Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Isolation of sea urchin embryo cell surface membranes on polycationic beads

  • 32 Accesses


Blastula cell surface membranes of the sea urchin, Strongylocentrotus purpuratus, were isolated on polycationic beads by a method modified from Jacobson and Branton (1977) and Jacobson (1980). This study represents the first application of this procedure to an embryonic system. Embryo cells were attached to polylysine-coated polyacrylamide beads and lysed, leaving the embryo cell surface membranes still attached to the beads, and cytoplasmic particles were washed free of the exposed inner surfaces of the membranes. Cell surface membrane sheets were desorbed from the beads and collected by centrifugation. Approximately 8% and 5% of the cell surface membranes of dissociated embryo cells were recovered on the beads and in the membrane pellet, respectively. Specific activities of [3H]concanavalin A-binding and of the cell surface marker enzymes, alkaline phosphatase and Na+/K+ ATPase, were 16-, 19-, and 32-fold higher, respectively, in the cell surface membrane fraction than in the embryo cell homogenate. Membranes were relatively free of cytoplasmic contaminants as judged from electron micrographs and enzyme analysis. Activities in the membrane fraction of the cytoplasmic marker enzymes, cytochrome c oxidase, catalase, acid phosphatase, NADP- and NADPH-cytochrome c reductase, and acetylcholinesterase, were substantially less than homogenate levels. The entire procedure can be completed in 4 h. Since this cell surface membrane isolation technique relies only on the tendency of a negatively charged cell to adhere to a positively charged surface, it is less likely than most other methods to exhibit species and developmental stage specificity and should prove useful in the study of the developmental role of embryonic stage-specific membrane components.

This is a preview of subscription content, log in to check access.


  1. Aebi H (1974) Catalase. In: Bergmeyer H (ed) Methods in enzymatic analysis, vol 2. Academic Press, New York, pp 674–678

  2. Akera T, Brody TM (1968) Inhibition of brain sodium- and potassium-stimulated adenosine triphosphatase activity by chlorpromazine free radical. Mol Pharmacol 4:600–612

  3. Ames B (1966) Assay of inorganic phosphate, total phosphate and phosphatase. In: Neufeld E, Ginsburg V (eds) Methods in enzymology, vol 8. Academic Press, New York, pp 115–118

  4. Appelmans F, Wattiaux R, de Duve C (1955) Tissue fractionation studies. 5. The association of acid phosphatase with a special class of cytoplasmic granules in rat liver. Biochem J 59:438–445

  5. Barber ML, Foy JE (1973) An enzymatic comparison of sea urchin egg ghosts prepared before and after fertilization. J Exp Zool 184:157–166

  6. Baufay H, Amar-Costesec A, Thines-Sempoux D, Wibo M, Robbi M, Berthet J (1974) Analytical study of microsomes and isolated subcellular membranes from rat liver. 3. Subfractionation of the microsomal fraction by isopycnic and differential centrifugation in density gradients. J Cell Biol 51:213–231

  7. Bonting SL, Simon KA, Hawkins NM (1961) Studies on sodium-potassium-activated adenosine triphosphatase. 1. Quantitative distribution in several tissues of the cat. Arch Biochem Biophys 95:416–423

  8. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

  9. Brown KB, Shaver JR (1987) Subcellular distribution of [3H]serotonin binding sites in blastula gastrula, prism, and pluteus sea urchin embryos. Comp Biochem Physiol (in press)

  10. Cestelli AM, Albeggiani G, Allotta J, Vittorelli ML (1975) Isolation of the plasma membrane from sea urchin embryos. Cell Differ 4:305–312

  11. Cohen DM, Kalish DK, Jacobson BS, Branton D (1977) Membrane isolation on polylysine-coated beads. Plasma membrane from HeLa cells. J Cell Biol 75:119–134

  12. de Duve C, Baudhuin P (1966) Peroxisomes (microbodies and related particles). Physiol Rev 46:323–357

  13. Dore D, Cousineau GH (1967) Acid phosphatase analysis in sea urchin eggs and blastulae. Exp Cell Res 48:179–182

  14. Duran A, Bowers B, Cabib E (1975) Chitin synthetase zymogen is attached to the yeast plasma membrane. Proc Natl Acad Sci (USA) 72:3952–3955

  15. Ellman GL, Courtney KD, Andrea V, Featherstone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95

  16. Fink RD, McClay DR (1985) Three cell recognition changes accompany the ingression of sea urchin primary mesenchyme cells. Dev Biol 107:66–74

  17. Giudice G, Mutolo V (1970) Reaggregation of dissociated cells of sea urchin embryos. Advan Morphogenesis 8:115–158

  18. Giudice G, Mutolo V, Donatum G, Bosco M (1969) Reaggregation of mixtures of cells from different developmental stages of sea urchin embryos. Exp Cell Res 54:279–281

  19. Gustafson T, Wolpert L (1967) Cellular movement and contact in sea urchin morphogenesis. Biol Rev Cambridge Philos Soc 42:442–498

  20. Hakomori S (1981) Glycolipids in cellular interaction, differentiation and oncogenesis. Ann Rev Biochem 50:733–764

  21. Herbst C (1904) Über die zur Entwicklung der Seeigellarven notwendigen anorganischen Stoffe, ihre Rolle und ihre Vertretbarkeit. 3. Die Rolle und ihre notwendigen anorganischen Stoffe. Roux's Arch Entwicklungmech 17:306–320

