Studies on NAD Nucleosidase in Erythrocytes

김형로,이강만,이민화 ( Hyung Rho Kim,Kang Man Lee,Min Wha Lee ) 한국생화학분자생물학회 (구 한국생화학회) 1969 BMB Reports Vol.2 No.2

적혈구의 막 단백에 관한 연구 1. 가토 적혈구 NAD Nucleosidase의 정제와 요소에 의한 부활현상

김형로,Kim, Hyung-Rho 생화학분자생물학회 1973 한국생화학회지 Vol.6 No.1

가토적혈구막 NAD nucleosidase (NADase)를 췌장 lipase로 가용화시켜 부분 정제하여 이 효소의 일부성상을 구영하였다. 췌장 lipase를 무손상가토적혈구에 작용시키면 적혈구의 파괴없이 막 NADase가 쉽게 가용화되어 효소활성이 소실되지 않고 유리되었다. 이는 NADase 분자전체가 적혈구막외연에 위치하고 있음을 시사해 주고 있다. 적혈구막 NADase를 lipase로 가용화시 킨 다음 26배 부분정제하여 그 성상을 관찰한 바, 이 효소는 저농도의 urea에 의하여 효소활성이 현저히 부활되었으나 가토의 다른 조직 NADase나 다른 포유동물의 적혈구막 NADase는 urea에 의하여 전혀 활성화되지 않았다. 0.2M urea는 NADase의 NAD에 대한 Michaelis 정수 (Km 치)와 최고반응속도 (Vmax)를 3배가량 동일한 율로 증가시켜 1/s에 대한 1/v의 double reciprocal plot에 있어서 urea 유무시의 양 직선이 서로 평행하였다. NADase의 상경성 저해제인 NADP와 nicotinamide mononucleotide (NMN)는 urea의 부활작용을 전혀 소멸시키지 못하였으나 비상경성 저해제인 nicotinamicle와 3-acetylpyridine은 urea의 부활작용을 거의 소멸시켰다. 이와같은 실험성적은 urea가 NADase의 uncompetitive activator로 작용하여 효소-기질복합체 형성반응에는 무관하고 효소-기질복합체의 분해 과정중에서 nicotinamide가 유리되는 단계를 촉전시키는 것이라고 시사되었다. When intact rabbit erythrocytes were treated with pancreatic lipase, the membrane-bound NAD nucleosidase (NADase) was partially solubilized without marked destruction of the cellular stucture, suggesting that the entire molecule of the rabbit erythrocyte NADase is located on the outer portion of the cell membrane. The lipase-solubilized NADase from rabbit erythrocyte membrance was partially purified 26-fold, and its properties were characterized. Among other properties the most characteristic one was that the rabbit erythrocyte NADase was activated by urea in low concentrations, whereas NADases from other sources were not affected. Urea (0.2 M) caused three-fold increases in both the Michaelis constant for NAD and the maximal velocity of NADase, thus making the reciprocal plot in the presence of urea parallel to the plot in the absence of urea. The competitive inhibitors such as NADP and nicotinamide mononucleotide did not eliminate the activation effect of urea, while nicotinamide and 3-acetylpyridine known as noncompetitive inhibitor eliminated the urea effect. These results strongly imply that the activation of NADase by urea in low concentrations is of uncompetitive nature, suggesting that the action of urea should enhance the rate of the enzyme-substrate complex breakdown involved in the liberation of nicotinamide and not earlier step concerned with the formation of the enzyme-substrate complex.

가토 적혈구막 NAD glycohydrolase 에 대한 urea 의 활성화 작용

김형로(Hyung Rho Kim) 전북대학교 의과학연구소 1978 全北醫大論文集 Vol.2 No.-

가토 간의 Adenosine Diphosphate Ribose Pyrophosphohydrolase 의 성장

김형로(Hyung Rho Kim),지은정(Eun Chung Jhee) 전북대학교 의과학연구소 1979 全北醫大論文集 Vol.3 No.-

인체 태반 Receptor 와 Insulin 의 상호 작용

김형로(Hyung Rho Kim) 전북대학교 의과학연구소 1977 全北醫大論文集 Vol.1 No.-

The Inhibition of Neutrophil Chemotaxis by Pertussis Toxin

김정수,김우현,노혜원,김종석,박진우,김형로,Kim, Jung-Soo,Kim, Uh-Hyun,Rho, Hye-Won,Kim, Jong-Suk,Park, Jin-Woo,Kim, Hyung-Rho 생화학분자생물학회 1989 한국생화학회지 Vol.22 No.2

