Outer arm dynein from trout spermatozoa. Purification, polypeptide composition, and enzymatic properties

• Published in: The Journal of biological chemistry 1989-07, Vol.264 (19), p.11450-11457
• Main Authors: GATTI, J.-L, KING, S. M, MOSS, A. G, WITMAN, G. B
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - isolation & purification; Adenosine Triphosphate - metabolism; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Centrifugation, Density Gradient; Chromatography; Dyneins - antagonists & inhibitors; Dyneins - isolation & purification; Dyneins - metabolism; electrophoresis; Electrophoresis, Polyacrylamide Gel; enzymatic activity; Enzymes and enzyme inhibitors; Freshwater; Fundamental and applied biological sciences. Psychology; Hydrogen-Ion Concentration; Hydrolases; Kinetics; Macromolecular Substances; Male; Microscopy, Electron; Molecular Weight; polypeptides; proteins; Salmo gairdneri; Salmonidae - metabolism; sperm; spermatozoa; Spermatozoa - enzymology; Substrate Specificity; Trout - metabolism; Vanadates - pharmacology
• ISSN: 0021-9258; 1083-351X

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.