Summary
In vitro biosynthesis of neurosecretory peptides specific to the X-organ sinus gland (XOSG) of the land crabCardisoma carnifex was studied. The neurosecretory somata comprising the X-organ were selectively exposed to saline containing3H-leucine, glucose and antibiotics for 5 h (pulse) followed by 19–24 h of superfusion with nutrient media (chase). Analysis of extracts of somata and terminal regions by high voltage paper electrophoresis demonstrated the uptake and incorporation of3H-leucine into protein by X-organ somata and its axonal transport to terminal regions. Tritiated-leucine incorporation was reduced 90% by 50 μg/ml puromycin, indicating ribosome-mediated synthesis. Axonal transport was blocked by ligation of the axons; this did not affect the rate of3H-leucine incorporation by the somata. Radiolabelled peptide hormones were identified by comigration of radiolabel with RPLC and/or SDS-PAGE determined regions of bioactivity and by comparison of RPLC elution patterns with those of sinus gland peptides purified and analyzed for amino acid composition by Newcomb (1983a). Erythrophore concentrating hormone (ECH) and at least two 6,000 Dalton peptides having crustacean hyperglycemic hormone activity (CHH) were identified among peptides biosynthetically radiolabelled. Comparison of XOSG systems receiving 5 h of incubation with those receiving a pulse-chase (5 h∶19 h) regime demonstrates the existence of polypeptide precursors to the XOSG peptide hormones.
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Abbreviations
- CHH :
-
crustacean hyperglycemic hormone
- ECH :
-
erythrophore concentrating hormone
- EP :
-
electrophoresis
- HEPES :
-
N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- HVPE :
-
high voltage paper electrophoresis
- PAGE :
-
polyacrylamide gel electrophoresis
- RPLC :
-
reverse phase high pressure liquid chromatography
- SDS :
-
sodium dodecyl sulfate
- SG :
-
sinus gland
- XO :
-
X-organ
- XOSG :
-
X-organ sinus gland
References
Abramowitz AA, Hisaw FL, Papandrea DN (1944) The occurrence of a diabetogenic factor in the eyestalks of crustaceans. Biol Bull 86:1–5
Aloyo VJ (1979) Scintillation counting of3H- and14C-containing gel slices: A one step method. Anal Biochem 99:161–164
Andrew RD, Saleuddin ASM (1979) Two-dimensional gel electrophoresis of neurosecretory polypeptides in crustacean eyestalk. J Comp Physiol 134:303–313
Arch S (1972) Biosynthesis of the egg-laying hormone (ELH) in the bag cell neurons ofAplysia californica. J Gen Physiol 60:102–119
Bliss DE, Welsh JH (1952) The neurosecretory system of brachyuran Crustacea. Biol Bull 103:157–169
Brownstein MJ, Russell JT, Gainer H (1980) Synthesis, transport and release of posterior pituitary hormones. Science 207:373–378
Chiu AY, Hunkapiller MW, Heller E, Stuart DK, Hood LE, Strumwasser F (1979) Purification and primary structure of the neuropeptide egg-laying hormone ofAplysia californica. Proc Natl Acad Sci USA 76:6656–6660
Cooke IM (1967) Potentials recorded intracellularly from neurosecretory terminals. Am Zool 7:732–733
Cooke IM (1977) Electrical activity of neurosecretory terminals and control of peptide hormone release. In: Gainer H (ed) Peptides in neurobiology. Plenum Press, New York, pp 345–374
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 205–391
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 70:125–149
Deanin GG, Gordon MW (1973) Chloramphenicol and cycloheximide-sensitive protein synthetic systems in brain mitochondrial and nerve-ending preparations. J Neurochem 20:55–68
Enami M (1951) The sources and activities of two chromatophorotropic hormones in crabs of the genusSesarma. II. Histology of incretory elements. Biol Bull 101:241–258
Fernlund P (1971) Chromactivating hormones ofPandalus borealis: isolation and purification of a light-adapting hormone. Biochim Biophys Acta 237:519–529
Fernlund P (1974) Structure of the red-pigment-concentrating hormone of the shrimp,Pandalus borealis. Biochim Biophys Acta 371:304–311
Fernlund P (1976) Structure of a light-adapting hormone from the shrimp,Pandalus borealis. Biochim Biophys Acta 439:17–25
Fernlund P, Josefsson L (1968) Isolation and purification of the ‘red-pigment-concentrating hormone’. Biochim Biophys Acta 158:262–273
Gainer H, Sarne Y, Brownstein MJ (1977) Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides. J Cell Biol 73:366–381
Gubler U, Seeburg P, Hoffman BJ, Gage LP, Udenfriend S (1982) Molecular cloning establishes proenkephalin as precursor of enkephalin containing peptides. Nature 295:206–208
Hanström B (1931) Neue Untersuchungen über Sinnesorgane und Nervensystem der Crustaceen. I. Z Morphol Ökol Tiere 23:80–236
Herp F Van, Buggenum HJM Van (1979) Immunocytochemical localization of hyperglycemic hormone (HGH) in the neurosecretory system of the eyestalk of the crayfishAstacus leptodactylus. Experientia 35:1527–1529
