Molecular cloning of the Na,K-ATPase alpha-subunit in developing brine shrimp and sequence comparison with higher organisms

We report here the molecular cloning, nucleotide sequence, and predicted amino acid sequence of an alpha-subunit of the developmentally useful model, Artemia. The amino acid sequence shows divergence from that of mammals, birds, Torpedo, and Drosophila. However, regions in the putative ATP binding and transmembrane domains show absolute or high levels of conservation. Major differences occur in the amino-terminal domain and several other hypervariable regions. These differences are consistent with the suggestion that the brine shrimp is a 'fast clock' organism which diverged from the precursors of vertebrates 0.5-1 billion years ago.     

Na,K-ATPase expression in the developing brine shrimp Artemia. Immunochemical localization of the alpha- and beta-subunits.

Developing brine shrimp are a good experimental model for study of gene expression during development. Development is initiated on suspension of brine shrimp cysts in seawater. Only 48 hr are required for progression from cyst to the larval stage. We have localize the alpha- and beta-subunits in different cells by immunostaining as development progresses. Both alpha- and beta-subunits are first detected in epidermal cells in the trunk region at the emergence 2 stage (16-hr incubation). At the nauplius 1 stage (24 hr) the enzyme appears in the brain and epidermal regions, as well as in mesenchymal cells, with weaker staining in the salt gland. After further development (nauplius 2 stage, 36 hr) stronger staining appears in the salt gland and in the epidermal region. At the nauplius 3 stage (48 hr) the enzyme appears in the midgut mucosa. Co-localization of the alpha- and beta-subunits appears in all positive cells during development. In the epidermal and salt gland cells the enzyme is mainly localized on the basolateral membrane. The basolateral localization of the Na,K-ATPase in epidermal and salt gland cells suggests that Na+ is actively transported into the epidermal and salt gland cells and passively diffuses out from the apical region.     

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.     

Photoaffinity labeling of the nucleotide binding site of actin

Rabbit skeletal muscle actin was photoaffinity-labeled by the nucleotide analogue 8-azidoadenosine 5'-triphosphate. In both G-actin and F-actin about 25% covalent incorporation was achieved. The labeled actins were digested with cyanogen bromide, and the labeled peptides were isolated and sequenced. In F-actin the label was bound primarily to Lys-336, while in G-actin the label was bound to Lys-336 or to Trp-356. The results indicate that the nucleotide binding site is near the phalloidin binding site of actin [Vanderkerckhove, J., Deboben, A., Nassal, M., & Wieland, T. (1985) EMBO J. 4, 2815-2818]. The binding of the azido group to Trp-356 in G-actin but not in F-actin may indicate that a change in the conformation of actin occurs in this region.     

Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [125I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.     

A protein whose binding to Na,K-ATPase is regulated by ouabain

Immunoprecipitation of Na,K-ATPase from kidney homogenate by antibodies against alpha1-subunit results in the precipitation of several proteins together with the Na,K-ATPase. A protein with molecular mass of about 67 kD interacting with antibodies against melittin (melittin-like protein, MLP) was found in the precipitate when immunoprecipitation was done in the presence of ouabain. If immunoprecipitation was done using antibodies against melittin, MLP and Na,K-ATPase alpha1-subunit were detected in the precipitate, and the amount of alpha1-subunit in the precipitate was increased after the addition of ouabain to the immunoprecipitation medium. MLP was purified from mouse kidney homogenate using immunoaffinity chromatography with antibodies against melittin. The addition of MLP to purified FITC-labeled Na,K-ATPase decreases fluorescence in medium with K+ and increases it in medium with Na+. The enhancement of fluorescence depends upon the MLP concentration. The N-terminal sequence of MLP determined by the Edman method is the following: HPPKRVRSRLNG. No proteins with such N-terminal sequence were found in the protein sequence databases. However, we revealed five amino acid sequences that contain this peptide in the middle part of the chain at distance 553 amino acids from the C-terminus (that corresponds to protein with molecular mass of about 67 kD). Analysis of amino acid sequence located between C-terminus and HPPKRVRSRLNG in all found sequences has shown that they were highly conservative and include WD40 repeats. It is suggested that the 67-kD MLP either belongs to the found protein family or was a product of proteolysis of one of them.     

