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S Lutsenko

Publications and source records attributed to S Lutsenko.

27 records · Page 2Linked to original sources
PubMed1995

Membrane disposition of the M5-M6 hairpin of Na+,K(+)-ATPase alpha subunit is ligand dependent.

Extensive proteolytic digestion of Na+,K(+)-ATPase (EC 3.6.1.37) by trypsin produces a preparation where most of the extramembrane portions of the alpha subunit have been digested away and the beta subunit remains essentially intact. The fragment Gln-737-Arg-829 of the Na+,K(+)-ATPase alpha subunit, which includes the putative transmembrane hairpin M5-M6, is readily, selectively, and irreversibly released from the posttryptic membrane preparation after incubation at 37 degrees C for several minutes. Once released from the membrane, the fragment aggregates but remains water soluble. Occlusion of K+ or Rb+ specifically prevents release of the Gln-737-Arg-829 fragment into the supernatant. Labeling of the posttryptic membrane preparation with cysteine-directed reagents revealed that Cys-802 (which is thought to be located within the M6 segment) is protected against the modification by Rb+ while this fragment is in the membrane but can be readily modified upon release. Cation occlusion apparently alters the folding and/or disposition of the M5-M6 fragment in the membrane in a way that does not occur when the fragment migrates to the aqueous phase. The ligand-dependent disposition of the M5-M6 hairpin in the membrane along with recent labeling studies suggest a key role for this segment in cation pumping by Na+,K(+)-ATPase.

Amino Acid Sequence↗
PubMed1994

Molecular events in close proximity to the membrane associated with the binding of ligands to the Na,K-ATPase.

The membrane-bound fraction of purified Na,K-ATPase was characterized following extensive proteolytic digestion in the presence of various physiological ligands which stabilize different conformational states of the sodium pump. There are distinctive conformational changes of the protein which are revealed by amino-terminal amino acid sequence analysis of the digests following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The changes in cleavage patterns result from alterations in domain-domain interactions of the protein. We provide evidence in the alpha-subunit for (i) tight interaction between part of the cytoplasmic ATP binding domain and the membrane-bound portion of the protein; (ii) involvement of the cytoplasmic loop between M2 and M3 in structural rearrangements upon phosphorylation or ion binding; (iii) generation of the same digested products when either ouabain or potassium (rubidium) is present. Similarly, evidence is provided for conformational sensitivity of the extracellular domain of the beta-subunit. The position of the tryptic cleavage point in the beta-subunit is altered depending on whether the alpha-subunit is phosphorylated or whether rubidium ions are occluded. Based upon the conformationally dependent patterns of exposure and protection of different tryptic cleavage sites in the alpha- and beta-subunits we propose a model for intraprotein interactions within the alpha-subunit and between alpha- and beta-subunits following the binding of physiological ligands to the Na,K-ATPase.

Adenosine Diphosphate↗
PubMed1994

Characterization of the Wilson disease gene encoding a P-type copper transporting ATPase: genomic organization, alternative splicing, and structure/function predictions.

Wilson disease is an autosomal recessive disorder of copper transport. Disease symptoms develop from the toxic build-up of copper primarily in the liver, and subsequently in the brain, kidney, cornea and other tissues. A candidate gene for WD (ATP7B) has recently been identified based upon apparent disease-specific mutations and a striking amino acid homology to the gene (ATP7A) responsible for another human copper transport disorder, X-linked Menkes disease (MNK). The cloning of WD and MNK genes provides the first opportunity to study copper homeostasis in humans. A preliminary analysis of the WD gene is presented which includes: isolation and characterization of the 5'-end of the gene; construction of a genomic restriction map; identification of all 21 exon/intron boundaries; characterization of extensive alternative splicing in brain; prediction of structure/function features of the WD and MNK proteins which are unique to the subset of heavy metal-transporting P-type ATPases; and comparative analysis of the six metal-binding domains. The analysis indicates that WD and MNK proteins belong to a subset of transporting ATPases with several unique features presumably reflecting their specific regulation and function. It appears that the mechanism of alternative splicing serves to regulate the amount of functional WD protein produced in brain, kidney, placenta, and possibly in liver.

