Rat brain and kidney cDNA libraries were constructed and screened with a cDNA insert corresponding to the mRNA for the sheep kidney Na+,K+-ATPase catalytic subunit. The alpha-subunit cDNAs isolated from the kidney library were derived from a single class of messenger RNA, and the brain cDNAs were derived from three classes of messenger RNA. The most abundant brain cDNA, which spans 5.1 kilobases, encodes the alpha(+) form of the enzyme. The second most abundant brain cDNA, which spans 3.65 kilobases, is identical with that of the kidney form and therefore encodes the alpha isoform. The third class of cDNA, which spans 3.55 kilobases, was present at low abundance and encodes an isoform of the alpha-subunit, designated alpha III, which has not been identified previously. The complete nucleotide sequence and deduced amino acid sequence for each of the brain and kidney cDNAs have been determined. In addition, we have identified a lysine-rich sequence that may function as a movable, ion-selective gate during cation binding and occlusion and have also identified several amino acid sequence variations that appear to explain some of the well-known species and tissue differences in cardiac glycoside sensitivity.

Molecular cloning of three distinct forms of the Na+,K+-ATPase alpha-subunit from rat brain.

Opening of the Mitochondrial Permeability Transition Pore Causes Depletion of Mitochondrial and Cytosolic NAD+and Is a Causative Event in the Death of Myocytes in Postischemic Reperfusion of the Heart

The role of humoral agents in volume expanded hypertension.

Publisher Summary This chapter discusses the molecular genetics of Na,K-ATPase The Na,K-ATPase is an integral membrane enzyme found in all cells of higher organisms and is responsible for the ATP-dependent transport of Na + and K + across the cell membrane The ion gradients formed by this enzyme are essential for the Na + -coupled transport of various nutrients, including glucose and amino acids, into cells movement of such ions as Ca 2+ and H + across the membrane, and osmotic balance and cell-volume regulation Protein synthesis and receptor-mediated endocytosis and recycling also depend on the resulting ion gradients Because two K + ions are pumped into the cell for every three Na + ions expelled, an electrochemical gradient is established In many instances, the information is more easily obtained using recombinant DNA techniques, and in some cases it can be obtained by no other means The power of this approach has clearly been demonstrated by recent advances in understanding Na, K-ATPase Na, K-ATPase also appears to play a role in determining the nutrient and ion composition of the cerebrospinal fluid and aqueous humor In addition, the enzyme is responsible for fluid movement across transport epithelia in the gastrointestinal tract, the nasotracheal lining, and the kidney Fluid reabsorption from the lung at birth also may depend in part on functioning of the Na, K-ATPase

Molecular genetics of Na,K-ATPase.

Na,K-ATPases from various species differ greatly in their sensitivity to cardiac glycosides such as ouabain. The sheep and human enzymes are a thousand times more sensitive than the corresponding ones from rat and mouse. To define the region of the alpha 1 subunit responsible for this differential sensitivity, chimeric cDNAs of sheep and rat were constructed and expressed in ouabain-sensitive HeLa cells. The construct containing the amino-terminal half of the rat alpha 1 subunit coding region and carboxyl-terminal half of the sheep conferred the ouabain-resistant phenotype to HeLa cells while the reverse construct did not. This indicates that the determinants involved in ouabain sensitivity are located in the amino-terminal half of the Na,K-ATPase alpha subunit. By use of site-directed mutagenesis, the amino acid sequence of the first extracellular domain (H1-H2) of the sheep alpha 1 subunit, Gln-Ala-Ala-Thr-Glu-Glu-Glu-Pro-Gln-Asn-Asp-Asn, was changed to that of the rat, Arg-Ser-Ala-Thr-Glu-Glu-Glu-Pro-Pro-Asn-Asp-Asp. When expressed in HeLa cells, this mutated sheep alpha 1 construct, like the rat/sheep chimera, was able to confer ouabain resistance to these cells. Furthermore, similar results were observed when HeLa cells were transfected with a sheep alpha 1 cDNA containing only two amino acid substitutions. This double mutation was a Gln-111----Arg and Asn-122----Asp change at the amino terminus and carboxyl terminus, respectively, of the H1-H2 extracellular region. The resistant cells, whether transfected with the rat alpha 1 cDNA, the rat/sheep chimera, or the mutant sheep alpha 1 cDNAs, exhibited identical biochemical characteristics including ouabain-inhibitable cell growth, 86Rb+ uptake, and Na,K-ATPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme.

