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P L Jorgensen

Publications and source records attributed to P L Jorgensen.

At least 19 recordsLinked to original sources
PubMed2003

Trafficking of Na,K-ATPase fused to enhanced green fluorescent protein is mediated by protein kinase A or C.

Fusion of enhanced green fluorescent protein (EGFP) to the C-terminal of rat Na,K-ATPase a1-subunit is introduced as a novel procedure for visualizing trafficking of Na,K-pumps in living COS-1 renal cells in response to PKA or PKC stimulation. Stable, functional expression of the fluorescent chimera (Na,K-EGFP) was achieved in COS-1 cells using combined puromycin and ouabain selection procedures. Na,K-pump activities were unchanged after fusion with EGFP, both in basal and regulated states. In confocal laser scanning and fluorescence microscopes, the Na,K-EGFP chimera was distributed mainly along the plasma membrane of COS cells. In unstimulated COS cells, Na,K-EGFP was also present in lysosomes and in vesicles en route from the endoplasmic reticulum to the plasma membrane, but it was almost absent from recycling endosomes labelled with fluorescent transferrin. After activation of protein kinase A or C, the density of co-localizing Na,K-EGFP and transferrin vesicles was increased 3-4-fold, while the ouabain-sensitive 86Rb uptake was reduced by 22%. Simultaneous activation of PKA and PKC had additive effects with a 6-fold increase of co-localization and a 38% reduction of 86Rb uptake. Responses of similar magnitude were seen after inhibition of protein phosphatase by okadaic acid. Reduction of the amount of Na,K-ATPase in surface plasma membranes through internalization in recycling endosomes may thus in part explain a decrease in Na,K-pump activity following protein kinase activation or protein phosphatase inhibition.

Animals↗
PubMed2003

Homology modeling of Na,K-ATPase: a putative third sodium binding site suggests a relay mechanism compatible with the electrogenic profile of Na+ translocation.

Identification of the third Na(+) binding site would be crucial in interpretation of the electrophysiological behavior of Na,K-ATPase. To address this question a three-dimensional homology model of Na,K-ATPase was built from the known crystallographic structure of Ca-ATPase (1EUL). Phe760, which is conserved in virtually all Ca-ATPases, is replaced by Ser768 in Na,K-ATPase, resulting in a small cavity between M4, M5, and M6. A partially hydrated Na(+) ion can be bound at this third site on the cytoplasmic side of cation binding sites 1 and 2. This leads to the proposal that the conductance of the "third Na(+)" ion across approximately 70% of the membrane dielectric may be achieved by adding up the passage of one Na(+) ion from the described cytoplasmic cavity to cation site 1 and the further conductance of the previously bound Na(+) ion from cation site 1 to the extracellular phase. This relay mechanism may therefore be compatible with the electrogenic profile of Na(+) translocation.

Amino Acid Sequence↗
PubMed2001

Structure-function relationships of Na(+), K(+), ATP, or Mg(2+) binding and energy transduction in Na,K-ATPase.

The focus of this article is on progress in establishing structure-function relationships through site-directed mutagenesis and direct binding assay of Tl(+), Rb(+), K(+), Na(+), Mg(2+) or free ATP at equilibrium in Na,K-ATPase. Direct binding may identify residues coordinating cations in the E(2)[2K] or E(1)P[3Na] forms of the ping-pong reaction sequence and allow estimates of their contributions to the change of Gibbs free energy of binding. This is required to understand the molecular basis for the pronounced Na/K selectivity at the cytoplasmic and extracellular surfaces. Intramembrane Glu(327) in transmembrane segment M4, Glu(779) in M5, Asp(804) and Asp(808) in M6 are essential for tight binding of K(+) and Na(+). Asn(324) and Glu(327) in M4, Thr(774), Asn(776), and Glu(779) in 771-YTLTSNIPEITP of M5 contribute to Na(+)/K(+) selectivity. Free ATP binding identifies Arg(544) as essential for high affinity binding of ATP or ADP. In the 708-TGDGVND segment, mutations of Asp(710) or Asn(713) do not interfere with free ATP binding. Asp(710) is essential and Asn(713) is important for coordination of Mg(2+) in the E(1)P[3Na] complex, but they do not contribute to Mg(2+) binding in the E(2)P-ouabain complex. Transition to the E(2)P form involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713) and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369).

