Outer-arm dynein from trout spermatozoa: substructural organization.
Outer-arm dynein purified from trout spermatozoa was disrupted by low-ionic-strength dialysis, and the resulting subunits were separated by sucrose density-gradient centrifugation. The intact 19 S dynein, containing the alpha- an beta-heavy chains, intermediate chains (ICs) 1-5 and light chains (LCs) 1-6, yielded several discrete particles: a 17.5 S adenosine triphosphatase (ATPase) composed of the alpha- and beta-chains ICs 3-5 and LC 1; a 9.5 S complex containing ICs 1 and 2 together with LCs 2, 3, 4, and 6; and a single light chain (LC 5), which sedimented at approximately 4 S. In some experiments, ICs 3-5 also separated from the heavy chain complex and were obtained as a distinct subunit. Further dissociation of the 17.5 S particle yielded a 13.1 S ATPase that contained the beta-heavy chain and ICs 3-5. The polypeptide compositions of the complexes provide new information on the intermolecular associations that occur within dynein. Substructural features of the trout dynein polypeptides also were examined. The heavy chains were subjected to vanadate-mediated photolysis at the V1 sites by irradiation at 365 nm in the presence of Mg2+, ATP, and vanadate. Fragment pairs of relative molecular mass (Mr) 245,000/185,000 and 245,000/170,000 were obtained from the alpha- and beta-heavy chains, respectively. Photolysis of these molecules at their V2 sites, by irradiation in the presence of vanadate and Mn2+, yielded fragments of Mr 160,000/270,000 and 165,000/250,000, respectively. These values confirm that the alpha- and beta-heavy chains have masses of 430,000 and 415,000 daltons, respectively. Immunological analysis using monoclonal antibodies revealed that one intermediate chain from trout dynein (IC 2) contains epitopes present in two different intermediate chains from Chlamydomonas dynein. This indicates that specific sequences within the dynein intermediate chains have been highly conserved throughout evolution.
King
S M
SM
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts.
Gatti
J L
JL
Moss
A G
AG
Witman
G B
GB
D000251:Adenosine Triphosphatases / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002499:Centrifugation, Density Gradient; D004398:Dyneins / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000276:immunology; D015151:Immunoblotting; D008297:Male; D008870:Microtubules / Q000032:analysis* / Q000502:physiology / Q000648:ultrastructure; D010782:Photolysis; D012487:Salmonidae / Q000033:anatomy & histology*; D013094:Spermatozoa / Q000032:analysis / Q000502:physiology / Q000648:ultrastructure*; D014337:Trout / Q000033:anatomy & histology*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D000251:Adenosine Triphosphatases; D004398:Dyneins