Chemical differences distinguish ciliary membrane and axonemal tubulins.
Tubulin was prepared by exhaustive dialysis solubilization from axonemal A and B subfibers and by detergent solubilization of the membrane of cilia from the scallop aequipecten irradians. The respective alpha and beta chains were isolated by preparative sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis and then compared by amino acid analysis and high-resolution two-dimensional tryptic peptide mapping. Minor amino acid and peptide differences distinguished A- and B-subfiber-derived tubulin subunits from each other, but far more significant amino acid differences distinguished the membrane-derive subunits from those of the axoneme. Peptide mapping revealed that each membrane tubulin subunit contained two major peptides not coincident with those of the axoneme and vice versa, but many corresponding peptides differed markedly in relative intensity. The alpha and beta subunits from these sources showed virtually identical isoelectric points. Certain NaDodSO4-polyacrylamide gel systems, sensitive to differential detergent binding, allowed the membrane-derived subunits to be distinguished from those of the axoneme. Under nondenaturing conditions, the membrane-derived tubulin bound both anionic and cationic detergents more strongly than axonemal tubulin. These data indicate that ciliary membrane tubulin is a distinct molecule whose subunits have the same molecular weight and isoelectric point as those of axonemal tubulin but differs chiefly in terms of nonpolar, conservative substitutions. These chemical differences argue against the artifactual origin of the protein from breakdown of the axoneme.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001369:Axons / Q000032:analysis*; D002462:Cell Membrane / Q000032:analysis; D002923:Cilia / Q000032:analysis*; D005880:Gills / Q000032:analysis; D046911:Macromolecular Substances; D008974:Mollusca; D010446:Peptide Fragments / Q000032:analysis; D014357:Trypsin; D014404:Tubulin
D000596:Amino Acids; D046911:Macromolecular Substances; D010446:Peptide Fragments; D014404:Tubulin; D014357:Trypsin