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J H Rasmussen

Publications and source records attributed to J H Rasmussen.

11 recordsLinked to original sources
PubMed1998

Contribution to Tl+, K+, and Na+ binding of Asn776, Ser775, Thr774, Thr772, and Tyr771 in cytoplasmic part of fifth transmembrane segment in alpha-subunit of renal Na,K-ATPase.

The sequence Y771TLTSNIPEIT781P in the fifth transmembrane segment of the alpha-subunit of Na,K-ATPase is unique among cation pump proteins. Here, in search of the molecular basis for Na,K specificity, alanine and conservative substitutions were directed to six oxygen-carrying residues in this segment. The contribution of the residues to cation binding was estimated from direct binding of Tl+ [Nielsen, et al. (1998) Biochemistry 37, 1961-1968], K+ displacement of ATP binding at equilibrium, and Na+-dependent phosphorylation from ATP in the presence of oligomycin. As an intrinsic control, substitution of Thr781 had no effect on Tl+(K+) or Na+ binding. There are several novel observations from this work. First, the carboxamide group of Asn776 is equally important for binding Tl+(K+) or Na+, whereas a shift of the position of the carboxamide of Asn776 (Asn776Gln) causes a large depression of Na+ binding without affecting the binding of Tl+(K+). Second, Thr774 is important for Na+ selectivity because removal of the hydroxyl group reduces the binding of Na+ with no effect on binding of Tl+(K+). Removal of the methyl groups of Thr774 or Thr772 reduces binding of both Tl+(K+) and Na+, whereas the hydroxyl group of Thr772 does not contribute to cation binding. Furthermore, the hydroxyl groups of Ser775 and Tyr771 are important for binding both Tl+(K+) and Na+. The data suggest that rotating or tilting of the cytoplasmic part of the fifth transmembrane segment may adapt distances between coordinating groups and contribute to the distinctive Na+/K+ selectivity of the pump.

Alanine↗
PubMed1998

Structure-function relationships of E1-E2 transitions and cation binding in Na,K-pump protein.

Fully active Na,K-ATPase and lethal mutations can be expressed in yeast cells in yields allowing for equilibrium ATP binding, occlusion of T1+, K+ displacement of ATP, and Na(+)-dependent phosphorylation with determinations of affinity constants for binding and constants for the conformational equilibria. Removal of the charge and hydrophobic substitution of the phosphorylated residue (Asp369Ala) reveals an intrinsic high affinity for ATP binding (Kd 2.8 vs. 100 nM for wild type) and causes a shift of conformational equilibrium towards the E2 form. Substitution of Glu327, Glu779, Asp804 or Asp808 in transmembrane segments 4, 5, and 6 shows that each of these residues are essential for high-affinity occlusion of K+ and for binding of Na+. Substitution of other residues in segment 5 shows that the carboxamide group of Asn776 is important for binding of both K+ and Na+. Differential effects of the relevant mutations identify Thr774 as specific determinant of Na+ binding in the E1P[3Na] form, whereas Ser775 is a specific participant of high-affinity binding of the E2[2K] form, suggesting that these residues engage in formation of a molecular Na+/K+ switch. The position of the switch may be controlled by rotating or tilting the helix during the E1-E2 transition.

Amino Acid Sequence↗
PubMed1998

Structure-function relationships based on ATP binding and cation occlusion at equilibrium in Na,K-ATPase.

This work evaluates the results of measurements of equilibrium binding of ATP and cations in lethal or partially active mutations of Na,K-ATPase that were expressed at high yield in yeast cells. ATP binding studies allowed estimation of the expense in free energy required to position the gamma-phosphate in proximity of the carboxylate groups of the phosphorylated residue Asp369 and the role of this residue in governing long range E1-E2 transitions. An arginine residue (Arg546) appearing to be involved in ATP binding has been identified. Wild type yeast enzyme was capable of occluding two T1(+)-ions per ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(T1+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. The substitutions to Glu327(Gln,Asp), Asp804(Asn,Glu), Asp808(Asn,Glu) and Glu779(Asp) completely abolished occlusion or severely reduced the affinity for T1+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to one T1+ ion per alpha 1 beta 1 unit with a 3-fold decrease of the apparent affinity for the ion (Kd(T1+) = 24 +/- 8 mM). These carboxylate groups in transmembrane segments 4, 5, and 6 therefore appear to be essential for high affinity occlusion of K(+)-ions.

