Identification of H+/K(+)-ATPase alpha,beta-heterodimers.

Glutaraldehyde treatment of the C12E8 solubilized H+/K(+)-ATPase crosslinks the catalytic subunit with an apparent molecular mass of 94 kDa in SDS polyacrylamide gels into two Coomassie stained particles migrating at approx. 147 and 173 kDa. The subunit composition of these particles was determined from the comparative distribution of FITC fluorescence, wheat germ agglutinin and anti-beta antibody reactivity in control and crosslinked preparations. FITC exclusively labelled the catalytic monomer of the native preparation and its fluorescence was initially distributed into two broad bands centered at approx. 147 and 173 kDa after crosslinking. These fluorescent bands coincided with the Coomassie stained particles. A glycoprotein(s) detected by wheat germ agglutinin reactivity was present in diffuse areas between 65 and 86 kDa and 95 to 134 kDa in the control preparation. This area was also labelled by the anti-beta antibodies. With crosslinking, the distribution of the wheat germ agglutinin reactive protein and anti-beta antibodies coincided with the crosslinked particles labelled by FITC. The presence of both the catalytic monomer and the beta subunit glycoprotein in the crosslinked particles indicated that these proteins were closely associated in the C12E8 solution. This suggests that the minimal structural particle of the H+/K(+)-ATPase is an alpha,beta-heterodimer.     

Combined allosteric and competitive interaction between extracellular Na(+) and K(+) during ion transport by the alpha(1), alpha(2), and alpha(3) isoforms of the Na, K-ATPase

A combined allosteric and competitive model describes the interaction between extracellular Na(+) and Rb(+) during ion transport mediated by the Na, K-ATPase. The model was developed from experiments based on (86)Rb uptake by whole cells transfected with rat isoforms of the enzyme. In the absence of Na(+), only a single transport site for extracellular Rb(+) exists. After the occupation of the Na(+)-specific allosteric site, the Rb(+) transport pocket opens to allow occupation by an additional Rb(+) and the subsequent transport of the two Rb(+) ions into the cells. Na(+) can also directly compete with Rb(+) for binding to at least one of the transport sites. While the model derived here applies to each of the three rat isoforms of the Na, K-ATPase expressed in HeLa cells, subtle differences exist among the isoforms. The alpha(3)* isoform has an increased intrinsic affinity for Rb(+) and a lower affinity for the allosteric Na(+) site than alpha(1) or alpha(2)*. The stimulation of uptake observed according to the best-fit model is due to the displacement by Rb(+) of inhibitory Na(+) bound to the transport site.     

Inactivation of chloroplast H(+)-ATPase by modification of Lys beta 359, Lys alpha 176 and Lys alpha 266.

Treatment of isolated, latent chloroplast ATPase with pyridoxal-5-phosphate (pyridoxal-P) in presence of Mg2+ causes inhibition of dithiothreitol-activated plus heat-activated ATP hydrolysis. The amount of [3H]pyridoxal-P bound to chloroplast coupling factor 1 (CF1) was estimated to run up to 6 +/- 1 pyridoxal-P/enzyme, almost equally distributed between the alpha- and beta-subunits. Inactivation, however, is complete after binding of 1.5-2 pyridoxal-P/CF1, suggesting that two covalently modified lysines prevent the activation of the enzyme. ADP as well as ATP in presence of Mg2+ protects the enzyme against inactivation and concomittantly prevents incorporation of a part of the 3H-labeled pyridoxal-P into beta- and alpha-subunits. Phosphate prevents labeling of the alpha-subunit, but has only a minor effect on protection against inactivation. The data indicate a binding site at the interface between the alpha- and beta-subunits. Cleavage of the pyridoxal-P-labeled subunits with cyanogen bromide followed by sequence analysis of the labeled peptides led to the detection of Lys beta 359, Lys alpha 176 and Lys alpha 266, which are closely related to proposed nucleotide-binding regions of the alpha- and beta-subunits.     

The Na(+)-K(+)-ATPase alpha2-subunit isoform modulates contractility in the perinatal mouse diaphragm

This study uses genetically altered mice to examine the contribution of the Na⁺-K⁺-ATPase ₂ catalytic subunit to resting potential, excitability, and contractility of the perinatal diaphragm. The ₂ protein is reduced by 38% in ₂-heterozygous and absent in ₂-knockout mice, and ₁-isoform is upregulated 1.9-fold in ₂-knockout. Resting potentials are depolarized by 0.8–4.0 mV in heterozygous and knockout mice. Action potential threshold, overshoot, and duration are normal. Spontaneous firing, a developmental function, is impaired in knockout diaphragm, but this does not compromise its ability to fire evoked action potential trains, the dominant mode of activation near birth. Maximum tetanic force, rate of activation, force-frequency and force-voltage relationships, and onset and magnitude of fatigue are not changed. The major phenotypic consequence of reduced ₂ content is that relaxation from contraction is 1.7-fold faster. This finding reveals a distinct cellular role of the ₂-isoform at a step after membrane excitation, which cannot be restored simply by increasing ₁ content. Na⁺/Ca²⁺ exchanger expression decreases in parallel with ₂-isoform, suggesting that Ca²⁺ extrusion is affected by the altered ₂ genotype. There are no major compensatory changes in expression of sarcoplasmic reticulum Ca²⁺-ATPase, phospholamban, or plasma membrane Ca²⁺-ATPase. These results demonstrate that the Na⁺-K⁺-ATPase ₁-isoform alone is able to maintain equilibrium K⁺ and Na⁺ gradients and to substitute for ₂-isoform in most cellular functions related to excitability and force. They further indicate that the ₂-isoform contributes significantly less at rest than expected from its proportional content but can modulate contractility during muscle contraction. Na⁺-K⁺-ATPase ₂ catalytic subunit; heterozygous mice; knockout mice; resting potential     

C(alpha)-hydroxymethyl methionine: synthesis, optical resolution and crystal structure of its (+)-N(alpha)-benzoyl derivative.

