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대두 유식물에서 Protein Kinase C 의 부분 정제
최윤희(Yoon Hi Choy),이준승(June Seung Lee) 한국식물학회 1993 Journal of Plant Biology Vol.36 No.2
Protein kinase C, a protein related in PI cascade, was partially purfied from the cytosol protein of etiolated plant of Glycine max by DEAE-52 cellulose chromatography and phenylsepharose chromatography. When the DEAE column was eluted with 0-0.8 M linear gradient KCl, two fractions were found that increased the phosphorylation of histon H1 about five and nine-fold in the presence of 5 ㎛/mL phosphatidylserine and 0.5 ㎛/mL diolein, respectively. These fraction were used as DEAE pool. The fraction eluted with relatively high concentration of KCl was loaded on phenylsepharose column with 5 mM CaCl_2 and eluted with 1 mM EGTA. A fraction contained the protein kinase C, which increased the phosphorylation of the histon H1 was fractionated. To determine the molecular weight of PKC, the fraction eluted from Phenylsepharose column was analyzed by 5∼15% polyacrylamide gel electrophoresis after concentrated with the Amicon membrane (YM10). That revealed two bands corresponding to 60 and 65 kD by silver staining of the gel, respectively.
대두 유식물에서 Protein Kinase C 에 의해서 인산화되는 단백질의 동정
최윤희(Yoon Hi Choy),허규정(Kyu Chung Hur),이준승(June Seung Lee) 한국식물학회 1992 Journal of Plant Biology Vol.35 No.1
The previous report (Chung and Lee, 1992) in our laboratory demonstrated that the protein kinase C(PKC) activator, TPA, promotes the elongation of corn coleoptiles significantly. To understand the role of TPA on the growth, substrates of PKC were investigated using PKC partially purified from soybean by DEAE-52 cellulose column. The enzyme activity increased about 5-fold in the presence of Ca^2+, phosphatidylserine and diolein compared with that in the absence of these reagents. Phosphorylation of both cytosol and membrane proteins by the purified PKC increased in the presence of Ca^2+ compared with that of EGTA treatment. However, the phosphorylation did not increase markedly by treatment with TPA of phosphatidylserine and diolein in the presence of Ca^2+ compared with Ca^2+ alone. The decrease in phosphorylation of 100, 61 and 43 Kd proteins of the cytosol, and 140, 11, 66, 47 and 32 Kd membrane proteins in hypocotyls, and 140, 110, 66, 47, 33, 31 and 16 Kd membrane proteins in the root was observed in the presence of PKC inhibitor staurosporine (STA). These results suggest that subatrates of PKC in soybean may be 110, 63 and 41 Kd proteins of the cytosol, and 140, 110, 66, 47 and 32 Kd membrane proteins in the subapical region of the hypocotyl, and 140, 110, 66, 47, 33, 31 and 16 Kd membrane proteins of the root.
대두 유식물에서 Protein Kinase C에 의해서 인산화되는 단백질이 동정
崔允僖,許圭晶,李埈承 이화여자대학교 생명과학연구소 1992 생명과학연구논문집 Vol.3 No.-
DEAE-52 cellulose column을 이용하여 부분적으로 분리한 PKC를 이용하여 대두 유 식물 하배축 정단부와 뿌리에서 각각 분리한 세포질 단백질과 막 단백질의 인산화 실험을 통해 PKC의 기질이 되는 단백질을 조사하여 보았다. PKC의 기질은 세포질 단백질의 경우 100, 63 그리고 41Kd 단백질들이며, 막 단백질 중 하배축 정단부의 경우는 140, 110, 66, 47 그리고 32Kd 단백질들이며, 뿌리의 경우는 140,110, 66, 47, 33, 31, 16Kd 단백질들이라고 생각된다. The previous report (Chung and Lee, 1992) in our laboratory demonstrated that the protein kinase C(PKC) activator, TPA, promotes the elongation of corn coleoptiles significantly. To understand the role of TPA on the growth, substrates of PKC were investigated using PKC partially purified from soybean by DEAE-52 cellulose column. The enzyme activity increased about 5-fold in the presence of Ca^2+ alone. The decrease in phosphorylation of 100, 61 and 43 Kd proteins of the cytosol, and 140, 110, 66, 47 and 32Kd membrane proteins in hypocotyls, and 140, 110, 66, 47, 33, 31 and 16 Kd membrane proteins in hypocotyls, and 140, 110, 66, 47, 33, 31 and 16Kd membrane proteins in the root was observed in the presence of PKC inhibitor staurosporine (STA). These results suggest that subatrates of PKC in soybean may be 110, 63 and 41Kd proteins of the cytosol, and 140,110, 66, 47 and 32Kd membrane proteins in the subapical region of the hypocotyl, and 140,110, 66, 47, 33, 31 and 16Kd membrane proteins of the root.
대두 유식물에서 Protein Kinase C의 부분 정제
崔允僖,李埈承 이화여자대학교 생명과학연구소 1993 생명과학연구논문집 Vol.4 No.-
대두(Glycine max L.) 유식물에서 PKC를 분리하고자 세포질 단백질만을 모아 DEAE-52 cellulose chromatography와 phenylsepharose chromatography를 수행하였다. DEAE-52 cellulose column에서 0~0.8M KCL linear gradient로 용출시켜 5㎍/mL phosphatidylserine과 0.5㎍/mL diolein의 존재하에서 histion H1의 이산화가 각각 5.4배화 9.2배가 증가하였던 분획들을 DEAE pool로 사용하였다. 비교적 고농도의 KCI에 의해 용출되었던 DEAE pool를 5mM Ca^2의 존재하에서 phenylsepharose에 loading하고 1 mM EGTA로 용출하였다. 이 phenylsepharose column의 분획들 중에서 protein kinase assay를 행하여 phosphatidylserine과 diolein이 있을 때 DEAE에서와 비슷한 정도의 histon H1 인산화의 증가를 보이는 분획을 찾을 수 있었다. 이 phenylsepharose pool을 Amicon membrane(YM10)으로 농축하여 5-15% polyacrylamide gel에서 전기영동하여 silver staining하였다. 이 때 나타난 두 band의 분자량은 각각 60과 65KD로 나타났다. Protein kinase C, a protein related in PI cascade, was partially purfied from the cytosol protein of etiolated plant of Glycine mas by DEAE-52 cellulose chromatography and phenylsepharose chromatography. When the DEAE column was eluted with 0-0.8M linear gradient KCI, two fractions were found that increased the phosphorylation of histon H1 about five and nine-fold in the presence of 5㎍/mL phosphatidylserine and 0.5㎍/mL diolein, respectively. These fractions were used as DEAT pool. The fraction eluted with relatively high concentration of KCI was loaded on phenylsepharose column with 5mM CaCl_2 and eluted with 1mM EGTA. A fraction contained the protein kinase C, which increased the phosphorylation of the histon H1 was fractionted. To determine the molecular weight of PKC, the fraction eluted from phenylsepharose column was analyzed by 5-15% polyacrylamide gel electrophoresis after concentrated with the Amicon membrane (YM10). That revealed two bands corresponding to 60 and 65KD by silver staining of the gel, respectively.