The Sodium-Potassium Adenosine Triphosphatase: Pharmacological, Physiological and Biochemical Aspects

For an adequate evaluation of mitochondria from diseased hearts, basic characteristics of isolation, storage, media, ultrastructure and type of assay were first determined using mitochondria from normal animals. A proteinase procedure yielded mitochondria from small laboratory animals, with low respiratory control and marked permeability changes. The isolation medium yielding the most stable mitochondria with the highest respiratory control contained 0.18M KCl, 10mM EDTA, and 0.5% to 1% bovine serum albumin at pH 7.2. Heart failure in guinea pigs and rabbits was produced by varying degrees of stenosis of the ascending aorta. An aberration in respiratory control was found in mitochondria from hearts in severe failure. The quantitative differences between normal and experimental respiratory control values were greatest when the highest possible normal respiratory control levels were obtained. The difference between mitochondria prepared by a proteinase method from control and failing hearts was minimal. No ...

Reevaluation of Oxidative Phosphorylation in Cardiac Mitochondria from Normal Animals and Animals in Heart Failure

Enzymatic studies on isolated nucleoli of rat liver.

1. Dissociation constants for sodium and potassium of a site that modulates the rate of ouabain-(Na(+)+K(+))-ATPase interaction were applied to models for potassium activation of (Na(+)+K(+))-ATPase. The constants for potassium (0.213 mM) and for sodium (13.7 mM) were defined, respectively, as activation constant, K(a) and inhibitory constant, K(i).2. Tests of the one- and the two-equivalent site models, that describe sodium and potassium competition, revealed that neither model adequately predicts the activation effects of potassium in the presence of 100 or 200 mM sodium.3. The potassium-activation data, obtained at low potassium and high sodium, were explained by a two-nonequivalent site model where the dissociation constants of the first site are 0.213 mM for potassium and 13.7 mM for sodium. The second site was characterized by dissociation constants of 0.091 mM for potassium and 74.1 mM for sodium.4. The two-nonequivalent site model adequately predicted the responses to concentrations of potassium between 0.25 and 5 mM in the presence of 100-500 mM sodium. At lower sodium concentrations the predicted responses formed an upper limit for the function of observed activities. This limit was reached at lower concentrations of potassium and higher concentrations of sodium, which inferred saturation of the sodium-activation sites with sodium.5. Sodium-activation data were corrected for sodium interaction with potassium-activation sites by use of the two-nonequivalent site model for potassium activation. Tests of equivalent site models suggested that the corrected data for sodium activation may be most consistent with a model that has three-equivalent sites. Other multiequivalent site models (n = 2, 4, 5 or 6), however, cannot be statistically eliminated as possibilities. The three-equivalent site activation model was characterized by dissociation constants of 1.39 mM for sodium and 11.7 mM for potassium. The system theoretically would be half-maximally activated by 5.35 mM sodium in the absence of potassium.6. Derivation of the model for sodium activation assumed that the affinities of these sites for sodium and potassium are independent of cation interactions with the potassium-activation sites. Therefore, the kinetic descriptions for sodium and potassium effects form a composite model that is consistent with simultaneous transport of sodium and potassium.7. Predictions of the composite equation are in reasonable agreement with data obtained by variation of sodium (potassium = 10 mM), variation of potassium (sodium = 100 mM) and by simultaneous variation of sodium and potassium (sodium:potassium = 10). Sodium-activation data (2.5-20 mM sodium) also agree with predictions of the model in the presence of potassium concentrations which are thought to be present at the sodium-activation sites in vivo.8. The kinetic description for sodium (three-equivalent sites) and potassium (two-nonequivalent sites) activation of the transport-ATPase is in accord with the probable stoichiometric requirements of the sodium pump. The model is also in general agreement with other studies on intact transporting systems and (Na(+)+K(+))-ATPase in fragmented membrane preparations with respect to potassium activation, although there is a quantitative disagreement. The model for sodium activation, though consistent with data obtained by other studies on fragmented (Na(+)+K(+))-ATPase preparations, is in apparent variance with much of the data obtained for intact transporting systems. The description for potassium activation suggests that the rates of ouabain binding to (Na(+)+K(+))-ATPase are modulated by competition between sodium and potassium for one of the two potassium-activation sites.

A kinetic description for sodium and potassium effects on (Na++K+)‐adenosine triphosphatase: a model for a two‐nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation

George E. Lindenmayer

Papers

The Sodium-Potassium Adenosine Triphosphatase: Pharmacological, Physiological and Biochemical Aspects

Reevaluation of Oxidative Phosphorylation in Cardiac Mitochondria from Normal Animals and Animals in Heart Failure

Enzymatic studies on isolated nucleoli of rat liver.

A kinetic description for sodium and potassium effects on (Na++K+)‐adenosine triphosphatase: a model for a two‐nonequivalent site potassium activation and an analysis of multiequivalent site models for sodium activation

Section of biological and medical sciences: respiratory control and calcium transport in heart mitochondria from the cardiomyopathic syrian hamster*,†