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.

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.

Vibrio vulnificus가 분비하는 Cytolysin에 관한 연구

김형로,김우현,노혜원,김강신,박인숙 의과학연구소 1988 全北醫大論文集 Vol.12 No.1

The extracellular cytolysin produced by Vibrio vulnificus isolated from a patient with primary sepsis was partially purified and its properties were studied. 1. The cytolysin production in culture supernatant reached to the maximum of 800HU/ml at 5 hours' culture and was reduced by the addition of NaCl in culture media. 2. The cytolysin was heat-labile and completely inactivated when stored at 4℃ for 24 hours but the stability was greatly enhanced by the addition of glycerol(50%) or bovine serum albumin(mg/ml). 3. The cytolysin in culture supernatant was partially purified 40 folds to the specific activity of 32570HU/mg protein by ammonium sulfate precipitation, calcium-phosphate gel adsorption and DEAE-cellulose chromatography. 4. The cytolysin was lethal for mouse(18HU/mouse), but pretreatment with verapamil or Tavegyl 1 hour before the injection of cytolysin significantly prevented the lethal effect of cytolysin.

알코홀 대사에 미치는 Nicotinamide의 영향

金炯魯,池垠政,金禹現 전북대학교 의과학연구소 1980 全北醫大論文集 Vol.4 No.2

家兎 淋巴球 NAD Glycohydrolase의 硏究 : 1. 胸腺細胞 NAD Glycohydrolase의 性狀 1. Properties of NAD Glycohydrolase in Thymocytes

金炯魯,金禹現 전북대학교 의과학연구소 1979 全北醫大論文集 Vol.3 No.-

NAD glycohydrloase bound to the membranes of the rabbit thymocytes was solubilized and some properties of the solubilized enzyme were compared to those of the bound enzyme. More than 90% of bound NADase was solubilized by the treatment with 0.5% Triton X-100 to the intact thymochtes, while only 55% was solubilized by the action of pancreatic lipase. NADase solubilized by Triton X-100 has different properties from the bound NADase in optimal pH, and in inhibition by nicotinamide or isonicotinic acid hydrazide (INH). Optimal pH shifted from 6.8 in bound enzyme to 6.0 in solubilized enzyme. The concentration of nicoti-amide giving 505 inhibiton was increased from 6mM in bound NADase to 8mM in solubilized form. INH inhibited the activity of solubilized NADase more strongly than bound enzyme and, accordingly, brought the lesser formation of INH analogue of NAD in case of the solubilized NADase.

Mg2+ Dependency of Adenosine Diphosphate Ribose Pyrophosphohydrolase in Rabbit Liver

김형로,지은정,김유현 생화학분자생물학회 1984 BMB Reports Vol.10 No.4

Previously, we reported that there were two kinds of organ specific ADPRase isoenzymes in rabbit tissues on the basis of their Mg^(2+) dependence. The present work is concerned with investigating the subcellular localization and properties of the liver ADPRase. ADPRase present in nuclei and mitochondria did not require Mg^(2+) for their activity, while cytoplasmic ADPRase showed absolute Mg^(2+) requirement but might be converted to Mg^(2+) independent form on storage at 4℃. These results suggest that newly synthesized cytoplasmic Mg^(2+)-dependent ADPRase be transferred and converted to nuclear and mitochondrial Mg^(2+)-independent ADPRase.

렛트 Adenosine Dearninase에 관한 연구

김우현,이정섭,이병국,박진우,김형로 의과학연구소 1989 全北醫大論文集 Vol.13 No.1

人體胎盤의 Adenosine Diphosphate Ribose Pyrophosphohydrolase에 關한 硏究

金炯魯,池垠政,金禹現 전북대학교 의과학연구소 1978 全北醫大論文集 Vol.2 No.-