Site of synthesis of the alpha and beta subunits of the Na,K-ATPase in brine shrimp nauplii

Developing nauplii (embryos) of the brine shrimp Artemia salina are an excellent model system for studying the biogenesis of the sodium- and potassium-activated adenosine triphosphatase (Na,K-ATPase). The nauplii exhibit a burst of Na,K-ATPase synthesis between 6 and 32 h of development (Peterson, G. L., Churchill, L., Fisher, J. A., and Hokin, L. E. (1982) J. Exp. Zool. 221, 295-308). We have now determined the sites of synthesis of the alpha and beta subunits of the Na,K-ATPase in developing A. salina nauplii. Membrane-bound and free polysomes were isolated from nauplii, and RNA was extracted from the polysomes. The polysomal RNA was translated in vitro in a rabbit reticulocyte lysate, and the translation products were immunoprecipitated by anti-subunit antisera. The immunoprecipitated proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. Our data show that the alpha subunit precursor is synthesized on membrane-bound polysomes and the beta subunit precursor is synthesized on free polysomes. In addition, the alpha subunit precursor appears as two separate peptides on sodium dodecyl sulfate-polyacrylamide gels, which suggests that the two alpha subunit forms seen in mature brine shrimp Na,K-ATPase are products of two distinct messenger RNAs. The beta subunit precursor appears as a single discrete band, unlike the mature beta subunit, which appears as a diffuse band.     

Differential expression of the alpha 2 and beta messenger RNAs of Na,K-ATPase in developing brine shrimp as measured by in situ hybridization.

We used in situ hybridization histochemistry with synthetic oligonucleotide probes to localize the mRNAs encoding the alpha 2- and beta-mRNAs of Na,K-ATPase during development of the brine shrimp Artemia. The mRNAs of the alpha 2- and beta-subunit were of low abundance in the cysts; in addition, less mRNA of the beta-subunit was localized. During emergence (12 hr), there was an increase in alpha 2-subunit mRNA in the gut mucosa, but there was a burst in beta-subunit mRNA throughout. As development progressed, the mRNAs of both the alpha 2- and beta-subunits showed a distinct pattern of expression in which the mRNA in the salt gland was of greatest abundance, followed by epidermal cells and gut mucosa. After 36 hr the alpha 2-subunit mRNA began to decrease in all positive cells but still remained highest in the salt gland and the brain region, while the mRNA of the beta-subunit kept increasing in the gut mucosa. Finally, the greatest abundance of the beta-subunit mRNA shifted from the salt gland to the antenna gland and the epidermal cells in the tail region, but the alpha 2-subunit mRNA did not. The more widespread distribution of the beta-mRNA than alpha 2-mRNA at certain stages (e.g., there was no alpha 2-mRNA in the antenna gland at the adult stage) is in all likelihood due to the marked drop in the alpha 2-subunit and a rise in alpha 1-subunit previously seen by Peterson et al. on polyacrylamide gel electrophoresis, as development progresses.     

Na,K-ATPase extracellular surface probed with a monoclonal antibody that enhances ouabain binding.

The Na,K-stimulated ATPase is inhibited by extracellular cardiac glycosides, which bind to the enzyme's alpha subunit. We used a monoclonal antibody, VG4, as a probe of the extracellular surface. The antibody was specific for Na,K-ATPase and bound to intact cells. The epitope was mapped to the first extracellular loop (H1-H2) of alpha, using a combination of techniques including trypsinolysis, N-terminal sequence of a fragment containing the determinant, and analysis of the effects of species-specific sequence differences. The antibody inhibited Na,K-ATPase activity under certain circumstances, indicating that the H1-H2 loop participates in conformational changes that are transmitted to the active site. Mutations in the H1-H2 loop have been shown by others to affect ouabain affinity. Ouabain and the antibody acted synergistically to inhibit the enzyme, which seemingly supported the hypothesis that the H1-H2 loop is an essential part of the cardiac glycoside binding site. Direct measurements of the binding of [3H]ouabain, however, indicated that VG4 enhanced rather than inhibited binding, presumably by promoting favorable conformation changes. The data suggest the possibility that the cardiac glycoside binding site may be intramembrane rather than extracellular.     

