (TNFR-1(-/-)), but not IFN-gamma knockout mice. Anti-CD3 mAb decreased mucosal Na(+)/K(+)-ATPase activity, which was blocked by anti-TNF mAb and occurred to a lesser degree in TNFR-1(-/-) mice. Neither alpha nor beta subunits of Na(+)/K(+)-ATPase decreased in abundance at 3 hours. Intestinal tissue from anti-CD3-treated mice exhibited increased permeability to mannitol at 1 hour and decreases in electroneutral Na(+) absorption, Na(+)-dependent glucose absorption, and cAMP-stimulated anion secretion at 3 hours. Furthermore, enteral fluid accumulation was observed in CFTR(-/-) mice, indicating a minor role of active anion secretion. These data suggest that diarrhea in IBD is due to TNF-mediated malabsorption rather than to secretory processes. T cell activation induces luminal fluid accumulation by increasing mucosal permeability and reducing epithelial Na(+)/K(+)-ATPase activity leading to decreased intestinal Na(+) and water absorption.

Pretreatment with cationic lipid-mediated transfer of the Na+K+-ATPase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema.

Resolution of pulmonary oedema is mediated by active absorption of liquid across the alveolar epithelium. A key component of this process is the sodium-potassium ATPase (Na+K+-ATPase) enzyme located on the basolateral surface of epithelial cells and up-regulated during oedema resolution. We hypothesised that lung liquid clearance could be further up-regulated by lipid-mediated transfer and expression of exogenous Na+K+-ATPase cDNA. We demonstrate proof of this principle in a model of high permeability pulmonary oedema induced by intraperitoneal injection of thiourea (2.5 mg/kg) in C57/BL6 mice. Pretreatment of mice (24 h before thiourea) by nasal sniffing of cationic liposome (lipid #67)-DNA complexes encoding the alpha and beta subunits of Na+K+-ATPase (160 microg per mouse), significantly (P<0.01) decreased the wet:dry weight ratios measured 2 h after thiourea injection compared with control animals, pretreated with an equivalent dose of an irrelevant gene. Whole lung Na+K+-ATPase activity was significantly (P<0.05) increased in mice pretreated with Na+K+-ATPase cDNA compared both with untreated control animals as well as animals pretreated with the irrelevant gene. Nested RT-PCR on whole lung homogenates confirmed gene transfer by detection of vector-specific mRNA in three of four mice studied 24 h after gene transfer. This demonstration of a significant reduction in pulmonary oedema following in vivo gene transfer raises the possibility of gene therapy as a novel, localised approach for pulmonary oedema in clinical settings such as ARDS and lung transplantation.

The β-Subunits of Na+,K+-ATPase and Gastric H+,K+-ATPase Have a High Preference for Their Own α-Subunit and Affect the K+ Affinity of These Enzymes

The alpha- and beta-subunits of Na+,K+-ATPase and H+,K+-ATPase were expressed in Sf9 cells in different combinations. Immunoprecipitation of the alpha-subunits resulted in coprecipitation of the accompanying beta-subunit independent of the type of beta-subunit. This indicates cross-assembly of the subunits of the different ATPases. The hybrid ATPase with the catalytic subunit of Na+,K+-ATPase and the beta-subunit of H+,K+-ATPase (NaKalphaHKbeta) showed an ATPase activity, which was only 12 +/- 4% of the activity of the Na+,K+-ATPase with its own beta-subunit. Likewise, the complementary hybrid ATPase with the catalytic subunit of H+,K+-ATPase and the beta-subunit of Na+,K+-ATPase (HKalphaNaKbeta) showed an ATPase activity which was 9 +/- 2% of that of the recombinant H+,K+-ATPase. In addition, the apparent K+ affinity of hybrid NaKalphaHKbeta was decreased, while the apparent K+ affinity of the opposite hybrid HKalphaNaKbeta was increased. The hybrid NaKalphaHKbeta could be phosphorylated by ATP to a level of 21 +/- 7% of that of Na+,K+-ATPase. These values, together with the ATPase activity gave turnover numbers for NaKalphabeta and NaKalphaHKbeta of 8800 +/- 310 min-1 and 4800 +/- 160 min-1, respectively. Measurements of phosphorylation of the HKalphaNaKbeta and HKalphabeta enzymes are consistent with a higher turnover of the former. These findings suggest a role of the beta-subunit in the catalytic turnover. In conclusion, although both Na+,K+-ATPase and H+,K+-ATPase have a high preference for their own beta-subunit, they can function with the beta-subunit of the other enzyme, in which case the K+ affinity and turnover number are modified.

