Lois K. Lane

A kinetic comparison of cardiac glycoside interactions with Na+,K+-ATPases from skeletal and cardiac muscle and from kidney.

Abstract The rates of association of [3H]ouabain to Na+,K+-ATPase and the rates of dissociation of the enzyme-ouabain complexes were determined for enzymes isolated from dog skeletal muscle, beef heart muscle, and lamb kidney medulla. The rates of association were strongly influenced by the presence of ligands such as magnesium, sodium, potassium, ATP, and inorganic phosphate. For a particular set of binding ligands, the rates of association did not vary much amongst the three enzymes studied, although enzyme from skeletal muscle was the fastest. In contrast, the rates of dissociation were relatively independent of the ligand conditions. The rates of dissociation also varied greatly amongst the enzyme sources, with skeletal muscle Na+,K+-ATPase being the fastest. Although the major determinant of the affinity of the Na+,K+-ATPase for ouabain is the rate of dissociation, the rate of association also plays a role. Since the binding of ouabain to the Na+,K+-ATPase in the presence of magnesium, ATP, sodium, and potassium is very slow, it is difficult to obtain an I50 (equilibrium) value for the inhibition of hydrolytic activity by ouabain. If measurements of activity are made after a long period of time (3 h), the affinity of the enzyme for ouabain, estimated from inhibition of Na+,K+-ATPase activity, approached the value calculated from [3H]ouabain binding. The ratio of the I50 value for ouabagenin to that for ouabain for the skeletal muscle enzyme was the same as that for cardiac muscle enzyme, indicating that the sugar moiety of ouabain was interacting with the receptor of both enzymes. It is apparent, therefore, that the absence of a sugar binding site in skeletal Na+,K+-ATPase is not the reason for the faster dissociation rate of this enzyme.

Large-Scale Purification of Na,K-ATPase and Its Protein Subunits from Lamb Kidney Medulla

Procedures are described for the large-scale isolation of purified Na,K-ATPase (EC 3.6.1.3) from frozen lamb kidney outer medulla and for the separation of its two protein subunits by hydroxyapatite chromatography in sodium dodecyl sulfate (SDS). The methods described permit the routine isolation of up to 800 mg of purified Na,K-ATPase in one week, which can subsequently be separated into 500 mg of mr = 95,000 catalytic subunit and 200 mg of glycoprotein with four SDS-hydroxyapatite column runs.

Characterization of two genes for the human Na,K-ATPase β subunit

Abstract A total of 29 human genomic DNA clones that hybridize with cDNAs for the sheep and rat Na,K-ATPase β subunits have been isolated, classified by restriction endonuclease mapping and Southern blot hybridization analysis, and sequenced. One class of clones, designated ATP1BL1, represents a processed pseudogene for the β subunit. The second class, designated ATP1B, includes 15 overlapping genomic clones and represents a functional gene for the human Na,K-ATPase β subunit. ATP1B spans about 26.7 kb of genomic DNA and includes 24 kb of intron sequence. The complete mRNA transcript for the human β subunit is encoded by six exons, ranging in size from 81 to 1427 bp. Primer extension and S1 nuclease protection experiments with human kidney RNA indicate the presence of two major transcription initiation sites at −510 and −201 to −191, with minor initiation sites at −268, −182 to −174, and −142. The distal initiation site at −510 is preceded by consensus sequences for CAAT and TATA boxes. The DNA sequence preceding the proximal heterogeneous initiation sites contains a CAAT box, but no TATA box. Two of the 12 GC boxes (GGCGGG and CCCGCC) located in the 5′ region of ATP1B are located between this CAAT box and the proximal clusters of transcription initiation sites.

