Effect of solution composition and proteolysis on the conformation of axonemal components.
The effect of solution composition and enzymic proteolysis on axonemes prepared from the sperm of sea urchins, Tripneustes gratilla, has been investigated. Aliquots of axonemes, prepared by treatment of sperm with Triton X-100 and differential centrifugation, were transferred to solutions of different composition with and without intervening tryptic proteolysis, and the particle conformations observed by dark-field and electron microscopy. In most solutions particles in partially digested preparations underwent conformational transformations to coiled or helix-like forms. Proteolysis was accompanied by an increase in the ATPase activity of the digest: by centrifuging down the insoluble digestion products it was shown that digestion resulted in the appearance of ATPase activity in the soluble phase with a concomitant decrease in ATPase activity in the pellet fraction. Gel electrophoresis showed this corresponded to the appearance of dynein in the supernatant and a decrease in dynein associated with the insoluble fraction. Supernatant dynein had a greater specific ATPase activity than dynein extracted from axonemes. Observations on specimens prepared for electron microscopy by thin sectioning allowed a rough correlation to be made between the dark-field observations, chemical analyses, and morphological alterations attendant with the proteolysis and solution conditions. It is concluded that in the intact axoneme the doublet tubules are under considerable tension and that proteolytic destruction of physical restraining elements allows spontaneous conformational alterations of the digestion products. In addition, proteolysis increases the specific ATPase activity of dynein and removes a portion of it from the axonemal structure.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002498:Centrifugation; D003720:Densitometry; D004591:Electrophoresis, Polyacrylamide Gel; D005407:Flagella / Q000187:drug effects; D005976:Glutaral / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008297:Male; D008853:Microscopy; D008854:Microscopy, Electron; D008870:Microtubules / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D012617:Sea Urchins; D013094:Spermatozoa / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D013501:Surface-Active Agents / Q000494:pharmacology; D014357:Trypsin / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000494:pharmacology
D011506:Proteins; D013501:Surface-Active Agents; D014361:Trypsin Inhibitors; D000255:Adenosine Triphosphate; D014357:Trypsin; D000251:Adenosine Triphosphatases; D005976:Glutaral