Adenosine Triphosphate↗
PubMed2001

Role of conserved TGDGVND-loop in Mg2+ binding, phosphorylation, and energy transfer in Na,K-ATPase.

In the P-domain, the 369-DKTGTLT and the 709-GDGVNDSPALKK segment are highly conserved during evolution of P-type E1-E2-ATPase pumps irrespective of their cation specificities. The focus of this article is on evaluation of the role of the amino acid residues in the P domain of the alpha subunit of Na,K-ATPase for the E1P[3Na]--> E2P[2Na] conversion, the K+-activated dephosphorylation, and the transmission of these changes to and from the cation binding sites. Mutations of residues in the TGDGVND loop show that Asp710 is essential, and Asn713 is important, for Mg2+ binding and formation of the high-energy MgE1P[3Na] intermediate. In contrast Asp710 and Asp713 do not contribute to Mg2+ binding in the E2P-ouabain complex. Transition to E2P thus involves a shift of Mg2+ coordination away from Asp710 and Asn713 and the two residues become more important for K+-activated hydrolysis of the acyl phosphate bond at Asp369. Transmission of structural changes between the P-domain and cation sites in the membrane domain is evaluated in light of the protein structure, and the information from proteolytic or metal-catalyzed cleavage and mutagenesis studies.

Adenosine Triphosphate↗
PubMed2001

Aspects of gene structure and functional regulation of the isozymes of Na,K-ATPase.

Gaps in our understanding of the complex regulated expression of isozymes of Na,K-ATPase and the diverse systems for posttranslational modification and short term regulation of active Na,K-transport in animals and humans are the main problems in comprehensive Na,K-pump physiology. In mammalian genomes, the genes of four alpha-subunit and at least three beta-subunit isoforms of Na,K-ATPase are identified and two gamma-subunits are expressed in kidney. The isoforms combine in a number of Na,K-ATPase isozymes that are expressed in a tissue and cell specific manner. Models of the molecular mechanism of regulation of these isozymes have become more reliable due to progress in understanding the three-dimensional protein structure and conformational transitions mediating transfer of energy from the P-domain to intramembrane Na+ and K+ binding sites.

Animals↗
PubMed2000

Importance of conserved alpha -subunit segment 709GDGVND for Mg2+ binding, phosphorylation, and energy transduction in Na,K-ATPase.

The segment (708)TGDGVNDSPALKK(720) in the alpha-subunit P domain of Na,K-ATPase is highly conserved among cation pumps, but little is known about its role in binding of Mg(2+) or ATP and energy transduction. Here, 11 mutations of polar residues are expressed at reduced temperature in yeast with preserved capacities for high affinity binding of ouabain and ATP, whereas the Thr(708) --> Ser mutation and alterations of Asp(714) abolish all catalytic reactions. In mutations of Asp(710) and Asn(713), ATP affinity is preserved or increased, whereas Na,K-ATPase activity is severely reduced. Assay of phosphorylation from ATP in the presence of oligomycin shows that Asp(710) contributes to coordination of Mg(2+) during transfer of gamma-phosphate to Asp(369) in the high energy Mg.E(1)P[3Na] intermediate and that Asn(713) is involved in these processes. In contrast, Asp(710) and Asp(713) do not contribute to Mg(2+) binding in the E(2)P.ouabain complex. Transition to E(2)P thus involves a shift of Mg(2+) coordination away from Asp(710) and Asn(713), and the two residues become more important for hydrolysis of the acyl phosphate bond at Asp(369). The Asp(710) --> Ala mutation blocks interaction with vanadate, whereas Asn(713) --> Ala interferes with phosphorylation from P(i) of the E(2).ouabain complex, showing that the GDGVND segment is required for stabilization of the transition state and for the phosphorylation reaction. The Asp(710) --> Ala mutation also interferes with transmission of structural changes to the ouabain site and reduces the affinity for binding of Tl(+) 2- to 3-fold, suggesting a role in transmission of K(+) stimulation of phospho-enzyme hydrolysis from transmembrane segment 5 to the P domain.

