Journal of Comparative Physiology B

, Volume 157, Issue 6, pp 717–729 | Cite as

Purification and characterization of calcium-binding conchiolin shell peptides from the mollusc,Haliotis rufescens, as a function of development

  • Marios A. Cariolou
  • Daniel E. Morse
Article

Summary

Conchiolin peptides of the molluscan shell are believed to determine structural organization and facilitate calcification during shell formation. Changes in patterns of conchiolin synthesis during development, and the possible contribution of these peptides to shell formation, have been investigated by purification and characterization of the soluble peptides extracted from the shell of the gastropod mollusc,Haliotis rufescens (red abalone), at various stages of development. Shell peptides were purified from young post-larvae, juveniles and adults by gel-filtration column chromatography in aggregation-reducing bicarbonate buffers. Calcium-binding domains were detected spectrophotometrically after reaction with a cationic carbocyanine dye. Juvenile and adult shell peptides were found to be heterogeneous, and rich in aspartic acid and glycine residues; in contrast, post-larval shells were found to contain one major glycine-rich component. The juvenile shell peptide population shares components from each of the other two populations, suggesting that the synthesis of the different shell peptides results from the differential expression of a multi-gene family, in a developmentally controlled progression. Enzymatic analyses suggest that calcium binds to the aspartic acid residues of the peptide core, rather than to satellite groups such as phosphate, sulfate or carbohydrate. The possibility is discussed that the aspartic acid residues found in shell peptides may play an important role in the calcification of the abalone shell matrix. The methods demonstrated here also should prove useful for the purification, characterization, and comparative analysis of calcium-binding proteins of connective tissues, extracellular matrices and support structures in many other systems.

Keywords

Molluscan Shell Aspartic Acid Residue Satellite Group Gastropod Mollusc Peptide Core 