Functional analysis and tissue-specific expression of Drosophila Na+,K+-ATPase subunits.

We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the beta subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase alpha subunit cDNA clone (PSalpha; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSalpha mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.

Transmembrane Topology of α- and β-Subunits of Na+,K+-ATPase Derived from β-Galactosidase Fusion Proteins Expressed in Yeast

Various models of the transmembrane topology of the Na+,K+-ATPase predict either 8 or 10 membrane spans for the alpha-subunit and one to three membrane spans for the beta-subunit. Structure/function analysis, however, requires precise knowledge about the folding of enzymes. Therefore, the intention of this work was to establish a transmembrane topology model for the subunits of Na+,K+-ATPase. The bacterial enzyme beta-galactosidase was fused to the C termini of truncated alpha- and beta-subunits of Na+,K+-ATPase. Fusions were generated at Arg60 (LTTAR60), Glu116 (AATEE116), Ala247 (VEGTA247), Leu311 (YTWEL311), Ala444 (VAGDA444), Ala789 (IFIIA789), Met809 (LGTDM809), Asp884 (RVTWD884), Ile946 (MKNKI946), and Arg972 (GVALR972) of the sheep alpha1-subunit and at Pro236 (LGGYP236) of the dog beta-subunit. The fusion constructs were expressed in yeast cells for studies on the localization of the fused reporter enzyme. Activity measurements of the reporter enzyme revealed that only intracellular fusion sites lead to active beta-galactosidase. Indirect immunofluorescence microscopy with cells expressing alpha1/beta-galactosidase and beta/beta-galactosidase hybrid proteins demonstrated that inactive beta-galactosidase is associated with the yeast plasma membrane and can be detected from the extracellular side. The data obtained suggest that Pro236 of the beta-subunit is located on the extracellular surface, corresponding to a model with one transmembrane segment, and that the alpha-subunit of the Na+,K+-ATPase consists of 10 membrane-associated spans. They also suggest that a stretch of the alpha1-subunit between membrane spans M7 and M8 might be hidden within the membrane, surrounded by the other hydrophobic spans, in analogy to the P-loop of Na+ or K+ channels and to the "hourglass" structure of water channels.

Intersubunit and Intrasubunit Contact Regions of Na+/K+-ATPase Revealed by Controlled Proteolysis and Chemical Cross-linking

To identify interfaces of alpha- and beta-subunits of Na+/K(+)-ATPase, and contact points between different regions of the same alpha-subunit, purified kidney enzyme preparations whose alpha-subunits were subjected to controlled proteolysis in different ways were solubilized with digitonin to disrupt intersubunit alpha,alpha-interactions, and oxidatively cross-linked. The following disulfide cross-linked products were identified by gel electrophoresis, staining with specific antibodies, and N-terminal analysis. 1) In the enzyme that was partially cleaved at Arg438-Ala439, the cross-linked products were an alpha,beta-dimer, a dimer of N-terminal and C-terminal alpha fragments, and a trimer of beta and the two alpha fragments. 2) From an extensively digested enzyme that contained the 22-kDa C-terminal and several smaller fragments of alpha, two cross-linked products were obtained. One was a dimer of the 22-kDa C-terminal peptide and an 11-kDa N-terminal peptide containing the first two intramembrane helices of alpha (H1-H2). The other was a trimer of beta, the 11-kDa, and the 22-kDa peptides. 3) The cross-linked products of a preparation partially cleaved at Leu266-Ala267 were an alpha,beta-dimer and a dimer of beta and the 83-kDa C-terminal fragment. Assuming the most likely 10-span model of alpha, these findings indicate that (a) the single intramembrane helix of beta is in contact with portions of H8-H10 intramembrane helices of alpha; and (b) there is close contact between N-terminal H1-H2 and C-terminal H8-H10 segments of alpha; with the most probable interacting helices being the H1,H10-pair and the H2,H8-pair.