Amino Acid Sequence↗
PubMed1999

Pullulanase type I from Fervidobacterium pennavorans Ven5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinant enzyme.

The gene encoding the type I pullulanase from the extremely thermophilic anaerobic bacterium Fervidobacterium pennavorans Ven5 was cloned and sequenced in Escherichia coli. The pulA gene from F. pennavorans Ven5 had 50.1% pairwise amino acid identity with pulA from the anaerobic hyperthermophile Thermotoga maritima and contained the four regions conserved among all amylolytic enzymes. The pullulanase gene (pulA) encodes a protein of 849 amino acids with a 28-residue signal peptide. The pulA gene was subcloned without its signal sequence and overexpressed in E. coli under the control of the trc promoter. This clone, E. coli FD748, produced two proteins (93 and 83 kDa) with pullulanase activity. A second start site, identified 118 amino acids downstream from the ATG start site, with a Shine-Dalgarno-like sequence (GGAGG) and TTG translation initiation codon was mutated to produce only the 93-kDa protein. The recombinant purified pullulanases (rPulAs) were optimally active at pH 6 and 80 degrees C and had a half-life of 2 h at 80 degrees C. The rPulAs hydrolyzed alpha-1,6 glycosidic linkages of pullulan, starch, amylopectin, glycogen, alpha-beta-limited dextrin. Interestingly, amylose, which contains only alpha-1,4 glycosidic linkages, was not hydrolyzed by rPulAs. According to these results, the enzyme is classified as a debranching enzyme, pullulanase type I. The extraordinary high substrate specificity of rPulA together with its thermal stability makes this enzyme a good candidate for biotechnological applications in the starch-processing industry.

Amino Acid Sequence↗
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

Importance of intramembrane carboxylic acids for occlusion of K+ ions at equilibrium in renal Na,K-ATPase.

Site-directed mutagenesis and assay of Rb+ and Tl+ occlusion in recombinant Na,K-ATPase from yeast were combined to establish structure-function relationships of amino acid side chains involved in high-affinity occlusion of K+ in the E2[2K] form. The wild-type yeast enzyme was capable of occluding 2 Rb+ or Tl+ ions/ouabain binding site or alpha 1 beta 1 unit with high apparent affinity (Kd(Tl+) = 7 +/- 2 microM), like the purified Na,K-ATPase from pig kidney. Mutations of Glu327(Gln,Asp), Asp804(Asn, Glu), Asp808(Asn, Glu) and Glu779(Asp) abolished high-affinity occlusion of Rb+ or Tl+ ions. The substitution of Glu779 for Gln reduced the occlusion capacity to 1 Tl+ ion/alpha 1 beta 1-unit with a 3-fold decrease of the apparent affinity for the ion (Kd(Tl+) = 24 +/- 8 microM). These effects on occlusion were closely correlated to effects of the mutations on K0.5(K+) for K+ displacement of ATP binding. Each of the four carboxylate residues Glu327, Glu779, and Asp804 or Asp808 in transmembrane segments 4, 5, and 6 is therefore essential for high-affinity occlusion of K+ in the E2[2K] form. These residues either may engage directly in cation coordination or they may be important for formation or stability of the occlusion cavity.

Adenosine Triphosphate↗
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

Regulation of Ca2+-activated K+ channel from rabbit distal colon epithelium by phosphorylation and dephosphorylation.

