인간의 c - Ha - ras 발암성 단백질에 결합되어 있는 GDP 와 GTPγ의 형태 비교

하종명,Gota Kawai,Tatsuo Miyazawa,Shigeyuki Yokoyama ( Jong Myung Ha ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.1

A normal ras protein and two of the mutant proteins were prepared to investigate the biochemical properties of ras proteins produced by ras genes. The methods for the preparation of ras proteins include 1) the truncation of c-Ha-ras protein at the position 18 from carboxy-terminal producing [ras(G12/1-171) protein], 2) the replacement of glycine by valine at position 12 of the ras protein from amino-terminal followed by the truncation of c-Ha-ras protein at the position 18 from carboxy-terminal [ras(V12/1-171) protein], 3) the replacement of glutamine by leucine at the position 61 followed by the truncation of c-Ha-ras protein at the position 18 from carboxy-terminal [ras(L61/1-171) protein]. The proton resonances of H8(guanine) and H1`, H2`, H3`, H4`, H5` (ribose) of the GDP and the GTP_γS [Guanosine 5`-0-(3-thiotriphosphate)] bound to ras(1-171) proteins were identified. By numerical simulation of these irradiated time-dependent NOE profiles, the conformations of the protein-bound GDP and GTP S were elucidated. The guanosine moieties of GDPs bound to three ras(1-171) proteins take the anti form about the N-glycosidic bond with a dihedral angle of χ= -120° and the ribose ring take the C2`-endo forms that were independent with point mutation. In contrast to the conformation of the bound GDP, the guanosine moieties of GTP_γSs bound to ras(1-171) proteins had anti-C3`-endo form and a dihedral angle of χ=-80°, and were not affected by the point mutation of ras(1-171) protein. The role of the interaction between the bound guanine nucleotide and effector region is discussed with regard to the mechanism of the ras proteins.

인간의 c-Ha-ras 발암성 단백질에 결합되어 있는 GDP와 $GTP{\gamma}S$의 형태 비교

하종명,Ha, Jong-Myung 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.1

ras발암유전자의 산물인 ras단백질에 결함한 구아닌 누콜레오티드의 형태를 규명하고자, 1종의 정상 ras단백젤과 2종의 변이 ras단백질을 조제하였다. 즉, 1) 정상세포의 ras단백질의 카르복시 말단의 18아미노산 잔기를 잘라낸 c-Ha-ras단백질 [ras(G12/1-171)단백질], 2) 아미노 말단에서 12번째의 글라선을 발린으로 바꾸어 발암활성형의 ras단백질로 만들고 동시에 카르복시 말단의 18아미노산 잔기를 잘라낸 c-Ha-ras변이단백질 [ras(V12/1-171)단백질], 3) 아미노 말단에서 61번째의 글루타민을 로이신으로 바꾸어 발암활성형의 ras단백질로 만들고 동시에 카르복시 말단의 18아미노산 잔기를 잘라낸 c-Ha-ras변이단백질 [ras(L61/1-171)만백질] 등이다. 각각의 ras(1-171)단백질에 결합하고 있는 GDP와 $GTP{\gamma}S$의 형태를 결정하기 위하여, 핵자기공명 스펙트럼으로부터 결합된 GDP와 $GTP{\gamma}S$의 프로톤 피크들을 귀속하고, 핵자기공명의 측정방법 중의 하나인 조사시간에 의존하는 NOE의 결과를 시뮬레이션하였다. 세 종류의 ras(1-171)만 백질에 결합하고 있는 GDP의 구아노선 부분은ras(1-171)단백질의 점 돌연변이와 무관하게 anti-C2'-endo 형태를 취하고, 글리코실결합의 2면각 X(O4'-C1'-N9-C4)는 $-120^{\circ}$이였다. ras(1-171) 단백질에 결합하고 있는 $GTP{\gamma}S$는 GDP의 경우와는 달리 anti-C3'-endo 형태를 취하고, X는 $-80^{\circ}$이였으며 ras(1-171)단백질의 점 돌연변이에 의한 영향을 받지 않았다. 이 결과로부터 ras(1-171)단백질에 결합하고 있는 구아닌 누클레오티드의 교환에 의한 형태 변화가 ras(1-171)단백질의 효과인자 부위에 미치는 상호작용에 대하여 논하였다. A normal ras protein and two of the mutant proteins were prepared to investigate the biochemical properties of ras proteins produced by ras genes. The methods for the preparation of ras proteins include 1) the truncation of c-Ha-ras protein at the position 18 from carboxy-terminal producing [ras(G12/1-171) protein], 2) the replacement of glycine by valine at position 12 of the ras protein from amino-terminal followed by the truncation of c-Ha-ras protein at the position 18 from carboxy-terminal [ras(V12/1-171) protein], 3) the replacement of glutamine by leucine at the position 61 followed by the truncation of c-Ha-ras protein at the position 18 from carboxy-terminal [ras(L61/1-171) protein]. The proton resonances of H8(guanine) and H1', H2', H3', H4', H5' (ribose) of the GDP and the $GTP{\gamma}S$ [Guanosine 5'-O-(3-thiotriphosphate)] bound to ras(1-171) proteins were identified. By numerical simulation of these irradiated time-dependent NOE profiles, the conformations of the protein-bound GDP and $GTP{\gamma}S$ were elucidated. The guanosine moieties of GDPs bound to three ras(1-171) proteins take the anti form about the N-glycosidic bond with a dihedral angle of X=$-120^{\circ}$ and the ribose ring take the C2'-endo forms that were independent with point mutation. In contrast to the conformation of the bound GDP, the guanosine moieties of $GTP{\gamma}Ss$ bound to ras(1-171) proteins had anti-C3'-endo form and a dihedral angle of X=$-80^{\circ}$, and were not affected by the point mutation of ras(1-171) protein. The role of the interaction between the bound guanine nucleotide and effector region is discussed with regard to the mechanism of the ras proteins.