Adenosine Triphosphatases↗
PubMed1993

An essential role for the extracellular domain of the Na,K-ATPase beta-subunit in cation occlusion.

The role of the Na,K-ATPase beta-subunit in stabilization of ion-binding sites has been investigated. Treatment of the purified renal Na,K-ATPase with 0.25 M DTT at 40 degrees C for 1 h resulted in 50% loss of Rb occlusion, which correlates with partial reduction of S-S bridges in the extracellular portion of the beta-subunit; both of these effects were prevented by the presence of 20 mM RbCl. To clarify the role of the extracellular portion of the beta-subunit, "19-kDa membranes" (Na,K-ATPase posttryptic residues, which have been shown to possess many of the cation-binding properties) were used. Incubation of the "19-kDa membranes" with 0.2 M DTT for 1 h at 37 degrees C abolished 70-80% of the 86Rb occlusion capacity. This was accompanied by accumulation of 16- and 17-kDa peptides (in SDS-PAGE of the membranes) and release of a 45-kDa band derived from the Na,K-ATPase beta-subunit to the supernatant. The appearance of the 45-kDa fragment of the beta-subunit in the supernatant confirms the existence of only one transmembrane fragment in this subunit. N-Terminal sequence analysis of the 16- and 17-kDa bands revealed the same structure, A-K-E-E-G-, which corresponds to the beta-subunit sequence beginning at Ala5. The simultaneous presence of 25 mM RbCl (but not 25 mM choline chloride) during DTT treatment prevents almost all (85%) of the loss of Rb occlusion, the appearance of 16- and 17-kDa bands, and reduction and release of the 45-kDa fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Transmembrane organization of the Na+,K(+)-ATPase molecule.

The organization of the hydrophobic domain of the Na,K-ATPase in the E1 and E2 form of the enzyme has been studied by labelling with two hydrophobic photoactivable reagents 3-(trifluoromethyl)-3-(m-[125I]iodophenyl) diazirine ([125I]TID) and 1-palmitoyl-2-[11-[4-[3-(trifluoromethyl) diazirinyl] phenyl] [2-3H]undecanoyl]-sn-glycero-3-phosphorylcholine ([3H]PTPC/11). The incorporation of the reagents into the alpha-subunit but not into the beta-subunit in the E1-conformation was shown to be lower than that in the E2 form. This indicated the structural rearrangement of the alpha-subunit, which resulted in a change in the accessibility of the membrane-bound fragments for the hydrophobic labels. The set of the [125I]TID-labelled peptides of the alpha-subunit was shown to be the same for the E1 and E2 form of the enzyme: Asp68-Lys142, Ile265-Lys341, Val545-Lys589, Ser770-Lys826, Leu842-Arg880, Asn936-Arg972 and Met973-Arg999, which points to the different level of modification of the same fragments. The first results of molecular modelling of the spatial organization of the intramembrane part of Na+,K(+)-ATPase are also presented.

Amino Acids↗
PubMed1990

ATP binding site of mitochondrial creatine kinase. Affinity labelling of Asp-335 with C1RATP.

The ATP binding site of mitochondrial creatine kinase from chicken heart has been studied by modifying the purified enzyme with a 14C-labelled ATP analogue, C1RATP, in which the reactive label was covalently bound to the gamma-phosphate group of ATP. The modified enzyme was digested by pepsin, and a single radioactive nonapeptide was isolated by HPLC. Amino acid analysis and direct sequence determination revealed that the isolated peptide corresponds to amino acids 335-343 within the C-terminal region of Mi-CK, this peptide being highly preserved throughout evolution. Asp-335 is very likely the site of modification by C1RATP. The specificity of the ATP analogue for the active site of creatine kinase was demonstrated by the inhibition of the enzymatic activity of Mi-CK by C1RATP and by the prevention of this inhibition bij ADP.

Adenosine Triphosphate↗