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

Abstract

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 α‐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 α‐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.

Advances in Na+,K+‐ATPase studies: from protein to gene and back to protein

“…Various models imply the a-subunit polypeptide chain to traverse the membrane bilayer 6-8 times 2 [ 14-161. Limited proteolysis of the membrane-bound enzyme [25] and immunochemical analysis of the LYsubunit topography in the membrane [26] showed that the a-subunit polypeptide chain spans the lipid bilayer seven times. The location of the N-terminus of the protein in the cytoplasmic region of the cell is generally accepted [5].…”

Immunodetection of Na,K‐ATPase α3‐isoform in renal and nerve tissues

“…As the o~3-isoform mRNA content is substantial in renal [7] and nerve tissues [6], the protein is assumed to enter the enzyme isolated from kidney as a minor component. The membrane-bound Na,K-ATPase from pig kidney outer medulla was subjected to ELISA with various dilutions of aKLH-I, aBSA-I and a-c~p999 [9]. (Only two amino acid substitutions Ile(ce)/Leu(a3) and Arg(ce)/Asn(~3) allowed supposition that the antibodies will recognize both enzyme isoforms equally well.)…”

“…Two peptides corresponding to the a3-subunit regions were synthesized following the solid-phase technique [9]. Peptide I was coupled to Keyhole limpet hemocyanin (KLH) and bovia© serum albumin (BSA) using glutaraldehyde and peptide II, coupled to KLH with N-succinimidyl 3-(2-pyridyldithio)prrpionate as described in [10].…”

“…Antibody binding to the peptides (1-2/zg/ml) or Na,KATPase (5-10/,g/ml) was detected by an indirect solid-phase ELISA [9]. SDS-PAGE (4-15 ~0) and immunoblotting was carried out as in [9].…”

“…SDS-PAGE (4-15 ~0) and immunoblotting was carried out as in [9]. The blots were treated with biotinylated goat anti-rabbit IgG (1:1000) followed by incubation with streptavidin (1 : 1000) and horseradish-peroxidase (HRP)-conjugated biotin (1:500).…”