N-acetylimidazole inactivates renal Na,K-ATPase by disrupting ATP binding to the catalytic site

Treatment of renal Na,K-ATPase with N-acetylimidazole (NAI) results in loss of Na,K-ATPase activity. The inactivation kinetics can be described by a model in which two classes of sites are acetylated by NAI. The class I sites are rapidly reacting, the acetylation is prevented by the presence of ATP (K0.5 congruent to 8 microM), and the inactivation is reversed by incubation with hydroxylamine. These data suggest that the class I sites are tyrosine residues at the ATP binding site. The second class of sites are more slowly reacting, not protected by ATP, nor reversed by hydroxylamine treatment. These are probably lysine residues elsewhere in the protein. The associated K-stimulated p-nitrophenylphosphatase activity is inactivated by acetylation of the class II sites only; thus the tyrosine residues associated with ATP binding to the catalytic center are not essential for phosphatase activity. Inactivated enzyme no longer has high-affinity ATP binding associated with the catalytic site, although low-affinity ATP effects (inhibition of phosphatase and deocclusion of Rb) are still present. The inactivated enzyme can still be phosphorylated by Pi, occlude Rb+ ions, and undergo the major conformational transitions between the E1 Na and E2 K forms of the enzyme. Thus acetylation of the Na,K-ATPase by NAI inhibits high-affinity ATP binding to the catalytic center and produces inactivation.     

Photoaffinity labeling of the catalytic site of prenyltransferase.

Three photoreactive substrate analogues, o-azidophenethyl pyrophosphate, p-azidophenethyl pyrophosphate, and 3-azido-1-butyl pyrophosphate, have been synthesized as site-directed probes to label the catalytic site of prenyltransferase. Due to the relatively poor affinity of p-azidophenethyl pyrophosphate and 3-azido-1-butyl pyrophosphate for the enzyme, only o-azidophenethyl pyrophosphate (aryl azide) was utilized for photoaffinity labeling. This aryl azide has a UV absorption maximum at 250 nm. In the absence of activating light, binding studies demonstrate that the o-aryl azide competes for binding with both the natural substrates, isopentenyl pyrophosphate and geranyl pyrophosphate. More than 90% enzymatic activity is lost when enzyme is irradiated in the presence of the aryl azide as compared to irradiation in the absence of the azide, and the protein loses its capacity for substrate binding in direct proportion to photolabeling. A stoichiometry of 2 mol of affinity label covalently bound per mol of enzyme dimer was established with [1-3H]-o-azidophenethyl pyrophosphate. Since there are two catalytic sites per enzyme dimer, the o-aryl azide appears specifically to label the enzyme at its catalytic sites. Additional evidence that the reagent was specific for the catalytic site came from the observation that farnesyl pyrophosphate afforded complete protection against photoinactivation, while isopentenyl pyrophosphate provided partial protection. Gel isoelectric focusing verified this stoichiometry and indicated that the labeled enzyme has a more acidic isoelectric point than the native enzyme.     

Photoaffinity labeling of an antimycin-binding site in Rhodopseudomonas sphaeroides

Tritium-labeled 3-azidosalicyl-N-(n-octadecyl)amide was synthesized and used as a photoaffinity probe for the antimycin-binding site in both purified ubiquinone-cytochrome b-c1 oxidoreductase and chromatophore vesicles from the photosynthetic bacterium Rhodopseudomonas sphaeroides. In both systems, a prominently labeled protein had a molecular weight of 11,000. Binding to this protein was inhibited by preincubation of the reaction mixture with antimycin prior to addition of the radioactive analog and subsequent irradiation. The antimycin analog, 3-azidosalicyl-N-(n-octadecyl)amide, inhibited succinate-cytochrome c reductase activity in chromatophore vesicles by 50% at a concentration of 150 nmols/mg of protein.     

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.     

Characterization of gill Na/K-ATPase activity and ouabain binding in Antarctic and New Zealand nototheniid fishes

The effects of thermal acclimation in two Nototheniid species, the stenothermal Antarctic Trematomous bernacchii and the eurythermal New Zealand Notothenia angustata, were investigated. Serum osmolality, gill Na/K-ATPase activity, sodium pump density and ouabain affinity were determined. Both fish were acclimated at their upper and lower viable thermal temperatures. Warm acclimation (+4 degrees C) of the T. bernacchii significantly decreased their serum osmolality from 550 to 450 mOsm/kg compared to cold-acclimation (-1.5 degrees C) and this was accompanied by a two-fold increase in gill Na/K-ATPase activity. Warm-acclimation (+14 degrees C) of N. angustata did not significantly change their serum osmolality from 330 mOsm/kg or gill Na/K-ATPase activity compared to the cold-acclimated (+4 degrees C) N. angustata. Using [(3)H]ouabain binding techniques, the B(max) and K(d) values of gill Na/K-ATPase enzymes were determined. No difference in the B(max) or K(d) of the warm-acclimated T. bernacchii accounted for the increase in Na/K-ATPase activity. We conclude that the change in gill Na/K-ATPase activity in the warm-acclimated T. bernacchii is not mediated by an increase in the number of enzyme sites and is not reflected in a change in ouabain affinity for Na/K-ATPase.     

