별 불가사리(Asterina pectinifera) 조직별 초산추출물의 생리활성 탐색

고혜진 ( Hye Jin Go ),조미정 ( Mi Jeong Jo ),김군도 ( Gun Do Kim ),박남규 ( Nam Gyu Park ) 한국수산과학회 2014 한국수산과학회지 Vol.47 No.2

The present study was performed to examine the contraction and relaxation responses of the smooth muscles, and search for antimicrobial and antioxidant activities in the tissues, of the starfish Asterina pectinifera. Frozen samples were extracted with distilled water containing 1% acetic acid. Extracts from all tissues showed potent antimicrobial activity against Escherichia coli D31. Relatively high levels of antimicrobial activity were also detected in the body extracts. Liver, tube feet, and body extracts caused contraction responses in the dorsal retractor muscles (DRM) of the starfish. In contrast, all tissues examined exhibited contractile activity in the esophagus of squid Todarodes pacificus. In addition, liver and gonad extracts caused contraction responses upon application to the intestine of the puffer fish Takifugu pardalis. Relaxation effects on the DRM of starfish were identified in most of the extracts, while no relaxant activity was detected in body extracts. Extracts from all tissues examined also exhibited antioxidant activities. The results of this study suggest that starfish are a potential source of novel bioactive compounds.

가물치(Channa argus)로부터 평활근 수축활성 펩타이드의 정제

고혜진 ( Hye Jin Go ),박남규 ( Nam Gyu Park ) 한국수산과학회 2012 한국수산과학회지 Vol.45 No.2

A novel neuropeptide was isolated from the skin of the snakehead Channa argus using the dorsal retractor muscle (DRM) of a starfish Asterina pectinifera as a bioassay system. The amino acid sequence of the purified peptide was analyzed using automated sequencing and MALDI-TOF mass spectrophotometry. The primary structure of the purified peptide was determined to be Pro-Ala-Leu-Ala-Leu. To investigate the complete primary structure of this peptide, Pro- Ala-Leu-Ala-Leu-OH and Pro-Ala-Leu-Ala-Leu-NH2 were synthesized. The chemical and pharmacological properties of the synthetic peptides were compared with those of the native peptide. Both the native peptide and synthetic Pro-Ala- Leu-Ala-Leu-OH had identical behaviors on the reverse-phase and cation-exchange HPLC chromatograms. Synthetic Pro-Ala-Leu-Ala-Leu-OH showed contractile activity on the DRM, and the threshold concentration of this peptide was approximately 10-8 M. The maximal contractile effect (Emax) of this peptide was 294±45.4% at 10-5 M.

Synthesis and Biological Activities of Myomodulin E and its Analogs

Hye-Jin Go(고혜진),Jung-Kil Seo(서정길),Hae Jeom Seo(서혜점),Min Jeong Lee(이민정),Tae Hyun Park(박태현),Gun Do Kim(김군도),Nam Gyu Park(박남규) 한국생명과학회 2012 생명과학회지 Vol.22 No.4