NK-2의 Antagonist인 cyclo-[Gln-Trp-Phe- $\beta$Ala-Leu-Met]의 형태에 관한 연구

하종명,Ha, Jong Myung 대한화학회 1999 대한화학회지 Vol.43 No.5

Solution conformation of cyclo-($Gln^1-Trp^2-Phe^3-{\beta}Ala^4-Leu^5-Met^6$), new NK-2 antagonist in dimethyl sulfoxide solution, has been determined by the use of two-dimensional nuclear magnetic resonance spectroscopy combined with simulated annealing calculations. The peptide exhibited converged structures with the atomic root-mean-square difference for the backbone atoms ($N,\;C^{\alpha},\;C'$) of all residues being 0.02${\AA}$ in the 25 annealed structures. The analysis of the structures indicated that the cyclic peptide has three intramolecular hydrogen bonds between $Met^6NH$ and ${\beta}Ala^4CO$, ${\beta}Ala^4NH$ and $Met^6CO$, $Phe^3NH$ and $Met^6CO$, and contain a type-I ${\beta}$-turn with Gln and Trp and ${\gamma}$-turn with Leu. The addition of an extra methylene group to Gly, i.e. P-Ala residue, may relax some unfavorable restraints in the cyclic backbone structure, hence enabling an additional hydrogen bond, which results in stabilizing one conformation. 새로운 NK-2 antagonist이며 고리상 펩티드인 cycIo-($Gln^1-Trp^2-Phe^3-{\beta}Ala^4-Leu^5-Met^6$)의 DMSO 용액 중에서의 형태를 2차원 핵자기공명과 분자동력학적인 계산에 의하여 결정하였다. 조건을 만족시키는 25개의 구조는 모든 아미노산 잔기의 골격원자들 (N,$C^{\alpha}$, C')의 자승 평균 평방근이 $0.02{\AA}$이내로 수렴하였다. 이 고리상 펩티드의 구조는 $Met^6NH$와 $Ala^4CO$, $Ala^4NH$와 $Met^6CO$, $Phe^3NH$와 $Met^6CO$의 사이에 분자내 수소결합이, Gln과 Trp에 type-I ${\beta}$-turn, 그리고 Leu에서 ${\gamma}$-turn을 취하고 있었다. 알려져 있는 고리상 펩티드인 cyclo-($Gln^1-Trp^2-Phe^3-Gly^4-Leu^5-Met^6$)의 Gly이 ${\beta}$Ala으로 바뀜에 따라 ${\beta}$Ala의 여분의 메틸렌이 고리의 골격의 반발력을 완화시키고 수소결합들이 형태의 안정에 기여하고 있다고 생각된다.

