Journal of comparative physiology

, Volume 153, Issue 2, pp 207–221 | Cite as

Peptides in the sinus gland ofCardisoma carnifex: isolation and amino acid analysis

  • Robert W. Newcomb


Reversed phase liquid chromatography (RPLC) has been used to isolate most of the peptides in the sinus gland of the land crabCardisoma carnifex. The peptides accounted for at least 50% (5–10 μg) of the non-amino acid primary amine compounds of the sinus gland. Peptides were characterized by (1) amino acid analysis; (2) bioassay for erythrophore concentrating, melanophore dispersing and hyperglycemic activity; (3) a comparison of absorption at 280 nm with intrinsic fluorescence; (4) RPLC elution properties relative to the shrimp crythrophore concentrating hormone (ECH); and (5) post-column detection with the fluorescamine reagent. High sensitivity peptide maps were obtained by RPLC of the peptide fluoreseamine derivatives.

Peptides in theCardisoma sinus gland can be summarized as:
  1. 1.

    Relatively hydrophobic peptides which elute in three broad peaks, labeled J, K, and L, each of which is possibly heterogeneous. The amino acid compositions of these fractions are similar to those previously reported by others for the hyperglycemic hormones of several species and at least two possessed this activity.

  2. 2.

    Compounds which eluted before the J, K and L peptide fractions, and which both absorbed light at 280 nm and possessed intrinsic fluorescence, Because of these properties, these compounds were considered likely to be peptides. One is indistinguishable from the shrimp ECH by (a) chromatography on two separate columns, (b) amino acid analysis, (c) the ratio of absorbance at 280 nm to intrinsic fluorescence, and (d) bioactivity on the erythrophores ofOcypode pallidula.

  3. 3.

    Peptides (all novel, labeled C-I) which did not possess tyr, trp, val, ile or lys, and which were more polar than the J, K and L peptide fractions. By amino acid analysis, all of these could be explained by cleavage from the largest, H, of M.W. 3300 to 4000. On a mole basis, the C-I peptides accounted for almost all of the small (M.W. 500–4000) peptide material of theCardisoma sinus gland.



Peptide Primary Amine Amino Acid Analysis Intrinsic Fluorescence Reverse Phase Liquid Chromatography 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