군소인 Aplysia kurodai의 중추신경절로부터 발견된 myomodulin A (MMA, PMSMLRLamide)와 myomodulin E (MME, GLQMLRLamide)는 Mytilus edulis의 anterior byssus retractor muscle (ABRM)을 활성측정시스템으로 사용하여 정제되었다. . 정제된 MMA와 MME는 연체동물에서 발견된 myomodulin 계열의 펩타이드와 동일한 일차구조를 지닌다. MME의 구조와 활성간의 상관관계를 알아보기 위해서 MME, 유도체 및 다른 신경성 펩타이드들을 합성하였다. MME의 유도체인 Des[Gly¹]-MME, Des[Gly¹,Leu²]-MME 및 Des[Gly¹, Leu²,Gln³]-MME의 일차구조는 각각 LQMLRLamide, QMLRLamide 및 MLRLamide이다. 합성 물질들을 사용하여 ABRM에 대한 phasic -contraction을 측정하였다. MME는 1×10<SUP>-9</SUP> M 또는 더 높은 농도에서 ABRM의 phasic contraction을 저해하였다. 또한 MME는 1×10<SUP>-8</SUP> M에서 catch-tension에 대해 이완활성을 나타내었다. 합성 펩타이드들을 사용하여 Africa giant snail, Achatina fulica의 소낭과 penial retractor muscle에 대해서도 활성을 측정하였다. MME와 유도체들은 소낭에 대해서는 수축반응을 보였지만, penial retractor muscle에 대해서는 이완 활성을 나타내었다. 이러한 결과들은 MME와 그 유도체들은 연체동물의 다양한 조직에 대해 조절 효과를 가지고 있다는 것을 의미한다. 본 연구는 생체 내에서 발생하는 신경 및 circuit의 변화를 조절하는 작용 연구에 대한 기본적인 자료가 될 것이다. Previous work has characterized myomodulin A (MMA, PMSMLRLamide) and myomodulin E (MME, GLQMLRLamide) purified from the central nervous systems of the sea hare, Aplysia Kurodai, using the anterior byssus retractor muscle (ABRM) of the mussel, Mytilus edulis. The amino acid sequences of MMA and MME were the same as those of the myomodulin family peptide found in other mollusks. In this study, we synthesized MME, its derivatives, and other neuropeptides to investigate the relationship between the structure and biological activity of MME. The primary structures of MME’s derivatives, Des[Gly¹]-MME, Des[Gly¹,Leu²]-MME, and Des[Gly¹,Leu²,Gln³]-MME, were LQMLRLamide, QMLRLamide, and MLRLamide, respectively. MMA and synthetic peptides were tested on ABRM in M. edulis as well as muscle preparations in Achatina fulica. MME displayed an inhibitory effect on phasic contraction of the ABRM at 1×10<SUP>-9</SUP> M or higher. MME also had a relaxing effect on the catch-tension of AMRM at 1×10<SUP>-8</SUP> M. Both MMA and its analogs stimulated a contractile response on the crop and relaxed the catch-relaxing response on the penial retractor muscle of A. fulica. These results suggest that MME and its analogs have modulatory effects on various muscles of mollusks. This study has also laid the groundwork for future neural and circuit modulation studies during animal behavioral changes.

붕장어(Conger myriaster)의 피부로부터 평활근 수축작용을 지닌 신경성 펩타이드의 정제

고혜진 ( Hye Jin Go ),박남규 ( Nam Gyu Park ) 한국수산과학회 2012 한국수산과학회지 Vol.45 No.4

A novel neuropeptide was isolated from the skin of the conger eel Conger myriaster using hagfish Eptatretus burgeri intestine as a bioassay system. The sequence of the purified peptide was analyzed using automated amino acid sequencing and MALDI-TOF mass spectrophotometry. The molecula ion peak in the MALDI-TOF mass spectrum of the peptide was at m/z 962.89 (M+H)+. The sequence of the peptide was determined to be L-P-M-L-E-T-Q-M, and was tentatively named comyrin. To investigate the complete primary structure of comyrin, comyrin-OH and comyrin-NH2 were synthesized and the chemical and pharmacological properties of the synthetic peptides were compared with those of the native peptide. However, the elution time of synthetic peptides did not match that of the native peptide on the reverse-phase HPLC chromatogram. In addition, the synthetic peptides did not cause contractile activity in the intestinal smooth muscle of the hagfish. Based on these results, one possible reason for this disagreement may be the presence of a D-amino acid in comyrin.