Iwasaki S, Satow Y (1971) Sodium- and calcium-dependent spike potentials in the secretory neuron soma of the X-organ of the crayfish. J Gen Physiol 57:216–238
Jaros PP (1979) Immunocytochemical demonstration of the neurosecretory X-organ complex in the eyestalk of the crabCarcinus maenas. Histochemistry 63:303–310
Jaros PP, Keller R (1979) Immunocytochemical identification of hyperglycemic hormone-producing cells in the eyestalk ofCarcinus maenas. Cell Tissue Res 204:379–385
Keller R (1976) Electrophoretic analysis of neurosecretory substances from the sinus gland of decapod Crustacea. Col Int CNRS (Actualités sur les hormones d'invertébrés) 251:247–254
Keller R (1977) Comparative electrophoretic studies of crustacean neurosecretory hyperglycemic and melanophore-stimulating hormones from isolated sinus glands. J Comp Physiol 122:359–373
Keller R (1981) Purification and amino acid composition of the hyperglycemic neurohormone from the sinus gland ofOrconectes limosus and comparison with the hormone fromCarcinus maenas. J Comp Physiol 141:445–450
Keller R, Wunderer G (1978) Purification and amino acid composition of the neurosecretory hyperglycemic hormone from the sinus gland of the shore crab,Carcinus maenas. Gen Comp Endocrinol 34:328–335
Kickhofen VB, Westphal O (1952) Papierelektrophorese bei hohen Spannungen zur Trennung von Peptiden. Z Naturforsch 7b:655–659
Kleinholz LH (1975) Purified hormones from the crustacean eyestalk and their physiological specificity. Nature 258:256–257
Kleinholz LH (1976) Crustacean neurosecretory hormones and physiological specificity. Am Zool 16:151–166
Kleinholz LH, Keller R (1973) Comparative studies in crustacean neurosecretory hyperglycemic hormones. I. The initial survey. Gen Comp Endocrinol 21:554–564
Kleinholz LH, Kimball F, McGarvey M (1967) Initial characterization and separation of hyperglycemic (diabetogenic) hormone from the crustacean eyestalk. Gen Comp Endocrinol 8:75–81
Knowles FGW, Carlisle DB (1956) Endocrine control in Crustacea. Biol Rev 31:396–473
Land H, Schutz G, Schmale H, Richter D (1982) Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor. Nature 295:299–303
Lewis RV, Stern AS, Kimura S, Stein S, Udenfriend S (1980a) Enkephalin biosynthetic pathway: Proteins of 8,000 and 14,000 Daltons in bovine adrenal medulla. Proc Natl Acad Sci USA 77:5018–5020
Lewis RV, Stern AS, Kimura S, Rossier J, Stein S, Udenfriend S (1980b) An about 50,000-Dalton protein in adrenal medulla: a common precursor of [Met]- and [Leu]enkephalin. Science 208:1459–1461
Loh YP, Sarne Y, Gainer H (1975) Heterogeneity of proteins synthesized, stored and released by the bag cell ofAplysia californica. J Comp Physiol 100:283–295
Newcomb R (1983a) Peptides in the sinus gland ofCardisoma carnifex: isolation and amino acid analysis. J Comp Physiol 153:207–221
Newcomb R (1983b) Biochemical studies of neurosecretion in an Arthropod. PhD thesis, University of Hawaii Honolulu, Hawaii
Pantin CFA (1948) Notes on microscopical technique for zoologists. Revised printing. Cambridge University Press, London
Passano LM (1951) The X-organ-sinus gland neurosecretory system in crabs. Anat Rec 111:502
Passano LM (1953) Neurosecretory control of molting in crabs by the X-organ-sinus gland complex. Physiol Comp Oecol 3:155–189
Potter DD (1956) Observations on the neurosecretory system of portunid crabs. PhD Thesis, Harvard University. Cambridge, Massachusetts
Ramirez G (1973) Synaptic plasma membrane protein synthesis: selective inhibition by chloramphenicol in vivo. Biochem Biophys Res Commun 50:452–458
Sachs H (1959) Vasopressin biosynthesis. Biochim Biophys Acta 34:572–573
Sarne Y, Neale EA, Gainer H (1976) Protein metabolism in transected peripheral nerves of the crayfish. Brain Res 110:73–89
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 28:707–719
Skorkowski EF, Rykiert M, Lipinska B (1977) Hyperglycemic hormone from the eyestalk of the shrimpCrangon crangon. Gen Comp Endocrinol 33:460–466
Stuart DK, Chiu AY, Strumwasser F (1980) Neurosecretion of egg-laying hormone and other peptides from electrically active bag cell neurons ofAplysia. J Neurophysiol 43:488–498
Stuenkel E (1983) Peptide biosynthesis and secretion from a crab neurosecretory system. PhD thesis, University of Hawaii, Honolulu, Hawaii
Swank RT, Munkres KD (1971) Molecular weight analysis of oligopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Anal Biochem 39:462–477
Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412
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Stuenkel, E.L. Biosynthesis and axonal transport of proteins and identified peptide hormones in the X-organ sinus gland neurosecretory system. J Comp Physiol B 153, 191–205 (1983). https://doi.org/10.1007/BF00689623
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DOI: https://doi.org/10.1007/BF00689623