Effects of competitive sodium-like antagonists on Na,K-ATPase suggest that cation occlusion from the cytoplasmic surface occurs in two steps

Topology of the alpha-subunit of Na,K-ATPase has been analyzed utilizing proteolytic digestion. Evidence is presented for a model with 10 transmembrane segments and lability of the C-terminal domain (M7-M10). Using reconstituted proteoliposomes, inside-out oriented pumps were digested with trypsin at the cytoplasmic surface. Evidence was obtained for the M7/M8 pair and cytoplasmic splits between M8 and M9 and between M9 and M10. Because an extracellular split between M9 and M10 was also observed, using right-side-out oriented renal microsomes, we propose that the M9/M10 pair either is destabilized by cytoplasmic digestion or is intrinsically mobile. Using renal microsomes, extracellular digestion of the alpha-subunit by trypsin, chymotrypsin, or an endogenous protease has been observed, after incubation at 55 or at 45 degrees C with beta-mercaptoethanol (beta-ME) and n-butanol. Both perturbations inactivate enzyme activity. Rb ions protect against inactivation and digestion. At 45 degrees C, with beta-ME and n-butanol, trypsin and chymotrypsin cut between M7 and M8 and between M9 and M10, consistent with the 10-segment model. At 55 degrees C, the topological organization is altered, the M8/M9 connecting loop is exposed at the extracellular surface, and an additional split between M8 and M9 is observed. Extracellular digestion of the alpha-subunit is associated with digestion of the beta-subunit near the first extracellular S-S bridge. Rb ions protect the beta-subunit. Exposure to proteases of extracellular domains of both subunits appears to be caused by disruption of subunit interactions.

Section: Discussionmentioning

confidence: 99%

Topology of the .alpha.-Subunit of Na,K-ATPase Based on Proteolysis. Lability of the Topological Organization

Goldshleger¹,

Tal²,

Karlish³

1995

“…This latter result establishes a direct connection between functional integrity of occlusion and intact structure of the 19-kDa fragment. It should be noted that this susceptibility to trypsin degradation in the presence of Na+ is species dependent; the 19-kDa fragment is not degraded by trypsin in the presence of Na+ when the kidney enzyme is studied (Capasso et al, 1992;Or et al, 1993).…”

Section: Discussionmentioning

confidence: 99%

Structural Integrity of the Membrane Domains in Extensively Trypsinized Na,K-ATPase from Shark Rectal Glands

Esmann¹,

Karlish²,

Sottrup‐Jensen³

et al. 1994

Removal of extramembranous portions of the integral membrane protein Na,K-ATPase from shark salt glands by trypsin in the presence of Rb+ (a K+ congener) preserves the intramembranous association of the remaining membrane-spanning tryptic peptides. This is evidenced from comparison of the rotational mobility of native and trypsinized Na,K-ATPase using saturation transfer electron spin resonance spectroscopy (ESR) and from study of the lipid-protein interactions using conventional ESR spectroscopy. The interface between the lipids and the intramembranous domains is conserved on removal of the extramembranous parts of the protein, since the population of motionally restricted boundary lipids remains essentially the same in the native and trypsinized preparations. The ability to occlude Rb+ is also retained by the trypsinized membranes, as previously observed with pig kidney Na,K-ATPase. A 19-kDa fragment remaining when Na,K-ATPase is trypsinized in the presence of Rb+ is degraded further when the trypsinization is carried out in the presence of Na+ instead of Rb+. The rotational mobility of the tryptic fragments in the Na(+)-trypsinized membranes is lower than for the Rb(+)-trypsinized membranes, indicating rearrangement of the peptides. In addition, occlusion capacity is lost when trypsinization is carried out in Na+, suggesting a correlation between structure and function in the trypsinized membranes. The sequences of four membrane-spanning tryptic fragments of shark Na,K-ATPase are found to be almost identical to corresponding sequences in pig kidney Na,K-ATPase.

