Display Settings:


Send to:

Choose Destination
See comment in PubMed Commons below
J Cell Sci. 1976 Mar;20(2):405-39.

Flagellar doublet microtubules: fractionation of minor components and alpha-tubulin from specific regions of the A-tubule.


Proteins occurring minor amounts with purified sperm flagellar doublet microtubules were identified and studied by SDS-gel electrophoresis. Methods were developed to solubilize selectively these minor components; electron microscopy (EM) of the fractionated products revealed possible locations of these proteins in the tubule. Doublet microtubules were prepared from sea-urchin (Echinus esculentus and Stronglyocentrotus droebachiensis) and scallop (Pecten maximus) sperm by dialysing flagellar axonemes against 2 mM Tris-0-2 mM EDTA-0-5 mM DTT. EM indicates that these doublet tubule preparations retain at least 70% of their radial spokes; cross-sections show a globule or fibre applied to the inside wall of the A-tubule, across from the inner B-tubule junction. On SDS-gels these preparations separate into at least 10 minor bands, accounting for 20-30% of the total protein; the remaining 75 +/- 4% migrates as tubulin. For E. esculentus the molecular weights and relative amounts of these components are: Component Ee 8 (150000 Daltons; 1%), 11 (114000; 2-5%), 15 (89000; 2%), 16 (80000; 2-5%), 17 (74000; 2%), 18 (69000; 2%), 19 (66000; 2%), 21 (48000; 4-5%), 22 (45000; 3%) and 23 (44500; 3%). Treatment of sea-urchin tubules with 0-1-0-5% sarkosyl, 0-1-0-3 M KSCN or 0-3-0-6 M KI results in the selective solubilization of: first, component 8 and some B-subfibre tubulin; second, components 11 and 23 and the remaining B-subfire tubulin; third, most of the A-subfire tubulin and components 17, 18 and 19. Thermal fractionation extracts none of these components, suggesting they are principally associated with the A-tubule. Finally 25-35% of the original protein is resistant to solubilization, and appears in the EM as ribbons of 3 protofilaments with 16-nm axial repeats. The resistant ribbons contain components 15, 16, 21 and 22 (plus component 20 in S. droebachiensis) in addition to 25 +/- 4% of the total tubulin. The data support the existence of two stable moieties in each doublet tubule: (1) a ribbon of 3 protofilaments and (2) either a second ribbon of 3 protofilaments or an equivalent amount of tubulin in some other form. EM images suggest that one ribbon forms the lateral side of the A-tubule (e.g. protofilaments A1,2,3 or A13,1,2 in the model) and that the globule applied to A13 may be a multisubunit complex of remaining minor components. Treatment of scallop tubules with 0-3 M KSCN preferentially extracts alpha-tubulin, yielding ribbons 1-4 protofilaments wide. The significance of this finding is discussed.

[PubMed - indexed for MEDLINE]
Free full text

LinkOut - more resources

Full Text Sources

Molecular Biology Databases

PubMed Commons home

PubMed Commons

How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for HighWire
    Loading ...
    Write to the Help Desk