Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties.
Extraction of isolated axonemes from trout (Salmo gairdneri) sperm with 0.6 M NaCl removed 97% of the outer arms, approximately 12% of the protein, and approximately 50% of the MgATPase activity. Fractionation of this high salt extract by sucrose density gradient centrifugation yielded a single peak of ATPase activity with an apparent sedimentation coefficient of 19 S. Electrophoretic analysis showed that this 19 S particle was composed of two heavy chains (termed alpha and beta; Mr 430,000 and 415,000, respectively), five intermediate molecular weight chains (IC1-IC5; Mr 85,000, 73,000, 65,000, 63,000, and 57,000), and six light chains (LC1-LC6; Mr 22,000-6,000). A similar complex was obtained following further purification by DEAE-Sephacel column chromatography. Quantitative densitometry of Coomassie Blue-stained gels indicated that the heavy and intermediate chains were present in equimolar amounts. Electron microscopic examination of the 19 S particles revealed that it consisted of two globular heads joined together by a Y-shaped stem. The 19 S particle had a specific MgATPase activity of 1.1 +/- 0.3 mumol of phosphate released/min/mg and exhibited an apparent Km for MgATP2- of 40 +/- 16 microM. MnATP2- and CaATP2- were hydrolyzed at rates 100 and 80% that of MgATP2-, respectively. The Mg-ATPase activity was inhibited by vanadate, but not by ouabain or oligomycin, and exhibited a high activity between pH 7.0 and 10.0 with a maximum at pH 9.0-9.5. ATP was the preferred nucleotide, although GTP and CTP (but not ITP) did interact with the dynein to a minor extent. Based on its origin, sedimentation coefficient, polypeptide composition, and enzymatic properties, we conclude that this two-headed 19 S particle represents the entire trout sperm axonemal outer arm dynein. This dynein is probably exemplary of the outer arm dyneins of other vertebrates.
Gatti
J L
JL
Cell Biology Group, Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545.
King
S M
SM
Moss
A G
AG
Witman
G B
GB
D000251:Adenosine Triphosphatases / Q000302:isolation & purification*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D004398:Dyneins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008297:Male; D008854:Microscopy, Electron; D008970:Molecular Weight; D012487:Salmonidae / Q000378:metabolism*; D013094:Spermatozoa / Q000201:enzymology*; D013379:Substrate Specificity; D014337:Trout / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D046911:Macromolecular Substances; D014638:Vanadates; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004398:Dyneins