아무르불가사리 추출물의 생리활성

고혜진(Hye-Jin Go),조미정(Mi Jeong Jo),김태영(Tae Young Kim),홍용기(Yong-Ki Hong),김군도(Gun-Do Kim),이병우(Byung-Woo Lee),박남규(Nam Gyu Park) 한국생명과학회 2014 생명과학회지 Vol.24 No.5

아무르불가사리(Asterias amurensis)의 6가지의 조직 추출물들을 사용하여 항균활성, 항산화 활성 및 평활근에 대한 수축과 이완반응에 대한 활성을 측정하였다. 1% (v/v) 초산용액을 사용하여 동결 건조한 샘플들을 추출하여 각각의 활성측정에 사용한 결과, 껍질을 제외한 모든 조직 추출물들은 E. coli D31에 대해 강한 항균활성을 나타내었고, 근육 및 장관 추출물들은 별불가사리(Asterina pectinifera)의 DRM에 대해 수축반응을 나타냈다. 한편, 오징어(Todarodes pacificus) 식도에 대한 수축반응은 실험에 사용한 모든 조직 추출물들에서 검출되었다. 특히, 간 추출물의 수축활성은 다른 조직의 추출물의 활성에 비해 더 높았다. 뿐만 아니라 간, 근육 및 소화관 추출물은 졸복(Takifugu pardalis)의 장관에 대해서 수축반응을 나타냈다. 별불가사리의 DRM에 대한 이완반응은 실험에 사용한 모든 조직 추출물들에서 활성을 나타냈다. 항산화 활성은 장관, 간 및 껍질의 추출물들에서 강하게 나타났다. 따라서 아무르불가사리는 새로운 생체기능 조절물질의 발견에 대한 가능성을 지닌 자원이라는 것을 본 연구의 결과들은 제시한다. Nowadays, starfish is one of problems about interruption of marine ecosystem, so many researchers are focusing on application of the starfish (Asterias amurensis). In this study, we investigated the contractile and relaxant activity of the smooth muscles and the antimicrobial and antioxidant activity of six different tissues (muscle, gut, liver, tube feet, gonads, and body) of A. amurensis. Frozen samples were extracted with distilled water containing 1% acetic acid. Extracts from all the tissues, except the body tissue, showed potent antimicrobial activity against Escherichia coli D31. The dorsal retractor muscles (DRM) of muscle and gut extracts showed strong contraction responses. On the other hand, contractile activity on esophagus of squid Todarodes pacificus could be detected in all tissues tested. The contractile activity of the liver extract was higher than that of the other tissues. The body, tube feet, and liver extracts showed the contractile activity on the intestine of the panther puffer fish (Takifugu pardalis). Relaxation response on the DRM of starfish (A. pectinifera) was observed in all tissues tested. Increased antioxidant activity was observed in the gut, liver, and body extracts. The results suggest that the starfish (A. amurensis) is a potential source of novel bioactive compounds.

붕장어 (Conger myriaster)의 뇌로부터 Vasopressin-related Peptide, [Arg8]-vasotocin의 정제

고혜진 ( Hye Jin Go ),김찬희 ( Chan Hee Kim ),김은정 ( Eun Jung Kim ),김인혜 ( In Hye Kim ),박희연 ( Hee Yeon Park ),윤호동 ( Ho Dong Yoon ),홍용기 ( Yong Ki Hong ),박남규 ( Nam Gyu Park ) 한국수산과학회 2005 한국수산과학회지 Vol.38 No.1

N/A Vasopressin (VP)-related peptide was purified from the brain extract of conger eel (Conger rnvriaster) by reverse-phase, ion-exchange high performance liquid chromatography (HPLC). This peptide with a molecular weight of 1,051.2 Da was determined as H-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NH₂, whose Cys residues made an intramolecular disulfide bridge by the automated amino acid sequence analysis, MALDI-TOF mass spectrometry. It`s sequence was confirmed by identity of the elution position with the synthetic peptide in HPLC system. As a result of homology investigation, the primary structure of this peptide was the same as that of VP-superfamily member, [Arg^(8)]-vasotocin. The synthetic peptide showed a contractile activity at a minimal effective concentration of 10^(-10) M on the intestinal smooth muscle of goldfish.