“…The present work shows that detergent-solubilized "19 kDa membranes" are much less stable than intact "19 kDa membranes". Two strong indications for this conclusion are that, after solubilization, Rb occlusion is no longer maintained in the absence of Rb and ouabain, and even in the presence of Rb and ouabain, Rb occlusion of solubilized "19 kDa membranes" is inactivated at temperatures (e.g., 40 °C, see Figure 9) at which intact "19 kDa membranes" are quite stable (Or et al, 1993;Shainskaya & Karlish, 1994;Nesatyi, Shainskaya, and Karlish, unpublished data). The implication of all these findings is that protein-phospholipid or proteinprotein interactions within the membrane domain are normally important stabilizing factors.…”

Section: Discussionmentioning

confidence: 97%

“…The stabilization of Rb occlusion by the presence of both Rb ions and ouabain during solubilization of "19 kDa membranes" can be understood as a special case of a general mechanism whereby occluded Rb ions protect against structural perturbations. For example, Rb ions strongly protect "19 kDa membranes" against thermal inactivation of Rb occlusion (Or et al, 1993) and further proteolytic digestion (Karlish et al, 1990;Capasso et al, 1992;Shainskaya & Karlish, 1994). Recently, we have proposed (Shainskaya & Karlish, 1996) that inactivation of Rb occlusion following chymotrytpic truncation of the β subunit, or inactivation induced by DTT [see Lutsenko and Kaplan (1993)], is caused by reduction in Rb affinity, dissociation of occluded Rb ions, and then thermal inactivation.…”

Section: Discussionmentioning

confidence: 99%

“…A number of observations on thermal inactivation and proteolytic digestion of "19 kDa membranes" have led us to propose that the fragments interact as a cation-stabilized complex (Shainskaya & Karlish, 1994). At 37 °C, Rb occlusion is rapidly inactivated in the absence of occluded cations, while the presence of occluded cations (Rb, K, or Na) strongly protects against thermal inactivation (Or et al, 1993). Ouabain binding and Na binding associated with charge movement are thermally inactivated at the same rate, suggestive of disruption of the complex (Schwappach et al, 1994).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Solubilization of a Complex of Tryptic Fragments of Na,K-ATPase Containing Occluded Rb Ions and Bound Ouabain

et al. 1996

The nonionic detergent C12E10 (polyoxyethylene 10-laurylether) has been used to solubilize a complex of tryptic fragments of Na, K-ATPase containing occluded Rb ions and bound ouabain. The aim was to define which fragments are required to maintain Rb occlusion. The experiments utilize "19 kDa membranes" consisting of a 19 kDa and several smaller tryptic fragments (8-11.7 kDa) of the alpha subunit, which include trans-membrane segments M7/M10 and the pairs M1/M2, M3/M4, and M5/M6 [Capasso, J. M., et al (1992) J. Biol. Chem. 267, 1150-1158]. The beta subunit is partially split into a 16 kDa fragment and a glycosylated approximately 50 kDa fragment. Cation occlusion and ouabain binding are intact. After preincubation of "19 kDa membranes" with Rb (5 mM) and then ouabain (10 mM), 90-100% of occluded Rb was solubilized by C12E10 at 0 degrees C. All fragments of the alpha and beta subunits, and also the gamma subunit, were cosolubilized by C12E10, and were observed to sediment together on a sucrose density gradient as a complex containing occluded Rb ions. The soluble complex consists of a monomer containing one copy of each fragment, as indicated by size-exclusion HPLC, as well as estimates of specific Rb occlusion (20.0 +/- 1.2 nmol/mg of protein). In the absence of Rb ions and ouabain, the complex was unstable. Whereas the 19 kDa fragment (M7-M10) and beta subunit remained associated, the smaller fragments, containing M5/M6 and M3/M4 and M1/M2, and the subunit dissociated. Observations on the thermal inactivation of Rb occlusion, and effect of pH and ionic strength, suggest that the soluble complex is stabilized by multiple interactions, both within the lipid bilayer and in hydrophilic domains (e.g., salt bridges).