Assembly of a hybrid from the alpha subunit of Na+/K(+)-ATPase and the beta subunit of H+/K(+)-ATPase.

Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.     

A hybrid between Na+,K+-ATPase and H+,K+-ATPase is sensitive to palytoxin, ouabain, and SCH 28080

Na(+),K(+)-ATPase is inhibited by cardiac glycosides such as ouabain, and palytoxin, which do not inhibit gastric H(+),K(+)-ATPase. Gastric H(+),K(+)-ATPase is inhibited by SCH28080, which has no effect on Na(+),K(+)-ATPase. The goal of the current study was to identify amino acid sequences of the gastric proton-potassium pump that are involved in recognition of the pump-specific inhibitor SCH 28080. A chimeric polypeptide consisting of the rat sodium pump alpha3 subunit with the peptide Gln(905)-Val(930) of the gastric proton pump alpha subunit substituted in place of the original Asn(886)-Ala(911) sequence was expressed together with the gastric beta subunit in the yeast Saccharomyces cerevisiae. Yeast cells that express this subunit combination are sensitive to palytoxin, which interacts specifically with the sodium pump, and lose intracellular K(+) ions. The palytoxin-induced K(+) efflux is inhibited by the sodium pump-specific inhibitor ouabain and also by the gastric proton pump-specific inhibitor SCH 28080. The IC(50) for SCH 28080 inhibition of palytoxin-induced K(+) efflux is 14.3 +/- 2.4 microm, which is similar to the K(i) for SCH 28080 inhibition of ATP hydrolysis by the gastric H(+),K(+)-ATPase. In contrast, palytoxin-induced K(+) efflux from cells expressing either the native alpha3 and beta1 subunits of the sodium pump or the alpha3 subunit of the sodium pump together with the beta subunit of the gastric proton pump is inhibited by ouabain but not by SCH 28080. The acquisition of SCH 28080 sensitivity by the chimera indicates that the Gln(905)-Val(930) peptide of the gastric proton pump is likely to be involved in the interactions of the gastric proton-potassium pump with SCH 28080.     

Sequence motif in control regions of the H+/K+ ATPase alpha and beta subunit genes recognized by gastric specific nuclear protein(s).

A nuclear protein(s) from rat or pig stomach recognized a conserved sequence in the 5'-upstream regions of the rat and human H+/K(+)-ATPase alpha subunit genes. A gel retardation assay suggested that part of the binding site was located in the TAATCAGCTG sequence. No nuclear proteins capable of the binding could be detected in other tissues of rat (liver, brain, kidney, spleen and lung) or pig liver. The sequence motif (GATAGC) located 5'-upstream of the beta-subunit gene also seemed to be recognized by the same protein, because the binding of nuclear protein to the sequence motifs in the alpha and beta subunits was mutually competitive. Considering the sense-strand sequence of the binding motif in the alpha-subunit gene, we conclude that (G/C)PuPu(G/C)NGAT(A/T)PuPy is a core sequence motif for the gastric specific DNA binding protein (PCSF, parietal cell specific factor).     

Characterization of the 5' flanking region of the rat Na+/K(+)-ATPase beta 1 subunit gene.

We have isolated the 5' end of the rat Na+/K(+)-ATPase beta 1 subunit gene. A genomic fragment containing 817 bp of the 5' flanking sequence, exon 1 and 479 bp of intron 1 was sequenced. The 5' flanking region contains a potential TATA box and several putative CAAT and GC boxes. Potential binding sites for thyroid and glucocorticoid receptors were identified together with multiple sequence motifs which exhibit homology to calcium and serum responsive elements.     

The C-terminal 165 amino acids of the plasma membrane Ca(2+)-ATPase confer Ca2+/calmodulin sensitivity on the Na+,K(+)-ATPase alpha-subunit.