별불가사리 Asterina pectinifera의 유문맹낭 추출물로부터 새로운 2종류의 항균활성 펩타이드의 정제

고혜진(Hye-Jin Go),배윤정(Yun Jung Bae),박남규(Nam Gyu Park) 한국생명과학회 2014 생명과학회지 Vol.24 No.8

별불가사리(Asterina pectinifera)의 유문 맹낭 추출물로부터 새로운 항균활성 펩타이드를 정제하기 위해서 유문맹낭 추출물을 역상 HPLC와 이온 HPLC column에 주입하였다. 강한 항균활성을 나타내는 2개의 새로운 펩타이드가 유문 맹낭 추출물로부터 정제되었다. 이러한 물질들은 Pyloric caeca Asterina pectinifera peptides (PAP-1와 PAP-2)라 명명하였다. 정제한 물질들의 특성을 알아보기 위해서, 분자량 및 아미노산 서열 분석은 MALDI-TOF 질량분석기와 에드만 분해법으로 조사하였다. PAP-1과 PAP-2의 분자량은 각각 약 2952 Da 및 2980 Da이었다. PAP-1과 PAP-2의 부분적인 N-말단 서열은 다음과 같다. PAP-1, AIQNAGES; PAP-2, AIQNAAES. PAP-2는 PAP-1의 6번째 위치(glycine or alanine)에서 한 잔기만 다른 isoform에 해당된다. 지금까지 밝혀진 항균활성 펩타이드와의 분자량 및 N-말단 아미노산 서열을 비교한 결과, 이들 물질들은 다른 물질들과 동일성을 나타내지 않았다. 이러한 발견은 PAP-1과 PAP-2가 별불가사리의 유문맹낭의 선천성 방어계에 중요한 역할을 담당하고 있는것을 시사하고 있다. PAP-1, a novel antimicrobial peptide isolated from pyloric caeca extract of the starfish Asterina pectinifera was purified and characterized. First, the acidified pyloric caeca extract was put through Sep-Pak C18 solid phase extraction cartridge using a stepwise gradient. Among the eluents, RM 60 (retained materials at 60% methanol) showed good antimicrobial activity against Bacillus subtilis and Escherichia coli D31 and was purified in C18 reversed-phase and ion-exchange high-performance liquid chromatography columns. The purification steps yielded two novel peptides showing strong antimicrobial activities. These peptides were named pyloric caeca A. pectinifera peptide 1 and 2 (PAP-1 and PAP-2). For the characterization of the purified peptides, the molecular weights and amino acid sequences were determined by MALDI-TOF MS and Edman degradation. The molecular weights of PAP-1 and PAP-2 were about 2951.54 Da and 2980.15 Da respectively. The amino acid sequences of PAP-1 and PAP-2 were partially determined: AIQNAGES and AIQNAAES, respectively. PAP-2 is an isoform of PAP-1, differing merely by a single residue at position 6 (glycine or alanine). The comparison of the N-terminal amino acid sequences and molecular weights of the peptides with those of other known antimicrobial peptides revealed that PAP-1 and PAP-2 have no homology with any known peptides. These findings suggest that PAP-1 and PAP-2 play a significant role in the innate defense system of starfish pyloric caeca.

붕장어(Conger myriaster)의 뇌로부터 Oxytocin-related Peptide, Isotocin의 정제

고혜진 ( Hye Jin Go ),김찬희 ( Chan Hee Kim ),김은정 ( Eun Jung Kim ),김인혜 ( In Hae Kim ),안상현 ( Sang Hyun An ),손희영 ( Hee Young Sohn ),박진일 ( Jin Il Park ),박희연 ( Hee Yun Park ),윤호동 ( Ho Dong Yoon ),박남규 ( Nam Gyu 한국수산과학회 2005 한국수산과학회지 Vol.38 No.5

개불 라이소자임 유래 항균성 모델 펩타이드(Uu-ilys-CF)의 재조합 단백질 생산 및 항균 활성

오혜영(Hye Young Oh),고혜진(Hye-Jin Go),박남규(Nam Gyu Park) 한국생명과학회 2021 생명과학회지 Vol.31 No.1

