title: Structure of the alpha-, beta-, and gamma-heavy chains of 22 S outer arm dynein obtained from Tetrahymena cilia.
abstract: Here we document the UV-induced, vanadate-dependent cleavage of the alpha-, beta-, and gamma-heavy chains of 22 S outer arm dynein obtained from Tetrahymena cilia. All three polypeptides have a single site of photocleavage in the presence of Mg2+, ATP, and vanadate (termed V1 cleavage). The alpha-chain yields complementary fragments with masses of 232 and 185 kDa, the beta-chain has complementary fragments with masses of 225 and 195 kDa, and the gamma-chain has complementary fragments with masses of 242 and 161 kDa. In the absence of ATP, only the beta-chain undergoes V1 cleavage. All three polypeptides have one single site of V2 cleavage, which are unaffected by the presence of nucleotide and only require the presence of Mn2+ and vanadate. V2 cleavage always occurs on the larger V1 fragments and is separated from the V1 site by 52, 48, and 57 kDa for the alpha-, beta-, and gamma-heavy chains, respectively. We have also found a third type of UV-induced vanadate-dependent cleavage which we have termed VMT cleavage. VMT cleavage occurs when dynein is bound to microtubules in an ATP-sensitive manner under V1 solution conditions that should only support cleavage of the beta-chain (i.e. vanadate, Mg2+, and absence of ATP). Under these conditions V1 cleavage of the beta-chain and V2 cleavage of all three chains occur. This is the first documented evidence of V2 cleavage occurring under V1 solution conditions and implies a change in dynein structure when it binds to a microtubule. Using a combination of polyclonal and monoclonal antibodies, we have been able to construct linear polypeptide maps of all three heavy chains. Their relationship to the polypeptide maps previously obtained for heavy chains obtained from the dynein of Chlamydomonas and sea urchin axonemes is discussed.
authors.affiliation: Department of Molecular and Cell Biology, Pennsylvania State University, University Park 16803.
authors.forename: S P
authors.initials: SP
authors.lastname: Marchese-Ragona
authors.forename: K C
authors.initials: KC
authors.lastname: Facemyer
authors.forename: K A
authors.initials: KA
authors.lastname: Johnson
mesh_terms: D000251:Adenosine Triphosphatases / Q000302:isolation & purification; D000818:Animals; D002923:Cilia / Q000201:enzymology*; D004398:Dyneins / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D010782:Photolysis; D013768:Tetrahymena / Q000201:enzymology*
chemical_list: D046911:Macromolecular Substances; D010446:Peptide Fragments; D000251:Adenosine Triphosphatases; D004398:Dyneins