Determinants of oligomeric structure in the chicken liver glycoprotein receptor

The oligomeric state of the chicken liver receptor (chicken hepatic lectin), which mediates endocytosis of glycoproteins terminating with N-acetylglucosamine, has been investigated using physical methods as well as chemical cross-linking. Receptor isolated from liver and from transfected rat fibroblasts expressing the full-length polypeptide is a homotrimer immediately following solubilization in non-ionic detergent, but forms the previously observed hexamer during purification. These results are most consistent with the presence of a trimer of receptor polypeptides in liver membranes and in transfected cells. Analysis of truncated receptors reveals that the C-terminal extracellular portion of this type-II transmembrane protein does not form stable oligomers when isolated from the membrane anchor and cytoplasmic tail. The behaviour of chimeric receptors, in which the cytoplasmic tail of the glycoprotein receptor is replaced with the corresponding segments of rat liver asialoglycoprotein receptor or the beta-subunit of Na+,K(+)-ATPase, or with unrelated sequences from globin, indicates that the cytoplasmic tail influences oligomer stability. Replacement of N-terminal portions of the receptor with corresponding segments of influenza virus neuraminidase results in formation of tetramers, suggesting that the membrane anchor and flanking sequences are important determinants of oligomer formation.

Low K+ increases Na(+)-K(+)-ATPase alpha- and beta-subunit mRNA and protein abundance in cultured renal proximal tubule cells

Studies from this laboratory demonstrate that LLC-PK1/Cl4 cells, a cultured renal cell line, respond to incubation in low-K+ medium by coordinately increasing abundance of both alpha- and beta-subunits of Na(+)-K(+)-ATPase but increase only beta- and not alpha-mRNA levels (Lescale-Matys et al. J. Biol. Chem. 265: 17935-17940, 1990) and that alpha-abundance is likely increased as a result of increased efficiency of alpha-mRNA translation (L. Lescale-Matys and A. A. McDonough. J. Cell Biol. 111: 311A, 1990). The aim of this report was to determine if nontransformed kidney cells would respond to low K+ in a similar manner. We incubated primary cultures of rat proximal tubule cells in low K+ (0.25 mM) for up to 24 h to address this aim. Na(+)-K(+)-ATPase activity, measured enzymatically, and abundance of alpha- and beta-subunits, measured by immunoblot, were increased significantly and coordinately by 8 h of low K+, and, by 24 h of low K+, these parameters were increased to 2.17 +/- 0.34 (activity), 2.03 +/- 0.21 (alpha), and 2.39 +/- 0.48 (beta)-fold over control. Pretranslationally, beta-mRNA, measured by Northern blot analysis, increased to 1.76 +/- 0.35 after 3 h of low K+ and to 3.4 +/- 0.75-fold over control after 24 h of low K+. The increase in alpha-mRNA was smaller and delayed compared with the beta-mRNA response, but it was sufficient to account for the observed increase in alpha-protein and Na(+)-K(+)-ATPase activity at steady state: alpha-mRNA increased to 1.27 +/- 0.09 after 6 h and to 1.91 +/- 0.41-fold over control after 24 h in low K+. We conclude that the accumulation of sodium pumps in cultured renal proximal tubule cells, unlike LLC-PK1 cells, can be accounted for by increases in both alpha- and beta-subunit mRNA levels.

