Electron Microscope Analysis of Two-Dimensional Crystals of Membrane-Bound Na,K-ATPase

Maunsbach, Arvid B.; Skriver, Elisabeth; Herbert, Hans; Lethj⊘rgensen, Peter

doi:10.1016/s0070-2161(08)60558-4

Cited by 3 publications

(1 citation statement)

References 3 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Nevertheless, future studies should be done to see if an acylphosphate other than Asp ~9 can be identified in the phosphorylated 83-kDa peptide. That membrane-bound Na+,K+-ATPase is an oligomer with interacting c~,13-protomers has been established [5,[20][21][22][23][24][25][26][27][28] independent of the questions regarding its reaction mechanism and its half-site reactivity. Our data presented here, along with previous studies of our laboratory and others [5,[20][21][22]29,30], provide unambiguous evidence for its halfsite reactivity.…”

Section: Discussionmentioning

confidence: 99%

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na⁺, K⁺‐ATPase

et al. 1996

View full text Add to dashboard Cite

Purified kidney Na+,K+-ATPase whose a-subunit is cleaved by chymotrypsin at Leu~66-Ala 267, loses ATPase activity but forms the phosphoenzyme intermediate (EP) from ATP. When EP formation was correlated with extent of s-cleavage in the course of proteolysis, total EP increased with time before it declined. The magnitude of this rise indicated doubling of the number of phosphorylation sites after cleavage. Together with previous findings, these data establish that half of the ~-subunits of oligomeric membrane-bound enzyme are dormant and that interaction of the N-terminal domain of c¢-subunit with its phosphorylation domain causes this half-site reactivity. Evidently, disruption of this interaction by proteolysis abolishes overall activity while it opens access to phosphorylation sites of all c¢-subunits.Key words: Na+,K+-ATPase; Phosphoenzyme; Half-site reactivity; Oligomeric structure; Chymotrypsin Materials and methodsPurified membrane-bound Na+,K+-ATPase of canine kidney medulla, with specific activity in the range of 1000-1600 ktmol of ATP hydrolyzed/mg/h, was prepared and assayed as indicated [9,10]. The Na+-dependent treatment with tx-chymotrypsin [6,9], phosphorylation with [y-32p]ATP in the presence of Na + and Mg 2+ at 0°C [6,7,10], assay of total phosphoenzyme on filters [10], resolution and assays of 32P-labeled peptides on acid gels either by autoradiography [6,7] or by counting of gel slices [11,12], and analysis of N-terminal sequences [6,9] were done by procedures the details of which we have described before. The relative quantities of stained peptide bands on gels were determined by densitometry [7,9]. The total amount of enzyme protein applied to each gel was in the range of 1 10 I.tg. It was established experimentally that under these conditions there were linear relationships between the amount of native or cleaved enzyme applied to the gel and the densities of stained a-subunit, ~-subunit, and 83-kDa peptide bands. Results

show abstract

Section: Discussionmentioning

confidence: 99%

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na⁺, K⁺‐ATPase

et al. 1996

View full text Add to dashboard Cite

show abstract

The Na+, K+-Transporting Adenosine Triphosphatase

Glynn

1985

The Enzymes of Biological Membranes

241

215

View full text Add to dashboard Cite

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy

Fringeli

Apell

Fringeli

et al. 1989

Biochimica et Biophysica Acta (BBA) - Biomembranes

View full text Add to dashboard Cite

Na-/ K'-ATPase can be isolated from the outer medulla of mammalian kidney in the form of flat membrane fragments containing the emyme in a densidr of 103-101 protein molecules per pm2 (Deguchi et al. (lnT J, Cell. Biol. 75,. In this paper we show tlrat these membrane fragments can be bound to a germanium plate coated with a phospholipid bilayer. With this system infrared spectroscopic studies of the enz5rme have been carried out using the technique of attenuated total reflection (ATR). At a coverage of the lipid surface corresponding to 30-407o of a monolayer of membrane fragments, chamcteristic infrared bands of the protein such as the amide I and II bands can be resolved. About A% ol tli.e NH-groupß of the peptide backbone are found to be resistant to proton/deuterium exchange within a time period of several days. Evidence for orientation of the protein with respect to the supporting lipid layer is obtained from experiments with polarized light, the largest polarization effects being associated with the -COO band at 14{X) cm-r. Experiments with aqueous media of diflerent ionic composition indicate that the average orientation of transition moments changes when K+ in the medium is replaced by Tris + or Na+.

show abstract

Electron Microscope Analysis of Two-Dimensional Crystals of Membrane-Bound Na,K-ATPase

Cited by 3 publications

References 3 publications

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na⁺, K⁺‐ATPase

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na⁺, K⁺‐ATPase

The Na+, K+-Transporting Adenosine Triphosphatase

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy

Contact Info

Product

Resources

About

Electron Microscope Analysis of Two-Dimensional Crystals of Membrane-Bound Na,K-ATPase

Cited by 3 publications

References 3 publications

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na+, K+‐ATPase

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na+, K+‐ATPase

The Na+, K+-Transporting Adenosine Triphosphatase

Polarized infrared absorption of Na+/K+-ATPase studied by attenuated total reflection spectroscopy

Contact Info

Product

Resources

About

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na⁺, K⁺‐ATPase

Restoration of phosphorylation capacity to the dormant half of the α‐subunits of Na⁺, K⁺‐ATPase