Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.
The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Mocz
G
G
Pacific Biomedical Research Center, University of Hawaii, Honolulu 96822.
Farias
J
J
Gibbons
I R
IR
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D000431:Ethanol / Q000032:analysis; D005454:Fluorescence Polarization; D006868:Hydrolysis; D008297:Male; D009994:Osmolar Concentration; D011487:Protein Conformation; D012617:Sea Urchins; D013082:Sperm Tail / Q000201:enzymology*; D013696:Temperature; D014357:Trypsin; D014364:Tryptophan
D000431:Ethanol; D014364:Tryptophan; D000255:Adenosine Triphosphate; D014357:Trypsin; D004398:Dyneins