title: Isolation and cloning of the K+-independent, ouabain-insensitive Na+-ATPase.
abstract: Primary Na+ transport has been essentially attributed to Na+/K+ pump. However, there are functional and biochemical evidences that suggest the existence of a K+-independent, ouabain-insensitive Na+ pump, associated to a Na+-ATPase with similar characteristics, located at basolateral plasma membrane of epithelial cells. Herein, membrane protein complex associated with this Na+-ATPase was identified. Basolateral membranes from guinea-pig enterocytes were solubilized with polyoxyethylene-9-lauryl ether and Na+-ATPase was purified by concanavalin A affinity and ion exchange chromatographies. Purified enzyme preserves its native biochemical characteristics: Mg2+ dependence, specific Na+ stimulation, K+ independence, ouabain insensitivity and inhibition by furosemide (IC50: 0.5 mM) and vanadate (IC50: 9.1 μM). IgY antibodies against purified Na+-ATPase did not recognize Na+/K+-ATPase and vice versa. Analysis of purified Na+-ATPase by SDS-PAGE and 2D-electrophoresis showed that is constituted by two subunits: 90 (α) and 50 (β) kDa. Tandem mass spectrometry of α-subunit identified three peptides, also present in most Na+/K+-ATPase isoforms, which were used to design primers for cloning both ATPases by PCR from guinea-pig intestinal epithelial cells. A cDNA fragment of 1148 bp (atna) was cloned, in addition to Na+/K+-ATPase α1-isoform cDNA (1283 bp). In MDCK cells, which constitutively express Na+-ATPase, silencing of atna mRNA specifically suppressed Na+-ATPase α-subunit and ouabain-insensitive Na+-ATPase activity, demonstrating that atna transcript is linked to this enzyme. Guinea-pig atna mRNA sequence (2787 bp) was completed using RLM-RACE. It encodes a protein of 811 amino acids (88.9 kDa) with the nine structural motifs of P-type ATPases. It has 64% identity and 72% homology with guinea-pig Na+/K+-ATPase α1-isoform. These structural and biochemical evidences identify the K+-independent, ouabain-insensitive Na+-ATPase as a unique P-type ATPase.
authors.affiliation: Laboratorio de Fisiología Molecular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Rocafull
authors.forename: Freddy J
authors.initials: FJ
authors.lastname: Romero
authors.forename: Luz E
authors.initials: LE
authors.lastname: Thomas
authors.forename: Jesús R
authors.initials: JR
authors.lastname: del Castillo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055162:Biocatalysis / Q000187:drug effects; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004305:Dose-Response Relationship, Drug; D020895:Enterocytes / Q000201:enzymology*; D005665:Furosemide / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D006168:Guinea Pigs / Q000235:genetics* / Q000378:metabolism; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
chemical_list: D018076:DNA, Complementary; D007527:Isoenzymes; D008565:Membrane Proteins; D049994:Sodium Potassium Chloride Symporter Inhibitors; D014638:Vanadates; D010042:Ouabain; D005665:Furosemide; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium