Isolation of pyroGlu-Gly-Arg-Phe-NH2 (Antho-RFamide), a neuropeptide from sea anemones.

Abstract

A radioimmunoassay has been developed for peptides containing the carboxyl-terminal sequence Arg-Phe-NH2 (RFamide). Using this radioimmunoassay and applying cation-exchange chromatography and HPLC, we (2,3,8,9). This paper describes the development of a radioimmunoassay for the coelenterate RFamide-like peptides and the purification and sequencing of such a peptide from the sea anemone Anthopleura elegantissima. METHODSRadioimmunoassay. Four rabbits were immunized with RFamide (Bachem, Bubendorf, Switzerland) that was coupled by carbodiimide to bovine thyroglobulin (Sigma). The antisera were treated overnight at 4°C with bovine thyroglobulin at 1 mg/ml, followed by centrifugation, to remove antibody populations specific for the carrier protein. One antiserum (code 145IV) was selected for the present study.125I-labeled Tyr-Phe-Met-Arg-Phe-NH2 was used as a tracer in the radioimmunoassay. It was prepared by adding 5 1.d (0.5 mCi; 18 MBq) of Na125I (Amersham) to 15 ,ul of 0.2 M sodium phosphate buffer, pH 7.0, containing 0.22 ,ug of Tyr-PheMet-Arg-Phe-NH2 (Peninsula Laboratories, San Carlos, CA), followed by addition of 10 ul of phosphate buffer containing 1.4 ,g of chloramine-T. After mixing, the reaction was stopped after 20 sec with 500 1.l of 0.3% Na2S205. The mixture was immediately loaded on a methanol/watertreated Sep-Pak C18 cartridge (Waters Associates), which was quickly rinsed with 40 ml of twice-distilled water, to remove unreacted 1251. Labeled material was eluted with 2-ml portions of 40%, 60%, 80%, and 90% (vol/vol) methanol and three times with 2-ml portions of 100% methanol. Each fraction was collected in 6 ml ofRIA buffer, consisting of0.05 M sodium phosphate at pH 7.4 and 1% bovine serum albumin (Sigma). The major portion of the tracer was eluted at 90% or 100% methanol. The quality of an aliquot (3000 cpm) of the tracer fractions was tested by overnight incubation, at 40C, in

Molecular cloning and functional expression of the first insect FMRFamide receptor

“…Since then, FMRFamide and FMRFamide-related peptides have been isolated from a wide variety of invertebrate species, ranging from primitive metazoans, such as cnidarians, to higher invertebrates, such as insects (2)(3)(4)(5). FMRFamide-related peptides occur in all classes of cnidarians, suggesting that these peptides were among the first transmitters used in evolution (6).…”

Isolation of L-3-phenyllactyl-Leu-Arg-Asn-NH2 (Antho-RNamide), a sea anemone neuropeptide containing an unusual amino-terminal blocking group.

“…The RFamide radioimmunoassay was described earlier (10). For the Arg-Asn-NH2 (RNamide) radioimmunoassay, four rabbits were immunized with RNam-ide that was coupled to bovine thyroglobulin (Sigma) via carbodiimide.…”

“…10, except that instead of methanol, increasing concentrations of acetonitrile in 0.1% trifluoroacetic acid (TFA) were used for elution of the tracer. All further information concerning the radioimmunoassay is given in ref.…”

“…For the Arg-Asn-NH2 (RNamide) radioimmunoassay, four rabbits were immunized with RNam-ide that was coupled to bovine thyroglobulin (Sigma) via carbodiimide. The antisera were treated as described (10). Antiserum 197, bleeding VII (197 VII) was selected for the radioimmunoassay.…”

“…By means of immunocytochemistry with antisera to the sequence ArgPhe-NH2 (RFamide), RFamide-like peptides were demonstrated in the nervous systems of animals belonging to all classes of coelenterates (5)(6)(7)(8)(9). With a radioimmunoassay for RFamide, three RFamide-like peptides were isolated from the sea anemone Anthopleura elegantissima and sequenced: <Glu-Gly-Arg-Phe-NH2 (Antho-RFamide), <Glu-SerLeu-Arg-Trp-NH2 (Antho-RWamide I), and <Glu-GlyLeu-Arg-Trp-NH2 (Antho-RWamide II) (10)(11)(12). The general structure of these peptides can be described as <Glu ... Arg-Xaa-NH2, where Xaa is an aromatic amino acid.…”