백일해 독소(PT)는 phospholipase C와 연결된 신호전달 체계를 변형시켜 중성 백혈구의 chemotaxis를 억제한다고 알려져 왔다. fMLP 수용체에 연결되어 phospholipase C가 활성화 되는데는 adenylate cyclase 체계를 억제하는 guanine nucleotide의 결합 조절 단백질(Gi 단백질) 또는 상동성 단백질이 신호전달을 중계할 것이라고 생각되고 있다. 본 연구에서는 이 체계에 대한 PT의 역할이 A-protomer의 ADP-ribosyltransferase 작용에 의한 Gi 단백질의 변형 때문인지 또는 B-oligomer의 세포 표면 결합에 의한 이차적인 효과인지를 구명하였다. PT와 메틸화 PT(mPT)는 chemotaxis 억제 및 관련된 현상들에 서로 다른 생물학적 활성을 가진 독소의 두 부분중 어느 부분이 관여하는지를 알기 위하여 이용하였다. 조제한 mPT는 ADP-ribosyltransferase 활성을 유지하고 있음을 확인하였고 이 mPT는 chemotaxis 억제능력이 없음을 관찰함과 동시에 그에 수반되는 생화학적 사건들에서도 일관성 있는 결과를 얻었기에 PT에 의한 chemotaxis의 억제는 A-protomer보다는 B-oligomer의 역할과 관계되리라 사료된다. It has been suggested that pertussis toxin (PT) inhibits the chemotaxis of neutrophil by modifying signal transduction system coupled to phospholipase C. The PT substrate, probably the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (Gi) or an analogous protein, is hence proposed to mediate N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor-linked activation of the phospholipase C. The aim of this investigation was to determine whether the role played by PT in this system, is dependent upon the ability of the A-protomer of the toxin to ADP-ribosylate Gi or secondary to the binding of the B-oligomer of the toxin to the surface of target cells. PT and methylated PT (mPT) were employed in parallel to assess the involvement of two distinct biologic activities of the toxin in the inhibition of chemotaxis and its related events. Our preparation of mPT retained its ability to ADP-ribosylate Gi, but the pretreatment of mPT to neutrophils did show the inhibition of the chemotaxis as well as the other events related to the phospholipase C, indicating that the involvement of PT is not solely dependent on its capacity to ADP-ribosylate Gi, but rather related to the role of B-oligomer of the toxin.

백일해 독소에 의한 중성 백혈구 chemotaxis 의 억제에 관한 연구

김정수,김우현,노혜원,김종석,박진우,김형로 ( Jung Soo Kim,Uh Hyun Kim,Hye Won Rho,Jong Suk Kim,Jin Woo Park,Hyung Rho Kim ) 생화학분자생물학회 1989 BMB Reports Vol.22 No.2

It has been suggested that pertussis toxin (PT) inhibits the chemotaxis of neutrophil by modifying signal transduction system coupled to phospholipase C. The PT substrate, probably the inhibitory guanine nucleotide-binding regulatory component of adenylate cyclase (Gi) or an analogous protein, is hence proposed to mediate N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor-linked activation of the phospholipase C. The aim of this investigation was to determine whether the role played by PT in this system, is dependent upon the ability of the A-protomer of the toxin to ADP-ribosylate Gi or secondary to the binding of the B-oligomer of the toxin to the surface of target cells. PT and methylated PT (mPT) were employed in parallel to assess the involvement of two distinct biologic activities of the toxin in the inhibition of chemotaxis and its related events. Our preparation of mPT retained its ability to ADP-ribosylate Gi, but the pretreatment of mPT to neutrophils did show the inhibition of the chemotaxis as well as the other events related to the phospholipase C, indicating that the involvement of PT is not solely dependent on its capacity to ADP-ribosylate Gi, but rather related to the role of B-oligomer of the toxin.