crustacean hyperglycemic hormone


erythrophore concentrating hormone


octadecyl silica




pigment dispersing hormone


reversed phase high pressure liquid chromatography


trifluoroacetic acid




Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andrew RD, Salueddin ASM (1979) Two-dimensional gel electrophoresis of neurosecretory polypeptides in crustacean eyestalk. J Comp Physiol 134:303–313Google Scholar
  2. Arch S (1972) Polypeptide secretion from the isolated parietovisceral ganglion ofAplysia californica. J Gen Physiol 59:47–59Google Scholar
  3. Castell JV, Cervera M, Marco R (1979) A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A reexamination of the conditions of the reaction. Anal Biochem 99:379–391Google Scholar
  4. Chen RF, Costt C, Trepman E (1979) Fluorescence properties of o-phthaldialdehyde derivatives of amino acids. Biochem Biophys Acta 576:440–455Google Scholar
  5. Cooke IM, Sullivan RE (1982) Hormones and neurosecretion. In: Atwood H, Sandeman D (eds) The biology of Crustacea, vol 3. Academic Press, New York, pp 205–290Google Scholar
  6. Cooke IM, Haylett BA, Weatherby TM (1977) Electrically elicited neurosecretory and electrical responses of the isolated crab sinus gland in normal and reduced calcium salines. J Exp Biol 70:125–149Google Scholar
  7. Crestfield AM, Moore S, Stein W (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated protein. J Biol Chem 238:622–627Google Scholar
  8. Fernlund P (1974) Synthesis of the red-pigment-concentrating hormone of the shrimp,Pandalus borealis. Biochim Biophys Acta 371:312–322Google Scholar
  9. Fernlund P (1976) Structure of a light-adapting hormone from the shrimpPandalus borealis. Biochim Biophys Acta 439:17–25Google Scholar
  10. Fernlund P, Josefsson L (1971) Chromactivating hormones ofPandalus borealis: Isolation and purification of a lightadapting hormone. Biochim Biophys Acta 234:519–529Google Scholar
  11. Fernlund P, Josefsson L (1972) Crustacean color-change hormone: amino acid sequence and chemical synthesis. Science 177:173–175Google Scholar
  12. Fingerman M (1966) Neurosecretory control of pigmentary effectors in crustaceans. Am Zool 6:169–179Google Scholar
  13. Fingerman M, Fingerman SW, Hammond RD (1974) Comparison of red-pigment-concentrating hormones from the eyestalks of the fiddler crab,Uca pugilator, and the prawn,Palaemonetes vulgaris, with synthetic red pigment-concentrating hormone ofPandalus borealis. Gen Comp Endocrinol 23:124–126Google Scholar
  14. Gruber KA, Stein S, Brink L, Radhaleristwass A, Udenfriend S (1976) Fluorometric assay of vasopressin and oxytocin: A general approach to the assay of peptides in tissues. Proc Nat Acad Sci USA 73:1314–1318Google Scholar
  15. Kamemoto FI (1976) Neuroendocrinology of osmoregulation in decapod Crustacea. Am Zool 16:141–150Google Scholar
  16. Keller R (1977) Comparative electrophoretic studies of crustacean neurosecretory and melanophore stimulating hormones from isolated sinus glands. J Comp Physiol 122:259–373Google Scholar
  17. Keller R (1981) Purification and amino acid composition of the hyperglycemic neurohormone from the sinus gland ofOrconectes limosus and comparison with the hormone ofCarcinus maenas. J Comp Physiol 141:445–450Google Scholar
  18. Keller R, Wunderer G (1978) Purification and amino acid composition of the neurosecretory hyperglycemic hormone from the sinus gland,Carcinus maenas. Gen Comp Endocrinol 34:328–335Google Scholar
  19. Kleinholz LH (1970) A progress report on the separation and purification of crustacean neurosecretory pigmentory effector hormones. Gen Comp Endocrinol 14:578–588Google Scholar
  20. Kleinholz LH (1972) Comparative studies of crustacean melanophore stimulating hormones. Gen Comp Endocrinol 19:473–483Google Scholar
  21. Kleinholz LH (1975) Purified hormones from the crustacean eyestalk and their physiological specificity. Nature 258:256–257Google Scholar
  22. Kleinholz LH (1976) Crustacean neurosecretory hormones and physiological specificity. Am Zool 16:151–166Google Scholar
  23. Kleinholz LH, Keller R (1973) Comparative studies in crustacean neurosecretory hyperglycemic hormones. I. The initial survey. Gen Comp Endocrinol 21:554–564Google Scholar
  24. Leuven RSEW, Jaros PP, Van Herp F, Keller R (1982) Species or group specificity in biological and immunological studies of crustacean hyperglycemic hormone. Gen Comp Endocrinol 46:288–296Google Scholar
  25. Lindorth P, Mopper K (1979) High performance liquid chromatographic determination of subpicomole amounts of amino acids by precolumn fluorescence derivatization with ophthaldialdehyde. Anal Chem 51:1667–1674Google Scholar
  26. Mancillas JR, McGinty JF, Selverston AI, Karten H, Bloom FE (1981) Immunocytochemical localization of enkephalin and substance P in retina and eyestalk neurones of lobster. Nature 293:576–578Google Scholar
  27. McHugh W, Sandemann RA, Haney WG, Sood SP, Wittmer DP (1976) Characterization of selected fluorescamine-amino acid reaction products by high performance liquid chromatography. J Chromatogr 124:376–380Google Scholar
  28. Mordue W, Stone J (1976) Comparison of the biological activities of an insect and a crustacean neurohormone that are structurally similar. Nature 264:287–289Google Scholar
  29. Newcomb R (1983) Biochemical studies of neurosecretion in an Arthropod. PhD thesis, University of Hawaii, Honolulu, HawaiiGoogle Scholar
  30. Nice EC, Capp M, O'Hare MJ (1979) Use of hydrophobic interaction methods in the isolation of proteins from endocrine and paraendocrine cells by high performance liquid chromatography. J Chromatogr 185:413–427Google Scholar
  31. Scheller RH, Jackson JF, McAllister LB, Schwartz JH, Kandel ER, Axel R (1982) A family of genes that codes for ELH, a neuropeptide eliciting a stereotyped pattern of behavior inAplysia. Cell 28:707–719Google Scholar
  32. Stuenkel E (1983a) Biosynthesis and axonal transport of proteins and identified peptide hormones in the X-organ sinus gland neurosecretory system. J Comp PhysiolGoogle Scholar
  33. Stuenkel E (1983b) Peptide biosynthesis and secretion from a crab neurosecretory system. PhD thesis, University of Hawaii, Honolulu, HawaiiGoogle Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • Robert W. Newcomb
    • 1
  1. 1.Department of Biochemistry and Biophysics and Békésy Laboratory of NeurobiologyUniversity of HawaiiHonoluluUSA

Personalised recommendations