title: Molecular cloning and characterization of Na,K-ATPase from Hydra vulgaris: implications for enzyme evolution and ouabain sensitivity.
abstract: We have used molecular and biochemical techniques to analyze Na,K-ATPase from a simple metazoan, Hydra vulgaris. First we isolated and characterized cDNA clones encoding the Na,K-ATPase alpha subunit from a Hydra lambda gt11 cDNA library. The open reading frame predicts a protein of 1031 amino acids that bears a high degree of primary sequence and secondary structure similarity to mammalian, avian, and arthropod alpha subunits. The predicted Hydra alpha subunit contains charged residues at the termini of the H1-H2 extracellular domain, suggesting that the Hydra alpha subunit may be resistant to cardiac glycoside inhibition. Biochemical analysis of partially purified Hydra Na,K-ATPase reveals both high- and low-affinity components of ouabain-inhibitable ATPase activity. Our results suggest that the evolutionary ancestor of all metazoans possessed a Na,K-ATPase alpha subunit that was highly conserved with respect to its vertebrate counterparts. Further, expression of a ouabain-resistant Na,K-ATPase activity in Hydra suggests that cardiac glycoside resistance arose randomly during evolution of the Na,K-ATPase.
authors.affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510.
authors.forename: V A
authors.initials: VA
authors.lastname: Canfield
authors.forename: K Y
authors.initials: KY
authors.lastname: Xu
authors.forename: T
authors.initials: T
authors.lastname: D'Aquila
authors.forename: A W
authors.initials: AW
authors.lastname: Shyjan
authors.forename: R
authors.initials: R
authors.lastname: Levenson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D005075:Biological Evolution; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D006801:Humans; D006829:Hydra / Q000201:enzymology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010042:Ouabain / Q000494:pharmacology; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D013045:Species Specificity
chemical_list: D010042:Ouabain; D004247:DNA; D000254:Sodium-Potassium-Exchanging ATPase