Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties
Department of Cell Biology
Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Centrifugation, Density Gradient; Chromatography; Dynein ATPase; purification; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Male; Microscopy, Electron; Molecular Weight; Salmonidae; Spermatozoa; Substrate Specificity; Trout; Vanadates
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.
J Biol Chem. 1989 Jul 5;264(19):11450-7.
The Journal of biological chemistry
Gatti J, King SM, Moss AG, Witman GB. (1989). Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties. Cell and Developmental Biology Publications. Retrieved from https://escholarship.umassmed.edu/cellbiology_pp/70