개불 라이소자임(Uu-ilys)은 개불(Urechis unicinctus)로부터 정제된 무척추형 라이소자임으로 병원균에 대한 방어에 주요하게 작용하는 선천성 면역 물질이며, 비효소적 항균 활성을 가지고 있어 항균 활성을 지닌 유도체의 개발 가능성을 가지고 있다. 본 논문은 개불 라이소자임에서 유래된 항균 활성을 가지는 유도체의 디자인과 생산을 기술하고 있다. 여러 항균성 펩타이드(antimicrobial peptide, AMP) 데이터베이스에서 제공하는 항균성 펩타이드 예측 도구를 사용하여 개불 라이소자임에서 항균 활성을 가지는 부위를 예측하였다. 개불 라이소자임 C-말단의 절편이 항균 활성을 나타낼 것으로 예측되었으며, 이 펩타이드는 C-말단 절편, Uu-ilys-CF라 명명하였다. Uu-ilys-CF은 이형 발현 시스템인 TrxA-Uu-ilys-CF 퓨전 단백질을 사용하여 생산하였다. 만들어진 퓨전 단백질은 브롬화시안을 사용하여 메티오닌 잔기를 절단하였으며, 절단된 Uu-ilys-CF은 고성능액체크로마토그래피와 역상 컬럼인 CapCell-Pak C18을 사용하여 분리되었다. Uu-ilys-CF의 항균 활성을 조사하기 위해서 여러 균주(그람양성균 4개, 그람음성균7개, 진균 1개)를 사용하였다. Uu-ilys-CF의 항균 활성은 살모넬라에서 가장 높은 반응을 보였다. 비록 Uu-ilys-CF는 진균에 활성을 나타내지 않았지만, 사용한 균주들에서 넓은 범위의 항균 활성을 나타내었다. Uu-ilys, an i-type lysozyme from spoon worm (Urechis unicinctus), is an innate immune factor that plays an important role in the defense against pathogens. It also possesses non-enzymatic antibacterial activity. Thus, there is a possibility to develop an antimicrobial model peptide from Uu-ilys. In this study, we report the design, production, and antibacterial activity of an Uu-ilys analog that exhibits antibacterial activity. The Uu-ilys structure was fragmented according to its secondary structures to predict the regions with antimicrobial activity using antimicrobial peptide (AMP) prediction tools from different AMP databases. A peptide containing the C-terminal fragment was predicted to exert antimicrobial activity. The chosen fragment was designated as an Uu-ilys analog containing the C-terminal fragment, Uu- ilys-CF. To examine the possibility of developing an AMP using the sequence of Uu-ilys-CF, recombinant fusion protein (TrxA-Uu-ilys-CF) was produced in an expression system that was heterologous. The produced fusion protein was cleaved after methionine leaving Uu-ilys-CF free from the fusion protein. This was then isolated through high performance liquid chromatography and reverse phase column, CapCell-Pak C18. The antibacterial activity of Uu-ilys-CF against different microbial strains (four gram-positive, six gram-negative, and one fungal strain) were assessed through the ultrasensitive radial diffusion assay (URDA). Among the bacterial strains tested, Salmonella enterica was the most susceptible. While the fungal strain tested was not susceptible to Uu-ilys-CF, broad spectrum antibacterial activity was observed.

아무르 불가사리(Asterias amurensis)의 관족으로부터 신경성 펩타이드의 분리 및 정제

조미정 ( Mi Jeong Jo ),고혜진 ( Hye Jin Go ),김군도 ( Gun Do Kim ),박남규 ( Nam Gyu Park ) 한국수산과학회 2014 한국수산과학회지 Vol.47 No.2

Two neuropeptides were purified from the acidified tube feet extract of the starfish Asterias amurensis by C18 re- versed phase and size-exclusion high-performance liquid chromatography (HPLC). The tube feet extract and the purified peptides (AST-I and AST-II) showed potent contractile activity on dorsal retractor muscle (DRM) of the starfish Asterina pectinifera and intestine (smooth muscle) of the panther puffer Takifugu pardalis. Treatment of the purified peptides with dithiothreitol (DTT) for 60 min at 37°C significantly altered their retention times, suggesting that these compounds contained disulfide bonds. The molecular weights of AST-I and AST-II were determined to be 2064.2 Da and 6137.2 Da, respectively, by MALDI-TOF mass spectrometry.