Assignment ofAmog (adhesion molecule on glia) gene to mouse chromosome 11 nearZfp-3 andAsgr-1,2 and to human chromosome 17

AMOG, identified as an adhesion molecule that mediates neuron-astrocyte interaction, has structural similarity to the beta-subunit of Na,K ATPase. We have mapped the AMOG gene to human chromosome 17 and mouse chromosome 11 by somatic cell hybrid analysis. Recombinant inbred strain mapping has placed the Amog locus close to genes for zinc finger protein-3 and the asialoglycoprotein receptor in a region of mouse chromosome 11 that is homologous to human 17p.

Cross-resistance and biochemical characteristics of second-step mutants of HeLa cells resistant to cardiac glycosides

From ouabain-resistant (OuaR) mutants of HeLa cells which do not show any cross resistance to the digoxin analog SC4453, stable second-step mutants resistant to either SC4453 or those exhibiting increased resistance to digoxin have been isolated. The mutants obtained exhibited highly specific cross resistance towards different cardiac glycosides (CGs) and, based on their cross-resistance patterns, contained more than one type of genetic lesion. Biochemical studies with these mutants showed that cellular uptake of 86Rb was inhibited by specific CGs to which they showed increased resistance. The mutants showed reduced binding of [3H]ouabain and [3H]digoxin in comparison with the parental OuaR cells and about 50-60% of the Na+, K(+)-ATPase activity in the mutant cell extract was highly resistant to inhibition by ouabain and digoxin. In contrast to the above changes, these mutants showed no evidence of amplification, enhanced transcription, or gross alterations in the genes for the alpha or beta subunits of Na+, K(+)-ATPase. These observations indicated that these mutants involved a second-specific alteration in Na+, K(+)-ATPase. In contrast to these mutants, Chinese hamster ovary cells, which naturally exhibit comparable levels of resistance to CGs, showed no significant binding of either [3H]ouabain or [3H]digoxin and all of their Na+, K(+)-ATPase activity was resistant to inhibition by CGs.

Thyroid hormone induction of Na(+)-K(+)-ATPase and its mRNAs in a rat liver cell line

The expression of mRNAs encoding the alpha- and beta-subunits of Na(+)-K(+)-ATPase (Na(+)-K+ pump) was examined in a rat liver cell line, Clone 9, in various thyroidal states. Northern blot analysis of total RNA isolated from cells incubated in hypothyroid serum-containing medium revealed the expression of mRNAs encoding Na(+)-K(+)-ATPase alpha 1-(mRNA alpha 1) and beta- (mRNA beta) subunits; mRNAs encoding the alpha 2- and alpha 3-subunits were undetectable. There was a discrepancy in the abundance of mRNA alpha 1 relative to mRNA beta such that mRNA alpha 1 exceeded the sum of the multiple mRNA beta bands by approximately 35-fold. 3,3',5-Triiodothyronine (T3) produced a coordinate augmentation of mRNA alpha 1 and mRNA beta contents that was demonstrable within 2 h and preceded the stimulation of Na(+)-K(+)-ATPase activity. After incubation of cells with T3 for 48 h, Na(+)-K(+)-ATPase activity was stimulated by 1.32-fold, whereas mRNA alpha 1 and mRNA beta abundances were increased 1.46- and 2.87-fold, respectively. Treatment of cells for 6 h with 10 micrograms/ml cycloheximide, a concentration sufficient to inhibit protein synthesis by 95%, elicited a 3.5- and 5.1-fold increase in mRNA alpha 1 and mRNA beta content, respectively. Cycloheximide abrogated the stimulatory effect of T3 on mRNA beta abundance, whereas the T3-induced increase in mRNA alpha 1 content was not prevented.(ABSTRACT TRUNCATED AT 250 WORDS)

Primary sequence of Xenopus laevis Na+-K+-ATPase and its localization in A6 kidney cells