NaNO₃, NaHCO₃ 농도가 Arthrospira platensis 생장에 미치는 영향

최수정,하종명,이재화,Choi, Soo-Jeong,Ha, Jong-Myung,Lee, Jae-Hwa 한국생물공학회 2015 KSBB Journal Vol.30 No.6

Arthrospira platensis (A. platensis) is one of the most explored cyanobacteria and has been studied for proteins, vitamins, pigment (chlorophyll and carotenoids) and fatty acid. In this study, we tested the effect of $NaHCO_3$ and $NaNO_3$ on the microalgae growth under photoautothrophic culture in A. platensis. As a result, cell growth and dry cell weight were increased in proportion to the $NaHCO_3$ and $NaNO_3$ concentration. Pigment (chlorophyll and carotenoids) contents of A. platensis were increased with proportion to $NaHCO_3$ concentration. But, the content of pigment (chlorophyll and carotenoids) in 100% $NaNO_3$ medium of A. platensis was the highest, 40%, 140% and 200% $NaNO_3$ medium with pigment content of A. platensis was reduced. In conditions of $NaHCO_3$ (50%) or $NaNO_3$ (40%) limitation, A. platensis could accumulate lipids to high as 1.7-fold and 1.3-fold.

Neurokinin A의 구조 및 활성에 관한 연구

신송엽,하종명,하배진,Shin, Song-Yub,Ha, Jong-Myung,Ha, Bae-Jin 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.8

Neurokinin A(NKA)와 Neurokinin B(NKB)는 Tachykinin족 펩티드에 속하는 포유류 유래의 신경펩티드이다. NKA와 NKB의 생물활성의 차이를 화학구조의 면에서 해명하기 위하여 Neurokinin펩티드의 유사체들을 화학합성하고 이 유사체들의 생물활성을 rabbit pulmonary artery(RPA)의 평활근에 대한 근육수축활성을 지표로하여 측정하였다. NKA의 아미노 말단으로부터 제5위의 세린을 페닐알라닌으로 치환시키면 약 1/5의 활성을 보였고, NKB의 아미노 말단으로부터 제5위의 페닐알라닌을 세린으로 치환시키면 NKA로서의 활성이 상승하였다. 이 결과로부터, NKA의 생물활성발현에는 아미노말단으로부터 제5위의 아미노산인 세린과 세린을 포함하는 message segment가 중요한 역할을 담당하고 있음이 시사되었다. Neurokinin A (NKA) is neuropeptide belong to mammalian tachykinin family. To investigate the difference in biological properties between NKA and NKB from the chemical structure, the peptide derivatives of neurokinins were synthesized by solution phase method and their contractile activities were measured by contraction assay with the smooth muscle of rabbit pulmonary artery (RPA). The substituion of Ser for Phe at position 5 in NKA drastically reduced contractile activity. The replacement of Phe at 5 position in NKB with Ser showed increase in contractile activity. These results suggest that message segment including Ser at position 5 has a critical role in the biological activity of NKA.

Neurokinin 군 펩티드유사체들의 모르모트 기관 평활근 수축활성

신송엽,하종명,하배진,장태식,강신원,종상 영보 ( Song Yub Shin,Jong Myung Ha,Bae Jin Ha,Tae Sik Jang,Shin Won Kang,Eisuke Munekata ) 생화학분자생물학회 1994 BMB Reports Vol.27 No.5

Neurokinin peptides (Substance P; SP, Neurokinin A; NKA and Neurokinin B; NKB) are a neuropeptide belong to the mammalian tachykinin family. To elucidate the difference in biological properties among neurokinin peptides from chemical structure, the peptide derivatives of neurokinins were synthesized by the solution and solid-phase method and their contractile activities were measured by contraction assay with the smooth muscle of guinea pig trachea (GPT). This study demonstrated that the C-terminal heptapeptide containing two amino acid residues, Asp⁴ and Val^7, is indispensible for contracting activity in GPT. Further, structureactivity studies indicate that the amino acid residues (Ser and Phe) at position 5 from N-terminus play an important role in showing the difference of biological activity between NKA and NKB in GPT assay.