Neurospora crassa 의 NAD glycohydrolase messenger RNA 정제 및 in vitro translation

김우현,서정선,김형로 ( Uh Hyun Kim,Jeong Sun Seo,Hyung Rho Kim ) 생화학분자생물학회 1986 BMB Reports Vol.19 No.1

The in vitro translation products were obtained by translating the isolated Neurospora messenger RNA from Neurospora crassa cultured in Fries minimal medium and zinc and nitrogen deficient medium and the NAD glycohydrolase (NADase) (EC3.2.2.5) was identified in the translation products by the immunoprecipitation methods. The yields of total RNA and messenger RNA from Neurospora crassa grown in zinc and nitrogen deficient media were lower than those from control media but the function of the synthesis of proteins was not affected. The synthesis of NADase-specific mRNA seemed not to be directly proportional to the increase of the specific activity of the enzyme. The increase of NADase in Neurospora crassa grown in zinc and nitrogen deficient seemed not to be ascribed to the regulation in the level of transcription. The molecular weight and the isoelectric point of NADase in the translational products was around 6,000 dalton and pI 9.0, respectively which were different from the other data obtained from purified Neurospora NADase, suggesting that this translation product was not processed post-translational modification.

Isolation and in vitro translation of the messenger RNA coding for Neurospora crassa NAD glycohydrolase

김우현,서정선,김형로,Kim, Uh-Hyun,Seo, Jeong-Sun,Kim, Hyung-Rho 생화학분자생물학회 1986 한국생화학회지 Vol.19 No.1

Fries minimal 배지와 zinc-nitrogen 결핍 배지에서 각각 배양한 Neurospora crassa로부터 messenger RNA을 얻었다. cell-free reticulocyte system을 이용하여 Neurospora mRNA로부터 in vitro translation 산물을 얻고 면역침전법을 이용하여 이 단백합성 산물내에서 NAD glycohydrolase (EC 3.2.2.5)를 확인하였다. zinc-nitrogen 결핍배지에서의 Neurospora RNA와 mRNA양은 정상배지에서의 양에 비하여 감소하였으나 mRNA의 단백합성능력에는 이상을 보이지 않았다. zinc-nitrogen 결핍배지에서 자란 Neurospora에서 NAD glycohydrolase 특이적 mRNA의 합성을 효소의 비활성도의 증가에 비례하여 증가되지 않은 것으로 보아 NAD glycohydrolase의 증가는 transcription level에서의 조절이 아닌 것으로 사료된다. SDS-polyacrylamide gel 전기영동과 isoelectrofocusing에 의한 단백 합성 산물내의 NAD glycohydrolase 분자량과 pI는 각각 6,000 dalton과 9.0으로서 cell-free reticulocyte system에서의 in vitro translation에서는 post-translational modification이 일어나지 않았음을 시사해 주었다. The in vitro translation products were obtained by translating the isolated Neurospora messenger RNA from Neurospora crassa cultured in Fries minimal medium and zinc and nitrogen deficient medium and the NAD glycohydrolase (NADase) (EC3.2.2.5) was identified in the translation products by the immunoprecipitation methods. The yields of total RNA and messenger RNA from Neurospora crassa grown in zinc and nitrogen deficient media were lower than those from control media but the function of the synthesis of proteins was not affected. The synthesis of NADase-specific mRNA seemed not to be directly proportional to the increase of the specific activity of the enzyme. The increase of NADase in Neurospora crassa grown in zinc and nitrogen deficient seemed not to be ascribed to the regulation in the level of transcription. The molecular weight and the isoelectric point of NADase in the translational products was around 6,000 dalton and pI 9.0, respectively which were different from the other data obtained from purified Neurospora NADase, suggesting that this translation product was not processed post-translational modification.

원저 : Mg2+ 의존형 Adenosine Diphosphoribose Pyrophosphatase 의 Mg2+ 비의존형으로의 전환

김종석(Jong Suk Kim),류연창(Yun Chang Yu),서만욱(Man Wook Seo),김형로(Hyung Rho Kim) 전북대학교 의과학연구소 1991 全北醫大論文集 Vol.15 No.1