CHANGES IN STEADY-STATE CONFORMATIONAL EQUILIBRIUM RESULTING FROM CYTOPLASMIC MUTATIONS OF THE NA, K-ATPASE ALPHA -SUBUNIT

Mutations comprising either deletion of 32 amino acids from the NH2 terminus (α1M32) or a Glu233 → Lys substitution in the first M2-M3 cytoplasmic loop (E233K) of the α1-subunit of the Na,K-ATPase result in a shift in the steady-state E 1 ↔E 2 conformational equilibrium towardE 1 form(s). In the present study, the functional consequences of both NH2-terminal deletion and Glu233 substitution provide evidence for mutual interactions of these cytoplasmic regions. Following transfection and selection of HeLa cells expressing the ouabain-resistant α1M32E233K double mutant, growth was markedly reduced unless the K+concentration in the culture medium was increased to at least 10 mm. Marked changes effected by this double mutation included 1) a 15-fold reduction in catalytic turnover (V max/EPmax), 2) a 70-fold increase in apparent affinity for ATP, 3) a marked decrease in vanadate sensitivity, and 4) marked (≈10-fold) K+activation of the Na-ATPase activity measured at micromolar ATP under which condition the E 2(K) → →E 1 pathway is normally (α1) rate-limiting and K+ is inhibitory. The decrease in catalytic turnover was associated with a 5-fold decrease in V max and a compensatory ≈3-fold increase in expressed α1M32E233K protein. In contrast to the behavior of either α1M32 or E233K, α1M32E233K also showed alterations in apparent cation affinities.K′Na was decreased ≈2-fold andK′K was increased ≈2-fold. The importance of the charge at residue 233 is underscored by the consequences of single and double mutations comprising either a conservative change (E233D) or neutral substitution (E233Q). Thus, whereas mutation to a positively charged residue (E233K) causes a drastic change in enzymatic behavior, a conservative change causes only a minor change and the neutral substitution, an intermediate effect. Overall, the combined effects of the NH2-terminal deletion and the Glu233substitutions are synergistic rather than additive, consistent with an interaction between the NH2-terminal region, the first cytoplasmic loop, and possibly the large M4-M5 cytoplasmic loop bearing the nucleotide binding and phosphorylation sites.

Purification and characterization of an (Na+ + K+)-ATPase proteolipid labeled with a photoaffinity derivative of ouabain.

Highly purified lamb kidney (Na+ + K+)-ATPase was photoaffinity labeled with the tritiated 2-nitro-5-azidobenzoyl derivative of ouabain (NAB-ouabain). The labeled (Na+ + K+)-ATPase was mixed with unlabeled carrier enzyme. Two proteolipid (gamma 1 and gamma 2) fractions were then isolated by chromatography on columns of Sepharose CL-6B and Sephadex LH-60. The two fractions were interchangeable when rechromatographed on the LH-60 column, suggesting that gamma 1 is an aggregated form of gamma 2. The total yield was 0.8-1.5 mol of gamma component per mol of catalytic subunit recovered. This indicates that the gamma component is present in stoichiometric amounts in the Na+ + K+)-ATPase. The proteolipids that were labeled with NAB-ouabain copurified with the unlabeled proteolipids.

Effects of extracts of rat brain on the digitalis receptor.

There is a possibility that an endogenous substance exists which interacts with a ouabain binding site on Na+, K+-ATPase. Recently, several reports have appeared suggesting the presence of an endogenous digitalis-like substance in acid-acetone extracts of brain. We have demonstrated that in preparing an acid-acetone extract, peroxidized lipids and lysophospholipids are produced, both of which inhibit Na+, K+-ATPase, thereby complicating interpretation. Preliminary evidence suggests, however, that when rat brains are extracted with an aqueous-acetone mixture under nitrogen, a principle is obtained which specifically inhibits Na+, K+-ATPase.

Structural studies on H+, K+-ATPase: Determination of the NH2-terminal amino acid sequence and immunological cross-reactivity with Na+,K+-ATPase

The NH2-terminal amino acid sequence of the 100 kilodalton subunit of porcine gastric H+,K+-ATPase has been determined to be YKAENYELYQVELGPGP. Although the NH2-terminal region of this protein is not similar to the same region of the lamb kidney Na+,K+-ATPase catalytic subunit, other regions of these ATPase proteins appear to be homologous. Both monoclonal and polyclonal antibodies raised to lamb kidney Na+,K+-ATPase and its alpha, but not beta, subunit cross-react with the 100 kilodalton protein of H+,K+-ATPase.