The C-terminal 165 amino acids of the rat brain plasma membrane (PM) Ca(2+)-ATPase II containing the calmodulin binding auto-inhibitory domain was connected to the C-terminus of the ouabain sensitive chicken Na+,K(+)-ATPase alpha 1 subunit. Expression of this chimeric molecule in ouabain resistant mouse L cells was assured by the high-affinity binding of [3H]ouabain. In the presence of Ca2+/calmodulin, this chimeric molecule exhibited ouabain inhibitable Na+,K(+)-ATPase activity; the putative chimeric ATPase activity was absent in the absence of Ca2+/calmodulin and activated by Ca2+/calmodulin in a dose-dependent manner. Furthermore, this chimeric molecule could bind monoclonal IgG 5 specific to the chicken Na+,K(+)-ATPase alpha 1 subunit only in the presence of Ca2+/calmodulin, suggesting that the epitope for IgG 5 in this chimera is masked in the absence of Ca2+/calmodulin and uncovered in their presence. These results propose a direct interaction between the calmodulin binding auto-inhibitory domain of the PM Ca(2+)-ATPase and the specific regions of the Na+,K(+)-ATPase alpha 1 subunit that are structurally homologous to the PM Ca(2+)-ATPase. A comparison of the deduced amino acid sequences revealed several possible regions within the Na+,K(+)-ATPase that might interact with the auto-inhibitory domain of the PM Ca(2+)-ATPase.     

Location of the cytoplasmic epitope for a K(+)-competitive antibody of the (H+,K+)-ATPase.

The monoclonal antibody (mAb) 95-111 binds the alpha subunit of (H+,K+)-ATPase and inhibits the K(+)-ATPase activity. To map the epitope, all of the partial sequences of the alpha subunit were expressed in Escherichia coli HB101 using rabbit alpha subunit cDNA restriction fragments ligated into PuEx vector. Bacterial recombinant lysates were separated by sodium dodecyl sulfate-gel electrophoresis, and the epitope was detected by Western blotting. The antibody site was mapped between Cys529 and Glu561. This is close to the Lys517 that binds fluorescein isothiocyanate (FITC) and is considered to be between M4 and M5 close to the ATP binding domain. However, the mAb inhibition of ATPase is not ATP-competitive but is K(+)-competitive with a KI of 2 x 10(-9) M. The mAb also inhibits K+ quench of FITC fluorescence competitively with a KI of 8 x 10(-9) M. The K+ activation of ATPase activity and quench of FITC fluorescence are dependent on K+ binding to an E2 form of the enzyme from the extracytoplasmic surface. The mAb epitope is cytoplasmic since the K(+)-ATPase activity of ion-tight gastric vesicles is inhibited. The 125I-mAb 95-111 binds to a single class of sites with an apparent KD of 2.3 +/- 0.8 x 10(-9) M and K+ does not displace bound mAb. Hence, antibody binding to a cytoplasmic Cys529-Glu561 epitope allosterically competes with K(+)-dependent reactions at extracytoplasmic sites.     

The K(+) affinity of gastric H(+),K(+)-ATPase is affected by both lipid composition and the beta-subunit

It is generally assumed that negatively charged residues present in the alpha-subunit of gastric H(+),K(+)-ATPase are involved in K(+) binding and transport. Despite the fact that there is no difference between various species regarding these negatively charged residues, it was observed that the apparent K(+) affinity of the pig enzyme was much lower than that of the rat H(+),K(+)-ATPase. By determining the K(+)-stimulated dephosphorylation reaction of the phosphorylated intermediate K(0.5) values for K(+) of 0.12+/-0.01 and 1.73+/-0.03 mM were obtained (ratio 14.4) for the rat and the pig enzyme, respectively. To investigate the reason for the observed difference in K(+) sensitivity, both enzymes originating from the gastric mucosa were either reconstituted in a similar lipid environment or expressed in Sf9 cells. After reconstitution in K(+)-permeable phosphatidylcholine/cholesterol liposomes K(0.5) values for K(+) of 0.16+/-0.01 and 0.35+/-0.05 mM for the rat and pig enzyme respectively were measured (ratio 2.2). After expression in Sf9 cells the pig gastric H(+),K(+)-ATPase still showed a 4.1 times lower K(+) sensitivity than that of the rat enzyme. This means that the difference in K(+) sensitivity of the rat and pig gastric H(+), K(+)-ATPase is not only due to a different lipid composition but also to the structure of either the alpha- or beta-subunit. Expression of hybrid enzymes in Sf9 cells showed that the difference in K(+) sensitivity between the rat and pig gastric H(+),K(+)-ATPase is primarily due to differences in the beta-subunit.     

cDNA cloning and membrane topology of the rabbit gastric H+/K(+)-ATPase alpha-subunit.