  22. Hodges TK, Leonard RT (1974) Purification of plasma membrane bound adenosine triphosphatase from plant roots. In: Fleischer S, Packer L (eds) Methods in enzymology, vol 32, part B. Academic Press, New York, pp 392–406

  23. Jacobson BS (1977) Isolation of plasma membrane from eucaryotic cells on polylysine-coated polyacrylamide beads. Biochim Biophys Acta 471:331–335

  24. Jacobson BS (1980) Improved method for isolation of plasma membrane on cationic beads. Biochim Biophys Acta 600:769–780

  25. Jacobson BS, Branton D (1977) Plasma membrane: rapid isolation and exposure of cytoplasmic surface by use of positively charged beads. Science 195:302–304

  26. Kinoshita T, Nachman RL, Minick R (1979) Isolation of human platelet plasma membranes with polylysine beads. J Cell Biol 82:688–696

  27. Kobayashi T, Maudsley DV (1974) Recent advances in sample preparation. In: Stanley P, Scoggins B (eds) Liquid scintillation counting-recent developments. Academic Press, New York, pp 189–205

  28. Maggio R (1959) Cytochrome oxidase activity in the mitochondria of unfertilized and fertilized sea urchin eggs. Exp Cell Res 16:272–278

  29. Matsumoto E, Tonegawa Y, Ishihara K (1984) Studies on the localization and activities of concanavalin-A-reactive glycoproteins on the cell surface of sea urchin eggs. J Exp Zool 232:157–165

  30. McClay DR, Chambers AF (1978) Identification of four classes of cell surface antigens appearing at gastrulation in sea urchin embryos. Dev Biol 63:179–186

  31. McClay DR, Chambers AF, Warren RW (1977) Specificity of cellcell interactions in sea urchin embryos. Appearance of new cell surface determinants at gastrulation. Dev Biol 56:343–355

  32. McMahon E, Hoffman S, Fry W, West C (1975) In: McMahon D, Fox C (eds) Developmental biology: pattern formation, gene regulation. W.A. Benjamin, Palo Alto, CA, pp 60–75

  33. Moscona AA (1974) Surface specificity of embryonic cells: Lectin receptor, cell recognition and specific cell ligands. In: Moscona A (ed) The cell surface in development. John Wiley, New York, pp 67–69

  34. Numanoi J (1959) Studies on the fertilization substance. 7. Effect of acetylcholine esterase on development of sea urchin eggs. Sci Papers Coll Gen Edu Univ Tokyo 9:279–285

  35. Oppenheimer SB, Meyer JT (1982) Isolation of species-specific and stage-specific adhesion promotion component by disaggregation of intact sea urchin embryo cells. Exp Cell Res 137:472–476

  36. Ozaki H (1974) Localization and multiple forms of acetylcholinesterase in sea urchin embryos. Dev Growth Differ 16:267–279

  37. Parish RW, Muller U (1976) The isolation of plasma membranes from the cellular slime mold Dictyostelium discoideum using concanavalin A and triton X-100. FEBS Lett 63:40–44

  38. Reiskind JB, Aldrich HC (1982) Biochemical characterization and comparison of plasma membranes from compatible mating strains of Physarum. J Cell Biol 95:253a

  39. Ribot H, Decker SJ, Kinsey WH (1983) Preparation of plasma membranes from fertilized sea urchin eggs. Dev Biol 97:494–499

  40. Scarborough GA (1975) Isolation and characterization of Neurospora crassa plasma membranes. J Biol Chem 250:1106–1111

  41. Sottocasa GL, Kuylenstierna B, Ernster L, Bergstrand A (1967) An electron transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J Cell Biol 32:415–438

  42. Van Der Meulen JA, Emerson DM, Grinstein S (1981) Isolation of chromaffin cell plasma membranes on polycationic beads. Biochim Biophys Acta 643:601–615

  43. Varela JM (1975) Potentials in exploring the physiological role of acetylcholinesterase isozymes. In: Markert C (ed) Isozymes, physiological function, vol 2. Academic Press, New York, pp 315–342

  44. Walsh FS, Barber BS, Crumpton MJ (1976) Preparation of inside-out vesicles of pig lymphocyte plasma membrane. Biochemistry 15:3557–3563

  45. Warren L, Glick MC (1971) The isolation of the surface membranes of animal cells: a survey. Biomembranes 1:257–288

  46. Weis P (1958) Cell contact. Int Rev Cytol 7:391–423

  47. Wharton DC, Tzagaloff A (1967) Cytochrome oxidase from beef heart mitochondria. In: Estrabrook R, Pullman M (eds) Methods in enzymology,vol 10. Academic Press, New York, pp 245–250

Download references

Author information

Correspondence to Kenneth Michael Brown.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Helmly, R.B., Brown, K.M. Isolation of sea urchin embryo cell surface membranes on polycationic beads. Roux's Arch Dev Biol 196, 262–267 (1987). https://doi.org/10.1007/BF00376351

Download citation

Key words

  • Cell surface membrane isolation
  • Polycationic bead method
  • Sea urchin embryo