abstract

• The plasma membrane H%2b-ATPase was purified from the yeast K. lactis. The oligomeric state of the H%2b-ATPase is not known. Size exclusion chromatography displayed two macromolecular assembly states (MASs) of different sizes for the solubilized enzyme. Blue native electrophoresis (BN-PAGE) showed the H%2b-ATPase hexamer in both MASs as the sole/main oligomeric state—in the aggregated and free state. The hexameric state was confirmed in dodecyl maltoside-treated plasma membranes by Western-Blot. Tetramers, dimers, and monomers were present in negligible amounts, thus depicting the oligomerization pathway with the dimer as the oligomerization unit. H%2b-ATPase kinetics was cooperative (n~1.9), and importantly, in both MASs significant differences were determined in intrinsic fluorescence intensity, nucleotide affinity and Vmax; hence suggesting the large MAS as the activated state of the H%2b-ATPase. It is concluded that the quaternary structure of the H%2b-ATPase is the hexamer and that a relationship seems to exist between ATPase function and the aggregation state of the hexamer. © 2019 by the authors.

authors

• De La Cruz-Torres V.
• Ruiz-Granados Y.G.
• Sampedro Pérez José Guadalupe

publication date

• 2019-01-01

funding provided via

• UASLP-CA-263  Grant

published in

• Molecules  Journal

keywords

• Binding site affinity; Enzyme kinetics; Fluorescence; H+-ATPase; Hexamer; Macromolecular assembly proton transporting adenosine triphosphatase; cell membrane; chemistry; enzymology; Kluyveromyces; macromolecule; metabolism; size exclusion chromatography; Western blotting; Blotting, Western; Cell Membrane; Chromatography, Gel; Kluyveromyces; Macromolecular Substances; Proton-Translocating ATPases

Digital Object Identifier (DOI)

• 10.3390/molecules24050958

PubMed ID

• 30857224

start page

end page

volume

• 24

issue

• 5