Tryptic digestion of dynein 1 in low salt medium. Origin and properties of fragment A.
Dynein 1 was extracted from sperm flagella of the sea urchin Tripneustes gratilla with 0.6 M NaCl and dialyzed against 0.5 mM EDTA, 14 mM 2-mercaptoethanol, 5 mM imidazole/HCl buffer, pH 7.0, for 24-48 h. In some cases, fractions containing the alpha heavy chain and the beta/intermediate chain 1 complex (beta/IC1) were separated by density gradient centrifugation in the same solution. Treatment of the samples at a trypsin:protein ratio of 1:10 w/w for 32 min at room temperature yields a crude digest from which Fragment A is purified by density gradient centrifugation. The purified Fragment A consists of two principal peptides (Mr = 195,000 and 130,000) that cosediment with the peak of ATPase activity at 12.5 S, which is slightly faster than the 11 S of the original beta/IC1 complex. When digests of the separated alpha chain and of the beta/IC1 complex are followed as a function of time, the early cleavages of the two heavy chains (Mr = 428,000) resemble each other in that both lead to similarly sized peptides of Mr 316,000 and 296,000, but only in the beta/IC1 fraction does the digestion proceed to form Fragment A. The remainder of the beta chain, termed Fragment B, occurs as an Mr 110,000 peptide sedimenting at 5.7 S with no associated ATPase activity. Fragment A has a specific ATPase activity of 4.3 mumol Pi X min-1 X mg-1, with a Km of 29 microM in 0.1 M NaCl medium, and an apparent Ki for inhibition by vanadate of 1.2 microM in the absence of salt, and 22 microM in 0.6 M NaCl. Photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-triphosphate indicates that the ATP binding site on the beta chain of dynein 1 is located on the Mr 195,000 peptide of Fragment A. The possibility that Fragments A and B of the beta/IC1 complex may correspond to the head and tail regions of the tadpole-shaped particle seen by electron microscopy is discussed.
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001665:Binding Sites; D002499:Centrifugation, Density Gradient; D004398:Dyneins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000032:analysis*; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification / Q000378:metabolism*; D012617:Sea Urchins / Q000032:analysis*; D013094:Spermatozoa / Q000032:analysis*; D014357:Trypsin / Q000378:metabolism*; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D001386:Azides; D010446:Peptide Fragments; D014639:Vanadium; D014638:Vanadates; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D004398:Dyneins