Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon.
FMRFamide-like immunoreactivity (FLI) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using a combination of three anti-FMRFamide-like peptide (FLPs) monoclonal antibodies. Approximately 3000 small neuronal cell bodies in the lamina ganglionalis; 100 medium to large size at the ganglion between the medulla interna and the medulla terminalis; and 250 medium size around the medulla terminalis were stained intensely. The neuronal processes in neuropils of the medulla externa, medulla interna, medulla terminalis, sinus gland and some nerve fibers in the optic nerve were also recognized. The small cell bodies, approximately 1500 cells, anterior to the medulla externa were stained inconsistently and the neuronal processes were not observed from these cells. Isolation of FLPs from 9000 eyestalks was performed using methanol/acetic/water (90:1:9) extraction. After the extract was partially purified using C18 cartridges, it was further purified by five to seven steps of RP-HPLC using three kinds of columns: C18; C8; and cyano, and three solvent systems: acetonitrile/trifluoro acetic acid; aceonitrile/heptafluoro butyric acid; and acetonitrile/triethyl ammonium acetate. Dot-ELISA using the combination of the same antibodies was used to monitor FLPs in the fractions during purification processes. Seven new sequences of FLPs were identified which can be divided into four subgroups according to the primary structure of the C-terminus: (1) GDRNFLRFamide; (2) AYSNLNYLRFamide; (3) AQPSMRLRFamide, SQPSMRLRFamide, SMPSLRLRFamide and DGRTPALRLRFamide; and (4) GYRKPPFNGSIFamide. These data indicate the high complexity of this peptide family in which multiple forms are usually exist.
Sithigorngul
Paisarn
P
Department of Biology, Faculty of Science, Srinakharinwirot University, Sukhumvit 23, 10110, Bangkok, Thailand. paisarn@psm.swu.ac.th
Pupuem
Jirasak
J
Krungkasem
Chatchadaporn
C
Longyant
Siwaporn
S
Chaivisuthangkura
Parin
P
Sithigorngul
Weerawan
W
Petsom
Amorn
A
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005136:Eye Proteins / Q000302:isolation & purification / Q000378:metabolism*; D019835:FMRFamide / Q000302:isolation & purification / Q000378:metabolism*; D007124:Immunoenzyme Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000302:isolation & purification / Q000378:metabolism*; D033561:Penaeidae / Q000378:metabolism*; D017956:Photoreceptor Cells, Invertebrate / Q000737:chemistry / Q000378:metabolism*
D005136:Eye Proteins; D009842:Oligopeptides; D019835:FMRFamide