Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.
Article| December 01 1988
Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase.
W L Dentler
Department of Physiology and Cell Biology, University of Kansas, Lawrence 66044.
Online Issn: 1540-8140
Print Issn: 0021-9525
J Cell Biol (1988) 107 (6): 2679–2688.
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W L Dentler; Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase.. J Cell Biol 1 December 1988; 107 (6): 2679–2688. doi: https://doi.org/10.1083/jcb.107.6.2679
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