Purification and characterization of the ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon. 2010

Dimas C Belisario, and Miguel A Rocafull, and Jesús R del Castillo
Laboratorio de Fisiología Molecular, Centro de Biofísica y Bioquímica, Instituto Venezolano de Investigaciones Científicas (IVIC), Apartado 21827, Caracas 1020-A, Venezuela.

Distal colon absorbs K+ through a Na+-independent, ouabain-sensitive H+/K+-exchange, associated to an apical ouabain-sensitive H+/K+-ATPase. Expression of HKalpha2, gene associated with this ATPase, induces K+-transport mechanisms, whose ouabain susceptibility is inconsistent. Both ouabain-sensitive and ouabain-insensitive K+-ATPase activities have been described in colonocytes. However, native H+/K+-ATPases have not been identified as unique biochemical entities. Herein, a procedure to purify ouabain-sensitive H+/K+-ATPase from guinea-pig distal colon is described. H+/K+-ATPase is Mg2+-dependent and activated by K+, Cs+ and NH4+ but not by Na+ or Li+, independently of K+-accompanying anion. H+/K+-ATPase was inhibited by ouabain and vanadate but insensitive to SCH-28080 and bafilomycin-A. Enzyme was phosphorylated from [32P]-gamma-ATP, forming an acyl-phosphate bond, in an Mg2+-dependent, vanadate-sensitive process. K+ inhibited phosphorylation, effect blocked by ouabain. H+/K+-ATPase is an alpha/beta-heterodimer, whose subunits, identified by Tandem-mass spectrometry, seems to correspond to HKalpha2 and Na+/K+-ATPase beta1-subunit, respectively. Thus, colonic ouabain-sensitive H+/K+-ATPase is a distinctive P-type ATPase.

UI MeSH Term Description Entries
D007527 Isoenzymes Structurally related forms of an enzyme. Each isoenzyme has the same mechanism and classification, but differs in its chemical, physical, or immunological characteristics. Alloenzyme,Allozyme,Isoenzyme,Isozyme,Isozymes,Alloenzymes,Allozymes
D008297 Male Males
D008969 Molecular Sequence Data Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories. Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D009092 Mucous Membrane An EPITHELIUM with MUCUS-secreting cells, such as GOBLET CELLS. It forms the lining of many body cavities, such as the DIGESTIVE TRACT, the RESPIRATORY TRACT, and the reproductive tract. Mucosa, rich in blood and lymph vessels, comprises an inner epithelium, a middle layer (lamina propria) of loose CONNECTIVE TISSUE, and an outer layer (muscularis mucosae) of SMOOTH MUSCLE CELLS that separates the mucosa from submucosa. Lamina Propria,Mucosa,Mucosal Tissue,Muscularis Mucosae,Mucous Membranes,Membrane, Mucous,Membranes, Mucous,Mucosae, Muscularis,Mucosal Tissues,Propria, Lamina,Tissue, Mucosal,Tissues, Mucosal
D010042 Ouabain A cardioactive glycoside consisting of rhamnose and ouabagenin, obtained from the seeds of Strophanthus gratus and other plants of the Apocynaceae; used like DIGITALIS. It is commonly used in cell biological studies as an inhibitor of the NA(+)-K(+)-EXCHANGING ATPASE. Acocantherin,G-Strophanthin,Acolongifloroside K,G Strophanthin
D010750 Phosphoproteins Phosphoprotein
D010766 Phosphorylation The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety. Phosphorylations
D002462 Cell Membrane The lipid- and protein-containing, selectively permeable membrane that surrounds the cytoplasm in prokaryotic and eukaryotic cells. Plasma Membrane,Cytoplasmic Membrane,Cell Membranes,Cytoplasmic Membranes,Membrane, Cell,Membrane, Cytoplasmic,Membrane, Plasma,Membranes, Cell,Membranes, Cytoplasmic,Membranes, Plasma,Plasma Membranes
D003106 Colon The segment of LARGE INTESTINE between the CECUM and the RECTUM. It includes the ASCENDING COLON; the TRANSVERSE COLON; the DESCENDING COLON; and the SIGMOID COLON. Appendix Epiploica,Taenia Coli,Omental Appendices,Omental Appendix,Appendices, Omental,Appendix, Omental
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs

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