Neurokinin A 의 구조 및 활성에 관한 연구

신송엽,하종명,하배진,Munekata Eisuke ( Song Yub Shin,Jong Myung Ha,Bae Jin Ha,Munekata Eisuke ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.8

Neurokinin A (NKA) is neuropeptide belong to mammalian tachykinin family. To investigate the difference in biological properties between NKA and NKB from the chemical structure, the peptide derivatives of neurokinins were synthesized by solution phase method and their contractile activities were measured by contraction assay with the smooth muscle of rabbit pulmonary artery (RPA). The substituion of Ser for Phe at position 5 in NKA drastically reduced contractile activity. The replacement of Phe at 5 position in NKB with Ser showed increase in contractile activity. These results suggest that message segment including Ser at position 5 has a critical role in the biological activity of NKA.

Neurokinin군 펩티드유사체들의 모르모트 기관 평활근 수축활성

신송엽,하종명,하배진,장태식,강신원,무나카타 에이스케,Shin, Song-Yub,Ha, Jong-Myung,Ha, Bae-Jin,Jang, Tae-Sik,Kang, Shin-Won,Munekata, Eisuke 생화학분자생물학회 1994 한국생화학회지 Vol.27 No.5

Substance P(SP), Neurokinin A(NKA) 및 Neurokinin B(NKB)는 포유류 유래의 신경펩티드(neuropeptide)로서 Neurokinin군으로 분류되며, Tachykinin족에 속한다. SP, NKA 및 NKB의 생물활성의 차이를 화학구조의 면에서 해명하기 위하여 Neurokinin군 펩티드들의 아미노산 배열을 서로 바꾼 유사체들을 Boc-HF에 의한 고상법에 의하여 화학합성하고, 이 펩티드들의 생물활성을 모르모트 기관(Guinea Pig Trachea : GPT)의 평활근에 대한 근육수축활성을 지표로하여 측정하였다. 본 연구에서는, GPT에 대한 Neurokinin군 펩티드들의 평활근의 수축활성은 카르복실 말단측의 7개의 아미노산이 필수적임을 확인하였다. 또한, 제4위의 아스파르트산, 제7위의 발린이 활성발현에 중요하며, NKA와 NKB의 생물활성의 차이는 제5위의 아미노산(NKA; Ser, NKB; Phe)의 구조적인 차이에 의존함이 시사되었다. Neurokinin peptides (Substance P; SP, Neurokinin A; NKA and Neurokinin B; NKB) are a neuropeptide belong to the mammalian tachykinin family. To elucidate the difference in biological properties among neurokinin peptides from chemical structure, the peptide derivatives of neurokinins were synthesized by the solution and solid-phase method and their contractile activities were measured by contraction assay with the smooth muscle of guinea pig trachea (GPT). This study demonstrated that the C-terminal heptapeptide containing two amino acid residues, $Asp^4$ and $Val^7$, is indispensible for contracting activity in GPT. Further, structureactivity studies indicate that the amino acid residues (Ser and Phe) at position 5 from N-terminus play an important role in showing the difference of biological actvity between NKA and NKB in GPT assay.

