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Topology of Na + ,K + ‐ATPase Identification of the extra‐ and intracellular hydrophilic loops of the catalytic subunit by specific antibodies
Author(s) -
Ovchinnikov Yu.A.,
Luneva N.M.,
Arystarkhova E.A.,
Gevondyan N.M.,
Arzamazova N.M.,
Kozhich A.T.,
Nesmeyanov V.A.,
Modyanov N.N.
Publication year - 1988
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(88)80904-9
Subject(s) - monoclonal antibody , intracellular , microbiology and biotechnology , protein subunit , extracellular , peptide , membrane topology , chemistry , epitope , cytoplasm , biochemistry , antibody , membrane , biology , membrane protein , gene , immunology
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 VG 2 . Another MAb, IIC 9 , 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 IIC 9 and VG 2 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.