We have cloned and sequenced a cDNA for the rabbit gastric proton-potassium pump (H+/K(+)-ATPase) alpha-subunit. The deduced peptide contains 1035 amino acids (Mr 114,201) and shows 97% sequence identity with the respective rat and hog proteins. A monoclonal antibody 146-14 has been shown previously to react with the extracytoplasmic side of the catalytic H+/K(+)-ATPase subunit and here we show that the epitope is in the region between amino acids 855 and 902 (the numbering of the H+/K(+)-ATPase catalytic subunit throughout the paper refers to the rabbit sequence). The localization of this epitope in conjunction with previously observed trypsin cleavage sites in the C-terminal one third of the enzyme and the hydrophobicity plot of the deduced peptide sequence are evidence for a structural model for the alpha-subunit of the H+/K(+)-ATPase which contains at least ten membrane spanning segments, similar to that deduced for the Ca(2+)-ATPase of sarcoplasmic reticulum.     

Mitochondrial sequence migrated downstream to a nuclear V-ATPase B gene is transcribed but non-functional.

A promiscuous nuclear sequence containing a mitochondrial DNA fragment was isolated from rice. Nucleotide sequence analysis reveals that the cDNA clone #21 carries a mitochondrial sequence homologous to the 3' portion of the rps19 gene followed by the 5' portion of the rps3 gene. The mitochondrial sequence is present in an antisense orientation. Sequence comparison of the #21 cDNA with the original mitochondrial sequence shows 99% similarity, suggesting a recent transfer event. Moreover, evidence for a lack of an RNA editing event and retaining of the group II intron sequence strongly suggests that the sequence was transferred from mitochondrion to the nucleus via DNA rather than RNA as an intermediate. The upstream region to the mitochondria-derived sequence shows homology to part of the vacuolar H(+)-ATPase B subunit (V-ATPase B) gene. Isolation of a functional V-ATPase B cDNA and its comparison with the #21 cDNA reveal a number of nucleotide substitutions resulting in many translational stop codons in the #21 cDNA. This indicates that the #21 cDNA sequence is not functional. Analysis of genomic sequences shows the presence of five intron sequences in the #21 cDNA, whereas the functional V-ATPase B gene has 14 introns. Of these, three exons and their internal two introns are homologous to each other, suggesting a duplication event of V-ATPase B genomic DNA. The results of this investigation strongly suggest that the mitochondrial sequence was integrated in an antisense orientation into the pre-existing V-ATPase B pseudogene that can be transcribed and spliced. This represents a case of unsuccessful gene transfer from mitochondrion to the nucleus.     

Phosphorylation of H+/K(+)-ATPase by inorganic phosphate. The role of K+ and SCH 28080.

The effects of K+ on the phosphorylation of H+/K(+)-ATPase with inorganic phosphate were studied using H+/K(+)-ATPase purified from porcine gastric mucosa. The phosphoenzyme formed by phosphorylation with Pi was identical with the phosphoenzyme formed with ATP. The maximal phosphorylation level obtained with Pi was equal to that obtained with ATP. The Pi phosphorylation reaction of H+/K(+)-ATPase was, like that of Na+/K(+)-ATPase, a relatively slow reaction. The rates of phosphorylation and dephosphorylation were both increased by low concentrations of K+, which resulted in hardly any effect on the phosphorylation level. A decrease of the steady-state phosphorylation level was caused by higher concentrations of K+ in a noncompetitive manner, whereas no further increase in the dephosphorylation rate was observed. The decreasing effect was caused by a slow binding of K+ to the enzyme. All above-mentioned K+ effects were abolished by the specific H+/K(+)-ATPase inhibitor SCH 28080 (2-methyl-8-[phenyl-methoxy]imidazo-[1-2-a]pyrine-3-acetonitrile). Additionally, SCH 28080 caused a 2-fold increase in the affinity of H+/K(+)-ATPase for Pi. A model for the reaction cycle of H+/K(+)-ATPase fitting the data is postulated.     