Adenosine Triphosphate↗
PubMed1997

Identification of Asp804 and Asp808 as Na+ and K+ coordinating residues in alpha-subunit of renal Na,K-ATPase.

Mutations to Asp804 and Asp808 in the alpha-subunit almost abolish Na,K-ATPase activity, but high-affinity binding of [3H]ATP or [3H]ouabain at equilibrium and E1-E2 transitions are preserved. Titration of K+-ion displacement of [3H]ATP or [3H]ouabain shows that the mutations interfere with occlusion of K+ in the E2[2K] conformation. Reduced phosphorylation levels or affinities for Na+ in presence of oligomycin indicate that Asp804 and Asp808 also contribute to coordination of Na+ in the E1P[3Na] form. Demonstration of alternate interactions of Na+ or K+ with Asp804 and Asp808 support the notion of cation binding in a ping-pong sequence in catalytic models of Na,K-pumping.

Adenosine Triphosphate↗
PubMed1996

Consequences of mutations to the phosphorylation site of the alpha-subunit of Na, K-ATPase for ATP binding and E1-E2 conformational equilibrium.

Expression of Na, K-ATPase in yeast allowed targeting of alpha beta-units with lethal substitutions at the phosphorylation site alpha 1 (D369N) beta 1 and alpha 1 (D369A) beta 1 at the cell surface at the same concentration of alpha-subunit and [3H] ouabain binding sites as for wild type Na, K-ATPase. Phosphorylation and reaction with vanadate were abolished, and the mutations had no Na, K-ATPase or K-phosphatase activity. Binding of [3H]-ATP at equilibrium revealed an intrinsic high affinity of the D369A mutation for ATP (KD = 2.8 nM) that was 39-fold higher than for wild type Na, K-ATPase (KD = 109 nM). The affinities for ADP were unaffected, indicating that the negative charge at residue 369 determines the contribution of the gamma-phosphate to the free energy of ATP binding. Analysis of the K(+)-ATP antagonism showed that the reduction of charge and hydrophobic substitution at Asp369 of the alpha-subunit caused a large shift in conformational equilibrium toward the E2-form. This was accompanied by a large increase in affinity for [3H] ouabain in Mg2+ medium with KD = 4.9 nM for D369A compared to KD = 51 nM for D369N and KD = 133 nM for wild type, and [3H] ouabain binding (KD = 153 nM) to D369A was detectable even in absence of Mg2+. In addition to its function as receptor of the gamma-phosphate of ATP, Asp369 has important short-range catalytic functions in modulating the affinity for ATP and long-range functions in governing the E1-E2 transitions which are coupled to reorientation of cation sites and changes in affinity for digitalis glycosides.

Adenosine Triphosphate↗
PubMed1996

Expression in high yield of pig alpha 1 beta 1 Na,K-ATPase and inactive mutants D369N and D807N in Saccharomyces cerevisiae.

Studies of structure-function relationships in Na,K-ATPase require high yield expression of inactive mutations in cells without endogenous Na,K-ATPase activity. In this work we developed a host/vector system for expression of fully active pig Na,K-ATPase as well as the inactive mutations D369N and D807N at high levels in Saccharomyces cerevisiae. The alpha 1- and beta 1-subunit cDNAs were inserted into a single 2-microns-based plasmid with a high and regulatable copy number and strong galactose-inducible promoters allowing for stoichiometric alterations of gene dosage. The protease-deficient host strain was engineered to express high levels of GAL4 transactivating protein, thereby causing a 10-fold increase in expression to 32,500 +/- 3,000 [3H]ouabain sites/cell. In one bioreactor run 150-200 g of yeast were produced with 54 +/- 5 micrograms of Na,K-pump protein/g of cells. Through purification in membrane bound form the activity of the recombinant Na,K-ATPase was increased to 42-50 pmol/mg of protein. The Na,K dependence of ATP hydrolysis and the molar activity (4,500-7,000 min-1) were close to those of native pig kidney Na,K-ATPase. Mutations to the phosphorylation site (D369N) or presumptive cation sites (D807N), both devoid of Na,K-ATPase activity, were expressed in the yeast membrane at the same alpha-subunit concentration and [3H]ouabain binding capacity as the wild type Na,K-ATPase. The high yield and absence of endogenous activity allowed assay of [3H]ATP binding at equilibrium, demonstrating a remarkable 18-fold increase in affinity for ATP in consequence of reducing the negative charge at the phosphorylation site (D369N).

Adenosine Triphosphate↗