Topology of Na+,K+-ATPase. Identification of the extra- and intracellular hydrophilic loops of the catalytic subunit by specific antibodies.

To study the topology of Na+,K+-ATPase monoclonal antibodies (MAbs) specific for membrane-bound enzyme were produced. Using immunofluorescence staining of viable cells or smears of a pig kidney embryonic (PKE) cell line, two groups of MAbs were selected, namely those binding to extra- or intracellular portions of the alpha-subunit. The extracellular location of peptide loop 804-841 linking the Vth and VIth intramembrane hydrophobic segments was proved using MAb VG2. Another MAb, IIC9, interacting with PKE cells only after membrane perforation (4% formaldehyde and 0.1% Tween-20), was shown to bind to the hydrophilic loop 868-945. The antigenic determinants recognized by MAb IIC9 and VG2 are located in peptides 887-904 and 810-825, respectively. The C-terminus of the alpha-subunit molecule was positioned on the outer side of the cytoplasmic membrane utilizing affinity-purified antibodies to the synthetic peptide corresponding to fragment 999-1008.

Differentiated analysis of the secondary structure of hydrophilic and hydrophobic regions in alpha- and beta-subunits of Na+,K+-ATPase by Raman spectroscopy.

Raman spectra of active Na+,K+-ATPase from pig kidney and membrane-bound products of its two-stage trypsinolysis, including alpha-subunit hydrophobic regions as well as the intact beta-subunit and hydrophobic regions of alpha- and beta-subunits, were measured to calculate the secondary structure of hydrophilic and hydrophobic regions of the enzyme. Consequent comparison demonstrated unambiguously that (i) membrane-bound hydrophobic parts of polypeptide chains of Na+,K+-ATPase subunits are in the alpha-helical conformation; (ii) essential contents of the alpha-helix as well as beta-sheet are estimated to form the hydrophilic (mainly cytoplasmic) domain of the Na+,K+-ATPase alpha-subunit; (iii) the exoplasmic hydrophilic domain of the beta-subunit is shown to include several antiparallel beta-pleated sheets and a small amount of the alpha-helix and unordered conformations. The model of the secondary structure organization of hydrophilic domains as well as 8 hydrophobic transmembrane segments of the enzyme molecule was proposed on the basis of experimental results and predictional calculations.

Detailed structural analysis of exposed domains of membrane-bound Na+,K+-ATPase. A model of transmembrane arrangement.

Exposed regions of the alpha- and beta-subunits of membrane-bound Na+,K+-ATPase were in turn hydrolyzed with trypsin. Resistance of the beta-subunit to proteolysis was shown to be due mainly to the presence of disulfide bridge(s) in the molecule. A model for the spatial organisation of the enzyme in the membrane was proposed on the basis of detailed structural analysis of extramembrane regions of both subunits.

[A method of selective isolation of tryptophan- and cysteine-containing peptides by covalent chromatography. Analysis of the topography of the Na+,K+-ATPase alpha-subunit]. / Metod selektivnogo vydeleniia triptofan- i tsisteinsoderzhashchikh peptidov kovalentnoi khromatografiei. Analiz topografii alpha-sub''edinitsy Na+,K+-ATP-azy.

A procedure for highly selective isolation of tryptophan- and cysteine-containing peptides from protein hydrolysates has been developed on the basis of covalent chromatography. It includes incorporation of a thiol group into the tryptophan residues by sequential treatment of peptides with 2-nitrophenylsulfenyl chloride and beta-mercaptoethanol followed by immobilization on the corresponding supports via thiol-disulfide exchange. The technique is applicable to the analysis of the hydrolysate of the Na+, K+-ATPase alpha-subunit obtained by limited trypsinolysis of the membrane-bound enzyme. Fifteen tryptophan- and cysteine-containing tryptic peptides, which comprise the protein portions exposed outside the membrane, have been isolated in addition to those previously identified. This structural information allows unequivocal determination of boundaries of transmembrane segments of the alpha-subunit in the spatial model earlier proposed.

[Primary structure of the beta-subunit of Na+,K+-ATPase from the swine kidney. I. Analysis of products of trypsin hydrolysis of immobilized proteins]. / Pervichnaia struktura beta-sub''edinitsy Na+,K+-ATP-azy pochek svin'i. I. Analiz produktov tripticheskogo gidroliza immobilizovannogo belka.

The Na+, K+-ATPase's beta-subunit immobilized on thiol-glass was hydrolyzed with trypsin. Over 25 peptides covering ca. 90% of the protein polypeptide chain were isolated from the digest by HPLC and characterized. Structural analysis allowed us to localize the sites of attachment of all three carbohydrate chains of beta-subunit. Sequence data were used to design of oligonucleotide hybridization probes for gene cloning.

Pig kidney Na+,K+-ATPase. Primary structure and spatial organization.

cDNAs complementary to pig kidney mRNAs coding for alpha- and beta-subunits of Na+,K+-ATPase were cloned and sequenced. Selective tryptic hydrolysis of the alpha-subunit within the membrane-bound enzyme and tryptic hydrolysis of the immobilized isolated beta-subunit were also performed. The mature alpha- and beta-subunits contain 1016 and 302 amino acid residues, respectively. Structural data on the peptides from extramembrane regions of the alpha-subunit and on glycopeptides of the beta-subunit underlie a model for the transmembrane arrangement of Na+,K+-ATPase polypeptide chains.

[Primary structure of the alpha subunit of Na+,K+-ATPase. I. Analysis of hydrophilic fragments of the polypeptide chain]. / Pervichnaia struktura alpha-sub''edinitsy Na+,K+-ATP-azy. I. Analiz gidrofil'nykh fragmentov polipeptidnoi tsepi.

The selective tryptic digestion of the native membrane-bound enzyme was carried out under conditions that provide the extensive hydrolysis of hydrophilic regions of the alpha-subunit into small fragments and allow to preserve the integrity of the beta-subunit. Twenty-seven water-soluble peptides comprising approximately 40% of the total polypeptide chain were isolated by HPLC and their complete or partial amino acid sequence was determined. It led to general outline of the structural organisation of the alpha-subunit hydrophilic regions exposed from membrane. The information thus obtained was used in synthesis of specific oligonucleotide probes.