(R, S)-Methionine was transformed into C(alpha)-hydroxymethyl methionine by a route involving C(alpha)-hydroxymethylation of 2-phenyl-4-methylthioethyl-5-oxo-4,5-dihydro-1,3-oxazole. The absolute configuration of (-)-C(alpha)-hydroxymethyl methionine was elucidated to be (S) by chemical correlation with (S) (-)-C(alpha)-ethyl serine. Absolute structure determination (by single crystal X-ray diffraction) on N(alpha)-benzoyl-C(alpha)-hydroxymethyl methionine confirmed the (R)-configuration for the (+)-enantiomer. In addition, the X-ray diffraction analysis showed that the C(alpha,alpha)-disubstituted glycyl residue adopts the fully extended (C5) conformation.     

Postnatal changes in an alpha subunit isoform, alpha(S), of Na+,K(+)-ATPase in the submandibular gland of rats

The alpha and alpha(+) isoforms of Na+,K(+)-ATPase were isolated from the kidney and brain of rats and purified. Their antisera were raised to analyze the alpha isoforms in rat tissues. We found that the submandibular gland (SMG) contains a new immunoreactive alpha subunit isoform, designated alpha(S) in this report, in addition to alpha identical with those found in the kidney or brain. The new alpha(S) strongly reacted with anti-alpha-antiserum but to a much lesser extent with anti-alpha(+)-antiserum. The alpha(S) had a slightly lower molecular weight (approximately 90,000) than the brain and kidney alpha isoforms. Various fractions of SMG tissues were added to the SMG microsomes and incubated in order to test whether or not the alpha(S) is formed artificially; no increase of alpha(S) was observed by these treatments, suggesting that the alpha(S) was not the product formed from alpha during the preparation of microsome sample, but was rather a protein originally present in the SMG. The alpha(S) protein was not detected in the SMG of 2- or 5-week-old rats, but it gradually increased in rats older than 8 weeks, reaching the maximum in 30-week-old animals. The Na+,K(+)-ATPase activity in the SMG increased concomitantly with the increase of alpha(S), indicating that Na+,K(+)-ATPase comprising alpha(S) also shows enzyme activity; it is speculated that alpha(S) may have some unique and unknown function(s) in older rats.     

The Na(+)-K(+)-ATPase alpha2 gene and trainability of cardiorespiratory endurance: the HERITAGE family study.

The Na(+)-K(+)-ATPase plays an important role in the maintenance of electrolyte balance in the working muscle and thus may contribute to endurance performance. This study aimed to investigate the associations between genetic variants at the Na(+)-K(+)-ATPase alpha2 locus and the response (Delta) of maximal oxygen consumption (VO(2 max)) and maximal power output (W(max)) to 20 wk of endurance training in 472 sedentary Caucasian subjects from 99 families. VO(2 max) and W(max) were measured during two maximal cycle ergometer exercise tests before and again after the training program, and restriction fragment length polymorphisms at the Na(+)-K(+)-ATPase alpha2 (exons 1 and 21-22 with Bgl II) gene were typed. Sibling-pair linkage analysis revealed marginal evidence for linkage between the alpha2 haplotype and DeltaVO(2 max) (P = 0.054) and stronger linkages between the alpha2 exon 21-22 marker (P = 0.005) and alpha2 haplotype (P = 0.003) and DeltaW(max). In the whole cohort, DeltaVO(2 max) in the 3.3-kb homozygotes of the exon 1 marker (n = 5) was 41% lower than in the 8.0/3.3-kb heterozygotes (n = 87) and 48% lower than in the 8.0-kb homozygotes (n = 380; P = 0.018, adjusted for age, gender, baseline VO(2 max), and body weight). Among offspring, 10.5/10.5-kb homozygotes (n = 14) of the exon 21-22 marker showed a 571 +/- 56 (SE) ml O(2)/min increase in VO(2 max), whereas the increases in the 10.5/4.3-kb (n = 93) and 4.3/4.3-kb (n = 187) genotypes were 442 +/- 22 and 410 +/- 15 ml O(2)/min, respectively (P = 0.017). These data suggest that genetic variation at the Na(+)-K(+)-ATPase alpha2 locus influences the trainability of VO(2 max) in sedentary Caucasian subjects.     

Identifications of 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 beta-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 alpha-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 beta-tetrol in cerebrotendinous xanthomatosis

The bile alcohols present in the feces of a patient with cerebrotendinous xanthomatosis were studied. Three bile alcohols which are different from any known natural bile alcohol were isolated as minor components of the fecal bile alcohol fraction. The structures of these compounds were established as 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 23 beta-tetrol, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 alpha-tetrol, and 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 24 beta-tetrol by comparison with synthetic samples.