The Ca2+-activated maxi K+ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na+ reabsorption from the intestinal lumen. Previous measurements of these basolateral K+ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca2+ with a K0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca2+. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K+ channels lost their high sensitivity to Ca2+, yet they could still be activated by high concentrations of Ca2+ (10 micron). Furthermore, the high sensitivity to Ca2+ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30-120 sec for dephosphorylation and of 10-280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca2+ of the maxi K+ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca2+ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca2+ sensitivities for maxi K+ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems.

Animals↗
PubMed1995

Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei.

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl- buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 A. The crystals belong to the trigonal lattice type with the spacegroup P3(1)21 or P3(2)21, respectively, have the cell dimensions a = b = 96.8 A, c = 196.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals have a theoretical packing density of 2.7 A3/Da, assuming one molecule with a molecule weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset.

Archaea↗
PubMed1995

Isolation and characterization of a heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene in Escherichia coli.

The gene encoding an extremely heat-stable pullulanase from the hyperthermophilic archaeon Pyrococcus woesei was cloned and expressed in Escherichia coli. Purification of the enzyme to homogeneity was achieved after heat treatment of the recombinant E. coli cells, affinity chromatography on a maltotriose-coupled Sepharose 6B column, and anion-exchange chromatography on Mono Q. The pullulanase, which was purified 90-fold with a final yield of 15%, is composed of a single polypeptide chain with a molecular mass of 90 kDa. The enzyme is optimally active at 100 degrees C and pH 6.0 and shows 40% activity at 120 degrees C. Enzyme activation up to 370% is achieved in the presence of calcium ions and reducing agents such as beta-mercaptoethanol and dithiothreitol, whereas N-bromosuccinimide and alpha-cyclodextrin are inhibitory. The high rigidity of the heat-stable enzyme is demonstrated by fluorescence spectroscopic studies in the presence of denaturing agents such as sodium dodecyl sulfate. At temperatures above 80 degrees C, the enzyme seems to switch from the compact to the unfolded form, which is accompanied by an apparent shift in the molecular mass from 45 to 90 kDa.

Archaea↗
PubMed1981

Conformational changes of renal sodium plus potassium ion-transport adenosine triphosphatase labeled with fluorescein.

We studied conformational changes of purified renal sodium plus potassium ion-transport adenosine triphosphatase (ATP phosphohydrolase, EC 3.6.1.3) labeled with fluorescein isothiocyanate. Fluorescein covalently binds to the alpha-subunit of the enzyme and inhibits the ATPase but not the p-nitrophenylphosphatase activity. Four unphosphorylated and three phosphorylated conformations were distinguished by the level of fluorescence and by the rate of its change (relative fluorescence is shown in percentages). Fluorescence of the ligand-free form (E1, 100%) was increased by Na+ (E1.Na form, 103%) and quenched by K+ (E2.K, 78%) at a site of high affinity (K0.5 for K+ = 0.07 mM). Mg2+ did not alter fluorescence of E1 or E1.Na but raised that of E2.K (E2.K.Mg form, 85-90%). Addition of excess Na+ to the E2.K.Mg form restored high fluorescence but the rate of transition from E2.K.Mg to E1.Na became progressively slower with increasing Mg2+ concentration. Two phosphorylated conformations, (E2-P).Mg (82%) and (E2-P).Mg.K (82%) were differentiated by a faster turnover of the latter form. A third conformation, (E2-P).Mg.ouabain, had the lowest fluorescence (56%) and its formation allowed the binding of ouabain to the phosphoenzyme. Reversible blocking of sulfhydryl groups with thimerosal inhibited the formation of E2.K and (E2-P).Mg.ouabain but not that of the other conformations of the fluorescein-enzyme. The thimerosal-treated fluorescein-enzyme retained K+-p-nitrophenylphosphatase activity, inhibition of this activity by ouabain and ouabain binding. The unphosphorylated enzyme had low (K0.5 = 1.2 mM) and the phosphoenzyme had high affinity (K0.5 = 0.03 - 0.09 mM) for Mg2+ in the absence of nucleotides. Since low and high affinity for Mg2+ alternates as the enzyme turns over, Mg2+ may be bound and released sequentially during the catalytic cycle.

Animals↗