Nucleotide binding to Na,K-ATPase: pK values of the groups affecting the high affinity site

Investigation of the ionic strength effect on the interactions between nucleotides (ATP and ADP) and Na,K-ATPase in a broad pH range was aimed at revealing pK values of the charged groups of the interacting species. Ionic strength experiments suggested that an amino acid residue with a pK > 8.0 is part of the protein binding site. A combination of equilibrium and transient experiments at various pH values allowed for the characterization of the groups electrostatically involved in either the association process (kon) or the stability of the preformed complexes (koff). Two groups (pK1 = 6.7 and pK2 = 8.4) appear to be important for the proper organization of the binding site and, therefore, the association reaction. Moreover, deprotonation of the basic group completely precludes association. pH dependencies of the dissociation rate constants for ATP and ADP are very different. An increase in pH from 5 to 9.5 induces a 9-fold increase in koff for ATP, whereas koff for ADP decreases 4-fold between pH 5 and 8, and decreases further in the alkaline region. A comparison of the pH dependencies for koff for ATP and ADP suggests two effects: (1) at acidic pH, the value of the total negative charge of the nucleotide determines the tightness of binding; and (2) short-range interactions involving the terminal phosphate group are important for nucleotide dissociation from the site. The difference in the pH dependencies of koff for the nucleotides suggests the existence of positive charges in close proximity to Asp369, relieving the repulsion between the gamma-phosphate of ATP and Asp369.     

Chromosome-mediated transfer of the murine Na,K-ATPase alpha subunit confers ouabain resistance.

We transferred murine NIH 3T3 metaphase chromosomes into monkey CV-1 cells to investigate the different ouabain sensitivities of rodent and primate cells. In 16 ouabain-resistant transferents, the mouse Na,K-ATPase alpha 1 subunit gene was detected, suggesting that structural differences between the rodent and primate alpha 1 subunits determine the different ouabain sensitivities.     

Photoaffinity labeling identifies the substrate-binding site of mammalian squalene epoxidase

Squalene epoxidase (SE) catalyzes the conversion of squalene to (3S)-2,3-oxidosqualene. Photolabeling and site-directed mutagenesis were performed on recombinant rat SE (rrSE) in order to identify the location of the substrate-binding site and the roles of key residues in catalysis. Truncated 50-kDa rrSE was purified and photoaffinity labeled by competitive SE inhibitor (Ki=18.4 microM), [(3)H]TNSA-Dza. An 8-kDa CNBr/BNPS-skatole peptide was purified and the first 24 amino acids were sequenced by Edman degradation. The sequence PASFLPPSSVNKRGVLLLGDAYNL corresponded to residues 388-411 of the full-length rat SE. Three nucleophilic residues (Lys-399, Arg-400, and Asp-407) were labeled by [(3)H]TNSA-Dza. Triple mutants were prepared in which bulky groups were used to replace the labeled charged residues. Purified mutant enzymes showed lower enzymatic activity and reduced photoaffinity labeling by [(3)H]TNSA-Dza. This constitutes the first evidence as to the identity of the substrate-binding site of SE.     

The effect of juvenile hormone III, methyl farnesoate, and methoprene on Na/K-ATPase activity in larvae of the brine shrimp, Artemia.

1. Ion transport enzyme (Na/K-ATPase) activity in stage III larvae of the brine shrimp, Artemia, remains elevated throughout the stadium when populations are exposed to methoprene in artificial seawater. 2. Infusion of methoprene, juvenile hormone, or methyl farnesoate causes increased Na/K-ATPase activity in homogenates of mid-stadium larvae that would otherwise exhibit low activity. 3. The sensitivity of the enzyme system to extremely low concentrations of the juvenoids suggests that this may be a common mode of action of these compounds. Additionally it suggests that the enzyme may be under the influence of a similar compound present in the larvae.