Polyclonal antibodies raised against the alpha- and beta-subunits of amphibian kidney Na+-K+-ATPase were used to screen an expression library from Xenopus laevis kidney epithelial cells (A6 cell line). cDNAs coding for each Na+-K+-ATPase subunit were identified and used to isolate near full-length cDNAs. The complete nucleotide sequence and the deduced amino acid sequence were determined. The alpha-subunit is an alpha (alpha I)-isoform. The alpha- and beta-subunits are more closely related to the mammalian and avian than the fish sequences. Antibodies raised against the fusion proteins produced by the two clones served to immunoprecipitate proteins from biosynthetically labeled or selectively surface-radioiodinated A6 cells grown on a porous substrate. The alpha- and the beta-subunits of Na+-K+-ATPase were found associated early in the course of biosynthesis and were restricted to the basolateral plasma membrane.

Absence of gene amplification in human cell mutants resistant to cardiac glycosides

In HeLa cells, four different types of mutants resistant to cardiac glycosides viz. ouabain and SC4453, which differ from each other in cross resistance pattern, have been isolated after single-step selections [J Biol Chem 260 (1985) 6843-6850; J Biol Chem 261 (1986) 2034-2040]. Using cloned cDNA probes specific for the alpha and beta subunits of Na+/K+ ATPase, these mutants have been investigated for amplification and/or increased transcription of Na+/K+ ATPase genes. Results from dot blots, Southern and Northern hybridizations provide evidence that these mutants do not involve any amplification or increased transcription or gross structural alterations in Na+/K+ ATPase genes of their transcripts. Similar results were obtained with the mutant cells grown either in the absence or presence of cardiac glycosides, the latter conditions of which cause 3-4-fold increase in the resistant form of the enzyme within the mutant cells. These results are consistent with the inference that resistance to cardiac glycosides in these mutants may be due to specific point mutations within the structural gene(s) of Na+/K+ ATPase leading to an altered enzyme that is resistant to inhibition by different cardiac glycosides.

Aldosterone regulation of Na+ transport and Na+-K+-ATPase in A6 cells: role of growth conditions.

The effects of aldosterone on transepithelial sodium transport (measured by the short-circuit current (SCC) and on Na+-K+-adenosine triphosphatase (ATPase) biogenesis have been studied in A6 kidney cells grown on collagen-coated filters in two different media. In medium A, base-line SCCA was close to zero but transmural electrical resistance (RA) was high. Aldosterone (100 nM, t24h) drastically increased SCCA and RA, but only after a 4-h latent period. In medium B, base-line SCCB and RB were significantly higher than in medium A. Aldosterone significantly enhanced SCCB and to a lesser extent RB after a much shorter latent period (approximately 45 min) than in medium A. In medium A, aldosterone elicited a fourfold increase in the relative rate of synthesis of alpha- and beta-subunits of Na+-K+-ATPase. A twofold increase was already observed within the observed latent period. This time course suggests that de novo synthesis of sodium pumps might be one of the critical factors underlying the increase in sodium transport in this growth medium. In medium B, aldosterone elicited a two- to fourfold increase in the relative rate of synthesis of the alpha- and beta-subunits of Na+-K+-ATPase that paralleled SCCB. Thus de novo synthesis of Na+-K+-ATPase is clearly not a prerequisite for the early mineralocorticoid response (t90 min - t180 min), but still could be part of the late mineralocorticoid response (t3 h - t24 h). In both media, the immunochemical cellular pool of Na+-K+-ATPase was apparently not modulated by aldosterone for up to 48 h of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection and localization of a cytoplasmic domain on the beta-subunit of Na+,K+-ATPase. A monoclonal antibody study.

