title: Fluorescence spectroscopic studies of pressure effects on Na+,K(+)-ATPase reconstituted into phospholipid bilayers and model raft mixtures.
abstract: To contribute to the understanding of membrane protein function upon application of pressure as relevant for understanding, for example, the physiology of deep sea organisms or for baroenzymological biotechnical processes, we investigated the influence of hydrostatic pressure on the activity of Na+,K+-ATPase enriched in the plasma membrane from rabbit kidney outer medulla using a kinetic assay that couples ATP hydrolysis to NADH oxidation. The data show that the activity of Na+,K+-ATPase is reversibly inhibited by pressures below 2 kbar. At higher pressures, the enzyme is irreversibly inactivated. To be able to explore the effect of the lipid matrix on enzyme activity, the enzyme was also reconstituted into various lipid bilayer systems of different chain length, conformation, phase state, and heterogeneity including model raft mixtures. To yield additional information on the conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined as well. Incorporation of the enzyme leads to a significant increase of the lipid chain order. Generally, similar to the enzyme activity in the natural plasma membrane, high hydrostatic pressures lead to a decline of the activity of the enzyme reconstituted into the various lipid bilayer systems, and in most cases, a multi-phasic behavior is observed. Interestingly, in the low-pressure region, around 100 bar, a significant increase of activity is observed for the enzyme reconstituted into DMPC and DOPC bilayers. Above 100-200 bar, this activity enhancement is followed by a steep decrease of activity up to about 800 bar, where a more or less broad plateau value is reached. The enzyme activity decreases to zero around 2 kbar for all reconstituted systems measured. A different scenario is observed for the effect of pressure on the enzyme activity in the model raft mixture. The coexistence of liquid-ordered and liquid-disordered domains with the possibility of lipid sorting in this lipid mixture leads to a reduced pressure sensitivity in the medium-pressure range. The decrease of ATPase activity may be induced by an increasing hydrophobic mismatch, leading to a decrease of the conformational dynamics of the protein and eventually subunit rearrangement. High pressures, above about 2.2 kbar, irreversibly change protein conformation, probably because of the dissociation and partial unfolding of the subunits.
authors.affiliation: Department of Chemistry, Physical Chemistry I, and Biophysical Chemistry, University of Dortmund, Otto-Hahn Strasse 6, D-44227 Dortmund, Germany.
authors.forename: Ewa
authors.initials: E
authors.lastname: Powalska
authors.forename: Sascha
authors.initials: S
authors.lastname: Janosch
authors.forename: Evamaria
authors.initials: E
authors.lastname: Kinne-Saffran
authors.forename: Rolf K H
authors.initials: RK
authors.lastname: Kinne
authors.forename: C F L
authors.initials: CF
authors.lastname: Fontes
authors.forename: Julio A
authors.initials: JA
authors.lastname: Mignaco
authors.forename: Roland
authors.initials: R
authors.lastname: Winter
mesh_terms: D015081:2-Naphthylamine / Q000031:analogs & derivatives / Q000737:chemistry; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002784:Cholesterol / Q000737:chemistry; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry; D007668:Kidney / Q000201:enzymology; D007848:Laurates / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D010713:Phosphatidylcholines / Q000737:chemistry; D011312:Pressure; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013109:Sphingomyelins / Q000737:chemistry; D013552:Swine
chemical_list: D005456:Fluorescent Dyes; D007848:Laurates; D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D013109:Sphingomyelins; D002784:Cholesterol; D015081:2-Naphthylamine; D000254:Sodium-Potassium-Exchanging ATPase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; C065580:laurdan