Abbreviations

Asp

aspartic acid

BSA

bovine serum albumin

Da

daltons

EDTA

ethylenediaminetetraacetic acid

GABA

γ-aminobutyric acid

HPLC

high-pressure liquid chromatography

ODS

octadecylsilane

OPA

o-phthaldialdehyde

SDS

sodium dodecyl sulfate

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References

  1. Addadi L, Weiner S (1985) Interactions between acidic proteins and crystals: Stereochemical requirements in biomineralization. Proc Natl Acad Sci USA 82:4110–4114Google Scholar
  2. Andrews P (1965) The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem J 96:595–606Google Scholar
  3. Bean RC, Shepherd WC, Kay RE, Walwick ER (1965) Spectral changes in a cationic dye due to interaction with macromolecules. III. Stoichiometry and mechanism of the complexing reaction. J Phys Chem 69:4368–4379Google Scholar
  4. Campbell KP, MacLennan DH, Jorgensen AO (1983) Staining of the calcium-binding proteins, calsequestrin, calmodulin, troponin C, and S-100, with the cationic carbocyanine dye ‘Stains-all’. J Biol Chem 258:11267–11273Google Scholar
  5. Cleveland DW, Fisher SG, Kirschner MW, Laemmli UK (1977) Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem 252:1102–1106Google Scholar
  6. Crenshaw MA (1972) The soluble matrix ofMercenaria mercenaria shell. Biomineralization 6:6–11Google Scholar
  7. Geisler N, Kaufmann E, Fischer S, Plessmann U, Weber K (1983) Neurofilament architecture combines structural principles of intermediate filaments with carboxy-terminal extensions increasing in size between triplet proteins. EMBO J 2:1295–1302Google Scholar
  8. Gysi JR, Chapman DJ (1978) Comparative biochemistry ofHaliotis pigmentation: unusual bilipeptides ofHaliotis cracherodii. J Biochem Physiol 63(B):355–361Google Scholar
  9. Green MR, Pastewka JV, Peacock AC (1973) Differential staining of phosphoproteins on polyacrylamide gels with a cationic carbocyanine dye. Anal Biochem 56:43–51Google Scholar
  10. Jones BN, Paabo S, Stein S (1981) Amino acid analysis and enzymatic sequence determination of peptides by an improvedo-phthaldialdehyde precolumn labeling procedure. J Liq Chrom 4:565–586Google Scholar
  11. Kaufmann E, Geisler N, Weber K (1984) SDS-PAGE strongly overestimates the molecular masses of the neurofilament proteins. FEBS Lett 170:81–84Google Scholar
  12. Kay RE, Walwick ER, Gifford CK (1964) Spectral changes in a cationic dye due to interaction with macromolecules. II. Effects of environment and macromolecule structure. J Phys Chem 68:1907–1916Google Scholar
  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond) 227:680–682Google Scholar
  14. Lindroth P, Mopper K (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivativation witho-phthaldialdehyde precolumn labeling procedure. J Liq Chrom 4:565–586Google Scholar
  15. Manahan DT, Davis JP, Stephens GC (1983) Bacteria-free sea urchin larvae take up neutral amino acids selectively from seawater. Science 220:204–206Google Scholar
  16. Morse A, Morse DE (1984) Recruitment and metamorphosis ofHaliotis larvae are induced by molecules uniquely available at the surfaces of crustose red algae. J Exp Mar Biol Ecol 75:191–215Google Scholar
  17. Morse DE, Duncan H, Hooker N, Morse A (1977) Hydrogen peroxide induces spawning in molluscs, with activation of prostaglandin endoperoxide synthetase. Science 196:298–300Google Scholar
  18. Morse DE, Hooker N, Duncan H, Jensen L (1979a) γ-Aminobutyric acid, a neurotransmitter, induces planktonic abalone larvae to settle and begin metamorphosis. Science 204:407–410Google Scholar
  19. Morse DE, Hooker N, Jensen L, Duncan H (1979b) Induction of larval abalone settling and metamorphosis by γ-aminobutyric acid and its congeners from crustose red algae: II. Applications to cultivation, seed production and bioassays; principle causes of mortality and interference. Proc World Maricul Soc 10:81–91Google Scholar
  20. Morse DE, Duncan H, Hooker N, Baloun A, Young G (1980) GABA induced behavioral and developmental metamorphosis in planktonic molluscan larvae. Fed Proc 39:3237–3241Google Scholar
  21. Paul M, Kafatos FC (1975) Specific protein synthesis in cellular differentiation. II. The program of protein synthetic changes during chorion formation by silkmoth follicles, and its implementation in organ culture. Dev Biol 42:141–159Google Scholar
  22. Sim GM, Kafatos FC, Jones CW, Koehler MD, Efstratiadis A, Maniatis T (1979) Use of a cDNA library for studies on evolution and developmental expression of the chorion multigene families. Cell 18:1303–1316Google Scholar
  23. Termine JD, Belcourt AB, Conn KM, Kleinman HK (1981) Mineral and collagen-binding proteins of fetal calf bone. J Biol Chem 256:10403–10408Google Scholar
  24. Weiner S (1979) Aspartic acid-rich proteins: Major components of the soluble organic matrix of the mollusc shells. Calcif Tissue Int 29:163–167Google Scholar
  25. Weiner S (1983) Mollusc shell formation: Isolation of two organic matrix proteins associated with calcite deposition in the bivalveMytilus californianus. Biochem 22:4139–4144Google Scholar
  26. Weiner S (1984) Organization of organic matrix components in mineralized tissues. Am Zool 24:945–951Google Scholar
  27. Weiner S, Hood L (1975) Soluble protein of the organic matrix of mollusk shells: a potential template for shell formation. Science 190:987–989Google Scholar
  28. Weiner S, Traub W (1984) Macromolecules in mollusc shells and their functions in biomineralization. Phil Trans R Soc Lond 304:425–434Google Scholar
  29. Weiner S, Lowenstam HA, Hood L (1977) Discrete molecular weight components of the organic matrices of mollusc shells. J Exp Mar Biol Ecol 30:45–51Google Scholar
  30. Wheeler AP, Sikes CS (1984) Regulation of carbonate calcification by organic matrix. Am Zool 24:933–944Google Scholar
  31. Wheeler AP, George JW, Evans CA (1981) Control of calcium carbonate nucleation and crystal growth by soluble matrix of oyster shell. Science 212:1397–1398Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Marios A. Cariolou
    • 1
  • Daniel E. Morse
    • 1
  1. 1.Marine Science Institute and Department of Biological SciencesUniversity of CaliforniaSanta BarbaraUSA

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