A monoclonal antibody directed against the beta-subunit of dog kidney Na+,K+-ATPase was generated. Immunoblots demonstrate that monoclonal antibody III 18A binds exclusively to the denaturated beta-subunit. Binding experiments with membranes and whole cells reveal that III 18A binds to membranes but not to whole cells, indicating that the antibody binds to a cytoplasmic domain on the native beta-subunit. To localize the antibody-binding epitope, purified membrane-bound enzyme was fragmented by protease treatment. Tryptic digestion yields a 30-kDa fragment of the beta-subunit, which still retains the binding capacity for the antibody. Thus III 18A probably does not bind to the NH2-terminal segment of the protein. On the other hand, fragmentation of the beta-subunit with low concentrations of papain, which is known to yield a 40-kDa NH2-terminal and a 16-kDa COOH-terminal fragment, results in a complete loss of III 18A binding. These results suggest that the antibody-binding epitope is localized at or near a papain cleavage site on the COOH-terminal part of the beta-subunit. This is inconsistent with a structure model of the beta-subunit containing only a single transmembrane hydrophobic segment with a cytoplasmic NH2-terminal portion, but agrees quite well with a hypothetical structure with four intramembrane segments.

Membrane insertion of alpha- and beta-subunits of Na+,K+-ATPase.

Insertion of the alpha- and beta-subunits of amphibian epithelial Na+,K+-ATPase into pancreatic microsomes in cell-free systems was shown to be the same as into membranes of intact cells. The glycoproteic beta-subunit was observed to be cotranslationally inserted into endoplasmic reticulum membranes and to adopt a different pattern of N-linked core and terminal sugars in two different amphibian species. The beta-subunit lacks a cleavable signal sequence but quantitative membrane integration required membrane addition at the start of synthesis. Proteolysis of beta-subunit assembled in vitro indicated a cleavable cytoplasmic domain of about 2000 daltons. The catalytic 98-kilodalton alpha-subunit was also membrane-associated during its synthesis in an alkali-resistant fashion and independent of newly synthesized beta-subunit. In contrast to the beta-subunit, membrane integration of the alpha-subunit was possible as late as a time point in its synthesis which corresponded to about 1/3-1/2 of completion of the nascent chain. A small 34 kDa trypsin-resistant fragment of the alpha-subunit was produced at an early stage of synthesis both in the intact cell and in the cell-free system. These results suggest that membrane insertion of both alpha- and beta-subunit occurs during their synthesis but with a different time course.

Receptor occupancy vs. induction of Na+-K+-ATPase and Na+ transport by aldosterone.

In the urinary bladder of the toad Bufo marinus aldosterone (between 0.8 and 100 nM) stimulates Na+ transport [half-maximal induction concentration (K1/2) = 6.5 nM]. At low hormone concentrations (0.8-8 nM), the increase of Na+ transport between 0.75 and 2.5 h is accompanied by a fall in transepithelial resistance (R). Higher hormone concentrations (30-800 nM) induce an additional resistance-independent fraction of Na+ transport within 2.5-8 h. From 6 h on, aldosterone (between 0.2 and 20 nM) stimulates in the same tissue the biosynthesis rate of the alpha- and beta-subunits of Na+-K+-ATPase (K1/2 = 3 and 1.5 nM, respectively). New pump synthesis is thus not a prerequisite for the early mineralocorticoid response but might be linked to the late transport event. The mineralocorticoid response is usually ascribed to interaction with the higher affinity type 1 receptor. In the present study we show, however, that at least 55% of the overall Na+ transport response is linked to nuclear occupation of the lower affinity type 2 receptors [dissociation constant (Kd) = 50 nM, maximum number of binding sites (Nmax) = 315 fmol/mg protein]. Distinct aldosterone effects, such as the fall in R and the increase in Na+-K+-ATPase synthesis, are more closely related to occupation of type 1 receptors (Kd = 0.3 nM, Nmax = 23 fmol/mg protein). At maximal induction of these latter parameters, only about 20% of type 2 receptors are occupied. These results suggest that both types of aldosterone receptors are involved in the mediation of the full mineralocorticoid response: type 1 in the early and late and type 2 particularly in the late tissue response.

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.