New Insights into the Role of the N Terminus in Conformational Transitions of the Na,K-ATPase

The deletion of 32 residues from the N terminus of the α1 catalytic subunit of the rat Na,K-ATPase (mutant α1M32) shifts the E1/E2 conformational equilibrium toward E1, and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E1 (Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998)J. Biol. Chem. 273, 23086–23092). To delimit the region of the cytoplasmic N terminus involved in these interactions, the consequences of a series of N-terminal deletions of α1 beyond Δ32 were evaluated. Criteria to assess shifts in conformational equilibrium were based on effects of perturbation of the entire catalytic cycle ((i) sensitivity to vanadate inhibition, (ii) K+ sensitivity of Na-ATPase measured at micromolar ATP, (iii) changes in K′ATP, and (iv) catalytic turnover), as well as estimates of the rates of the conformational transitions of phospho- and dephosphoenzyme (E1P → E2P and E2(K+) → E1 + K+). The results show that, compared with α1M32, the deletion of up to 40 residues (α1M40) further shifts the poise toward E1. Remarkably, further deletions (mutants α1M46, α1M49, and α1M56) reverse the effect, such that these mutants increasingly resemble the wild type α1. These results suggest novel intramolecular interactions involving domains within the N terminus that impact the manner in which the N terminus/M2-M3 loop regulatory domain interacts with the M4-M5 catalytic loop to effect E1 ↔ E2 transitions.

Ouabain-insensitivity of highly active Na+, K+-dependent adenosinetriphosphatase from rat kidney.

Abstract Highly purified preparations of Na + +K + -dependent adenosinetriphosphatase were isolated from rat kidney by two different procedures. The I 50 values for ouabain inhibition of the rat kidney enzyme at various stages of purification were determined to be essentially the same for all fractions tested (0.7 to 1.0 × 10 −4 M). These results suggest that the marked insensitivity of the rat enzyme to inhibition by cardiac glycosides is due to the primary structure of the enzyme, and not to some other component in the tissue.

Immunochemical comparison of cardiac glycoside-sensitive (lamb) and -insensitive (rat) kidney (Na+ + K+)-ATPase

The immunological cross-reactivity of the ouabain-sensitive lamb kidney and the ouabain-insensitive rat kidney (Na+ + K+)-ATPase (EC 3.6.1.37) was examined using polyclonal and monoclonal antibodies. Studies using rabbit antisera prepared against both the lamb kidney and rat kidney holoenzymes showed the existence of substantial antigenic differences as well as similarities between the holoenzymes and the respective denatured alpha and beta subunits of these two enzymes. Quantitation of the extent of cross-reactivity using holoenzyme-directed antibodies showed a 40-60% cross-reactivity. In addition, rabbit antisera monospecific to the purified, denatured alpha and beta subunits of the lamb kidney enzyme showed about a 50% cross-reactivity towards the respective subunit of the rat enzyme. In contrast to the cross-reactivity observed using the polyclonal antibodies, six monoclonal antibodies specific for the alpha subunit of the lamb holoenzyme exhibited no cross-reactivity with the rat holoenzyme. Four of these monoclonal antibodies, however, showed substantial cross-reactivity with rat alpha subunit as resolved by SDS-polyacrylamide gel electrophoresis. A fifth antibody did not bind to the denatured alpha subunit of either the lamb or the rat enzyme. Another monoclonal antibody (M7-PB-E9), which is specific for an epitope previously implicated in the regulation of both ATP and ouabain binding to (Na+ + K+)-ATPase (Ball, W.J., Jr. (1984) Biochemistry 2275-2281) was found to bind to the denatured lamb alpha but not to the rat alpha. This antibody has identified a region of the lamb alpha that has an altered amino acid sequence in the ouabain-insensitive rat enzyme. These immunological studies indicate that there are substantial antigenic differences between the lamb and rat kidney (Na+ + K+)-ATPases. The majority of these antigenic differences appear to be due to variations in the tertiary structures rather than to variations in the primary structures of the alpha subunits.