Abstract
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.
Full Text
The Full Text of this article is available as a PDF (4.2 MB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Fairbanks G., Steck T. L., Wallach D. F. Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry. 1971 Jun 22;10(13):2606–2617. doi: 10.1021/bi00789a030. [DOI] [PubMed] [Google Scholar]
- Gruenstein E., Rich A., Weihing R. R. Actin associated with membranes from 3T3 mouse fibroblast and HeLa cells. J Cell Biol. 1975 Jan;64(1):223–234. doi: 10.1083/jcb.64.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
- HIRAMOTO Y. MECHANICAL PROPERTIES OF SEA URCHIN EGGS. I. SURFACE FORCE AND ELASTIC MODULUS OF THE CELL MEMBRANE. Exp Cell Res. 1963 Oct;32:59–75. doi: 10.1016/0014-4827(63)90069-7. [DOI] [PubMed] [Google Scholar]
- Helenius A., Simons K. Solubilization of membranes by detergents. Biochim Biophys Acta. 1975 Mar 25;415(1):29–79. doi: 10.1016/0304-4157(75)90016-7. [DOI] [PubMed] [Google Scholar]
- Hiramoto Y. Mechanical properties of the surface of the sea urchin egg at fertilization and during cleavage. Exp Cell Res. 1974 Dec;89(2):320–326. doi: 10.1016/0014-4827(74)90796-4. [DOI] [PubMed] [Google Scholar]
- KANE R. E. THE MITOTIC APPARATUS. PHYSICAL-CHEMICAL FACTORS CONTROLLING STABILITY. J Cell Biol. 1965 Apr;25:SUPPL–SUPPL:144. doi: 10.1083/jcb.25.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
- KANE R. E. The mitotic apparatus: isolation by controlled pH. J Cell Biol. 1962 Jan;12:47–55. doi: 10.1083/jcb.12.1.47. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mooseker M. S., Tilney L. G. Organization of an actin filament-membrane complex. Filament polarity and membrane attachment in the microvilli of intestinal epithelial cells. J Cell Biol. 1975 Dec;67(3):725–743. doi: 10.1083/jcb.67.3.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pollard T. D., Korn E. D. Electron microscopic identification of actin associated with isolated amoeba plasma membranes. J Biol Chem. 1973 Jan 25;248(2):448–450. [PubMed] [Google Scholar]
- Pollard T. D., Weihing R. R. Actin and myosin and cell movement. CRC Crit Rev Biochem. 1974 Jan;2(1):1–65. doi: 10.3109/10409237409105443. [DOI] [PubMed] [Google Scholar]
- Spudich J. A. Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. II. Purification, properties, and membrane association of actin from amoebae of Dictyostelium discoideum. J Biol Chem. 1974 Sep 25;249(18):6013–6020. [PubMed] [Google Scholar]
- Tilney L. G., Detmers P. Actin in erythrocyte ghosts and its association with spectrin. Evidence for a nonfilamentous form of these two molecules in situ. J Cell Biol. 1975 Sep;66(3):508–520. doi: 10.1083/jcb.66.3.508. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tilney L. G., Hatano S., Ishikawa H., Mooseker M. S. The polymerization of actin: its role in the generation of the acrosomal process of certain echinoderm sperm. J Cell Biol. 1973 Oct;59(1):109–126. doi: 10.1083/jcb.59.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Tilney L. G., Mooseker M. Actin in the brush-border of epithelial cells of the chicken intestine. Proc Natl Acad Sci U S A. 1971 Oct;68(10):2611–2615. doi: 10.1073/pnas.68.10.2611. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
- YAGI K., MASE R., SAKAKIBARA I., ASAI H. FUNCTION OF HEAVY MEROMYOSIN IN THE ACCELERATION OF ACTIN POLYMERIZATION. J Biol Chem. 1965 Jun;240:2448–2454. [PubMed] [Google Scholar]
- ZIMMERMAN A. M., LANDAU J. V., MARSLAND D. Cell division: a pressure-temperature analysis of the effects of sulfhydryl reagents on the cortical plasmagel structure and furrowing strength of dividing eggs (Arbacia and Chaetopterus). J Cell Physiol. 1957 Jun;49(3):395–435. doi: 10.1002/jcp.1030490304. [DOI] [PubMed] [Google Scholar]