The Journal of Cell Biology, Vol 69, 73-89, Copyright © 1976 by The Rockefeller University Press
The polymerization of actin. III. Aggregates of nonfilamentous actin and its associated proteins: a storage form of actin
LG Tilney
When echinoderm sperm are treated with the detergent Triton X-100 at pH 6.4
in 10 mM phosphate buffer, the membranes are solubilized, but the actin
which is located in the periacrosomal region remains as a phase- dense cup.
These cups can be isolated free from the flagella and chromatin and can be
solubilized by increasing the pH to 8.0 and by changing the ionic strength
and type of buffer used. Since the actin does not exist in the "F" state in
unreacted sperm, and since the actin remains as a unit that does not
diffuse away, it must be present in the mature sperm in a bound or storage
state. The actin is, in fact, associated with a pair of proteins whose mol
wt are 250,000 and 230,000. When the isolated cups are digested with
trypsin, these high molecular weight proteins are digested, thereby
liberating the actin. The actin will polymerize if heavy meromyosin or
subfragment 1 is added to a preparation of isolated cups. Evidence is
presented that this pair of high molecular weight proteins is similar in
molecular weight and properties to erythrocyte spectrin. Attempts at
transforming the storage form of actin in the cup into filaments were only
moderately successful. The best conditions for filament formation involve
incubating the cup in ATP and divalent salts. Careful examination of these
cups reveals that the actin polymerized preferentially on either end of
oriented filaments that already exist in the cup, indicating that
self-nucleation is inefficacious. I conclude that the actin can exist in
the storage form by its association with spectrin-like molecules and that
the actin in this state polymerizes preferentially onto existing filaments.
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