An mRNA from human brain encodes an isoform of the B subunit of the vacuolar H(+)-ATPase

The B subunit (approximately 60 kDa) of the vacuolar H(+)-ATPase is one of the two major subunits comprising the hydrophilic catalytic complex of the enzyme. Using left and catalytic complex of the enzyme. Using left and right primers which bind two highly conserved sequences of the B subunit, an 836-base pair fragment was amplified from human brain cDNA by the polymerase chain reaction. The amplified fragment was used to probe a Northern blot and to screen a brain cDNA library. A single RNA band, 3.2 kilobases (kb) in length, was detected on Northern blots. A positive cDNA clone containing a 2.5-kb insert was isolated and sequenced. It included a long 3'-untranslated region (greater than 1.2 kb) and was missing a minor portion of the 5'-end of the coding region. The coding region of the brain cDNA sequence was 77% identical at the nucleotide level and 90% identical at the amino acid level to the previously reported sequence for the B subunit of the vacuolar H(+)-ATPase from human kidney (Sudhof, T. C., Fried, V. A., Stone, D. K., Johnston, P. A., and Xie, X.-S. (1989) Proc. Natl. Acad. Sci, U. S. A. 86, 6067-6071). Within the coding region of the brain cDNA, which is 6 amino acid residues shorter at the 3'-end than the kidney sequence, an 11% difference in the GC content was calculated. The 3'-noncoding sequence of the brain cDNA was completely unrelated to that of kidney and was three times longer. We conclude that the B subunit cDNAs from human kidney and brain represent different isoforms. This is the first demonstration of an isoform of a vacuolar H(+)-ATPase subunit.     

The Arabidopsis thaliana plasma membrane H(+)-ATPase multigene family. Genomic sequence and expression of a third isoform

The plasma membrane of higher plants contains a H(+)-ATPase as its major ion pump. This enzyme belongs to the P-type family of cation-translocating enzymes and generates the proton-motive force that drives solute uptake across the plasma membrane. In Arabidopsis thaliana the plasma membrane H(+)-ATPase is encoded by a multigene family (Harper, J. F., Surowy, T. K., and Sussman, M. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1234-1238). The complete genomic sequence of a third Arabidopsis H(+)-ATPase isoform (referred to as AHA2) is presented here, and the predicted protein sequence is compared with previously published AHA1, AHA3, and tobacco Nicotiana plumbaginifolia NP1 isoforms. The AHA2 gene is most similar to AHA1, with predicted proteins containing 95% amino acid identity. The mRNA start site and 5'-untranslated sequence for AHA2 were determined from cDNA amplified by the polymerase chain reaction. The 5' region contains a 23-base pair (bp) polypyrimidine sequence and a short upstream reading frame. In comparison with the 16 introns reported in AHA3, AHA2 is missing one intron in the 5'-untranslated region and a second intron in the C-terminal coding region. An unusually large intron for Arabidopsis (greater than 1000 bp) is present at the beginning of the coding sequence of both AHA2 and AHA3. In the 3'-untranslated sequence of AHA1 and AHA2 but not AHA3, there is a 65-bp region of 85% identity and a second shorter region of 16-bp identity harboring an unusual putative poly(A) addition signal (dTTTGAAGAAACAAGGC). Northern blot analysis indicates that AHA2 mRNA relative to total cellular RNA is expressed at significantly higher levels in root tissue as compared with shoot tissue.     

Epitope mapping by amino-acid-sequence-specific antibodies reveals that both ends of the alpha subunit of Na+/K(+)-ATPase are located on the cytoplasmic side of the membrane.

Right-side-out vesicles of pig kidney microsomes and amino-acid-sequence-specific antibodies were used to probe the sidedness of the C-terminus and the N-terminus of the catalytic alpha subunit of Na+/K(+)-ATPase. Polyclonal antibodies were raised in rabbits against the peptide corresponding to the N-terminal sequence GRDKYEPAAVSE (peptide 1-12) and against peptides corresponding to the C-terminal sequences IFVYDEVRKLIIRRR (peptide 991-1005) and RPGGWVEKETYY (peptide 1005-1016). These antibodies were purified by affinity chromatography on the respective peptide-Sepharose columns. Moreover, antibodies against the N-terminal dodecapeptide GRDKYEPAAVSE were obtained by affinity purification from heteroclonal antibodies against the alpha subunit of pork kidney Na+/K(+)-ATPase. These antibodies reacted with native as well as SDS-denaturated Na+/K(+)-ATPase. When the antibodies were used to probe the sidedness of the sequences in right-side-out vesicles of pig kidney microsomes, the N-terminal peptide 1-12 as well as the C-terminal peptides 991-1005 and 1005-1016 were found on the cytosolic side. Concanavalin A, however, which interacts with the beta subunit, a glycoprotein, reacted with the outside of right-side-out vesicles.