Purification and properties of ghrelin from the intestine of the goldfish, Carassius auratus.
In goldfish, intraperitoneal (IP) or intracerebroventricular (ICV) administration of synthetic ghrelin consisting of 12- or 19-amino-acid residues, deduced from its precursor cDNA, with an octanoic acid modification at the third N-terminal serine residue (Ser(3)), stimulates growth hormone release and food intake. However, native ghrelin generated from its precursor has not yet been identified in this species. Therefore, we purified ghrelin from the goldfish intestine using acid extraction, cation-exchange and reverse-phase high-performance liquid chromatography combined with immune-affinity purification. In order to confirm ghrelin activity in the fractions at each purification step, we examined the effect of each fraction on intracellular Ca(2+) mobilization in rat growth hormone secretagogue-receptor (GHS-R)-expressing cells. We characterized the goldfish ghrelin as 11 molecular forms consisting of 14-, 17-, 18- and 19-amino-acid residues with acylation at Ser(3), and the 17-residue form was predominant. We then synthesized 17-residue forms with octanoic acid modification (octanoyl ghrelin17) and without acylation (des-acyl ghrelin17) at Ser(3), and examined their biological activity. Octanoyl ghrelin17, but not des-acyl ghrelin17, increased the intracellular Ca(2+) concentration in rat GHS-R-expressing cells with a potency similar to those of synthetic ghrelin consisting of 12 residues (octanoyl ghrelin12) and octanoyl rat ghrelin. IP and ICV administration of octanoyl ghrelin17 and octanoyl ghrelin12, but not des-acyl ghrelin17, increased food intake in goldfish. The present findings indicate that native goldfish ghrelin consists of 11 molecular variants, the major form being a 17-residue peptide. This dominant form with acylation is implicated in the regulation of food intake in goldfish.
Miura
Tohru
T
University of Toyama, Gofuku, Japan.
Maruyama
Keisuke
K
Kaiya
Hiroyuki
H
Miyazato
Mikiya
M
Kangawa
Kenji
K
Uchiyama
Minoru
M
Shioda
Seiji
S
Matsuda
Kouhei
K
D000818:Animals; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D005247:Feeding Behavior / Q000187:drug effects; D054439:Ghrelin / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification* / Q000494:pharmacology; D006054:Goldfish; D007274:Injections, Intraperitoneal; D007276:Injections, Intraventricular; D007422:Intestines / Q000737:chemistry*; D012333:RNA, Messenger / Q000235:genetics
D054439:Ghrelin; D012333:RNA, Messenger