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  • Title: Major membrane protein differences in cilia and flagella: evidence for a membrane-associated tubulin.
    Author: Stephens RE.
    Journal: Biochemistry; 1977 May 17; 16(10):2047-58. PubMed ID: 861196.
    Abstract:
    The membrane of both sperm flagella and gill cilia of the scallop Aequipecten irradians may be selectively solubilized in 1% Triton X-100, 30 mM tris(hydroxymethyl)-aminomethane hydrochloride (Tris-HCl), pH8, and 3 mM MgCl2, leaving the axoneme totally intact. This membrane fraction represents about 20% of the total protein of the respective organelle. Analysis of the flagellar membrane by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis revealed one principal protein component, periodic acid-Schiff (PAS) positive and migrating with an apparent molecular weight of 250,000. The remaining minor proteins, none of them PAS positive, accounted for less than one-third of the total flagellar membrane fraction. Analysis of the ciliary membrane also revealed one major protein component, weakly PAS positive and migrating with an apparent molecular weight of 55,000. The remaining minor proteins represented about one-third of the total ciliary membrane fraction; two components with molecular weights of 100,000 and 40,000 predominated. The latter could be substantially reduced by purification of the cilia on a sucrose density gradient and was assumed to be actin, derived by vesiculation of the brush border during deciliation. The principal ciliary membrane protein, that of 55,000 daltons, was resolved into two equimolar components on NaDodSO4-Tris-glycine-polyacrylamide gels, comigrating with the alpha and beta chains of outer fiber tubulin. S-carboxymethylation caused increased splitting of the two components and concomitant migration with similarly treated ciliary tubulin. Preparative gel electrophoresis yielded separate components whose cyanogen bromide cleavage products were virtually identical in size distribution with those obtained from outer fiber alpha and beta chains; tryptic peptides corresponded almost exactly to those of authentic tubulin subunits but certain positional differences indicated possible side chain modification. At 25 degrees C both whole cilia and its solubilized membrane fraction bound colchicine while whole flagella and the 9 + 2 axoneme from either organelle did not. Thus certain molluscan flagellar membranes primarily contain a 250,000-dalton glycoprotein but ciliary membranes have a modified tubulin as the major protein component. At an electron microscopic level, flagellar membranes have a distinct trilamellar "unit membrane" structure while ciliary membranes appear thinner and considerably less distinct, perhaps reflecting the protein compositional differences in the membranes of these other wise morpholobically identical organelles.
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