오징어(Todarodes pacificus)의 뇌로부터 평활근 수축활성을 지닌 새로운 신경성 펩타이드의 정제 및 특성

Abstract

A novel neuropeptide with contractile activity was isolated from the brain of the squid, Todarodes pacificus using the esophagus of the squid as the bioassay system. To purify the neuropeptide the acidified extract was partially purified with Sep-Pak C18 solid phase extraction cartridges using stepwise gradient with increasing the percent of methanol (10 %, 60 % and 100 %) containing 0.1 % TFA. Contractile activity was recovered in the 60 % methanol fraction and this fraction was further purified by ion-exchange and C18 reversed-phase HPLC. Finally, a new peptide was obtained in isocratic condition of 21 % CH3CN at a flow rate of 0.5 ml/min. This peptide was analyzed by MALDI-TOF mass and Edman degradation method. The molecular weight of this peptide was 1559.9 Da and the peptide was tetradecapeptide. To compare the identity between native and synthetic peptide, the purified peptide was synthesized by the solid phase method. The C-terminal portion of this peptide turned out to be -NH2 form. From these results, the structure of this peptide was having the following primary structure: GFKDNVSNRIAHGFamide. We have named it squid brain tetradecapeptide (SBTP). SBTP showed a potent excitatory action on the esophagus of the squid. The threshold concentration of SBTP was between 5 x 10-10 M and 10-9 M, and Emax was 166.39 ± 26.09 % at 10-6 M. In order to investigate the structure-activity relationship of SBTP, various analogs of SBTP were synthesized. They are analogs with is deleted one by one amino acid residue in N- and C-terminus. Contractile activities of these peptides were measured by using the mid-esophagus of squid. N-terminal deleted analog, 1DES -SBTP, showed a similar contractile effect compared with that of SBTP-NH2, while, other analogs was inactive. These results indicate that 1DES-SBTP is the minimum structure required for the contractile activity on the esophagus of the squid.