Human Epidermal Growth Factor 유사체들의 고상합성과 생물활성

신송엽,하종명,Shin, Song-Yub,Ha, Jong-Myung 생화학분자생물학회 1993 한국생화학회지 Vol.26 No.3

Human Epidermal Growth Factor(h-EGF)는 세포의 증식과 분화를 촉진시키는 53개의 아미노산으로 이루어진 폴리펩티드이다. h-EGF의 생물활성의 발현에 중요한 잔기를 확인하기 위하여, h-EGF의 제 13, 15, 37 및 41위의 아미노산을 다른 아미노산으로 치환시킨 펩티드들을 Boc-HF strategy를 이용한 단계적 고상합성에 의하여 화학합성하였다. 모든 합성펩티드들의 순도는 역상 고속액체크로마토그래피(RP-HPLC), FAB-mass spectroscopy, 아미노산 분석 및 Thermolysin에 의한 효소분해법 등으로 확인하였고, NIH/3T3세포의 세포증식효과에 의하여 모든 합성펩티드들에 대한 생물학적 활성을 측정하였다. 그 결과, 제 13위와 37위의 티로신을 같은 방향족 아미노산인 페닐알라닌으로 치환시킨 경우 활성의 변화는 거의 없었으나, 같은 부위를 알라닌으로 치환시키면 각각 약 $5{\times}10^{-2}$배 및 $10^{-3}$배로 활성이 감소하였다. 이와 같은 결과는 h-EGF의 생물활성의 발현에는 제 13위와 37위의 방향족 곁사슬이 중요한 역할을 담당하고 있다는 것을 시사한다. 또한, 제 15위의 로이신을 알라닌으로 치환했을 때와 제 41위의 아르기닌을 리진으로 치환했을 때 각각 약 $10^{-4}$배 및 $10^{-5}$배의 심한 활성의 저하를 나타내었다. 따라서 제 15위의 hydrophobic bulky group과 제 41위의 guanidino group은 h-EGF의 기능의 발현에 필수적임을 시사한다. Human epidermal growth factor (h-EGF) is 53 amino acid polypeptide that stimulates proliferation and differentiation of various cells. To identify critical residues that govern biological activity of h-EGF, six analogues having specific amino acid substitution at 13, 15, 37 and 41 were synthesized by stepwise solid phase method using the Boc-HF strategy. Homogenity and purity of the synthesized peptides were performed by RP-HPLC, FAB-MS, amino acid analysis and thermolytic digestion. The biological activity of these h-EGF analogues was measured by mitogenic stimulation in NIH/3T3 cells. The substitution of aromatic amino acid (Phe) at position 37 and 13 had little effect on mitogenic activity of h-EGF. In contract, the replacement of Ala at these positions occurred a big loss of acitivity $(5{\times}10^{-2} \; and \; 10^{-3}-fold)$. This result indicate that aromatic side chain at position 13 and 37 play an important role in biological activity of h-EGF. The semiconservative substitution of Ala for Leu at position 15 and conservative change of Lys for Arg at position 41 also drastically reduced mitogenic activity $(10^{-4} \; and \; 10^{-5}-fold)$. Thus, the hydrophobic bulky group at postion 15 and the guanidino group at position 41 were essential for the biological activity of h-EGF.

Human Epidermal Growth Factor 유사체들의 고상합성과 생물활성

신송엽,하종명 ( Song Yub Shin,Jong Myung Ha ) 생화학분자생물학회 1993 BMB Reports Vol.26 No.3

Human epidermal growth factor (h-EGF) is 53 amino acid polypeptide that stimulates proliferation and differentiation of various cells. To identify critical residues that govern biological activity of h-EGF, six analogues having specific amino acid substitution at 13, 15, 37 and 41 were synthesized by stepwise solid phase method using the Boc-HF strategy. Homogenity and purity of the synthesized peptides were performed by RP-HPLC, FAB-MS, amino acid analysis and thermolytic digestion. The biological activity of these h-EGF analogues was measured by mitogenic stimulation in NIH/3T3 cells. The substitution of aromatic amino acid (Phe) at position 37 and 13 had little effect on mitogenic activity of h-EGF. In contract, the replacement of Ala at these positions occurred a big loss of acitivity (5×10^(-2) and 10^(-3) -fold). This result indicate that aromatic side chain at position 13 and 37 play an important role in biological activity of h-EGF. The semiconservative substitution of Ala for Leu at position 15 and conservative change of Lys for Arg at position 41 also drastically reduced mitogenic activity (10^(-4) and 10^(-5) `-fold). Thus, the hydrophobic bulky group at postion 15 and the guanidino group at position 41 were essential for the biological activity of h-EGF.