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분열 유발 인자에 의한 흰쥐 림프구 단백의 인산화

Other Titles
 Phosphorylated proteins of mitogen stimulated-rat peripheral blood lymphocytes 
Authors
 노덕현 
Issue Date
1991
Description
의학과/박사
Abstract
[한글]단백의 가역적인 인산화는 세포기능 조절의 공통된 최종 경로의 하나로 인산화/탈인산화의 순환이 가역적인 단백구조의 변화를 일으켜 탄수화물의 대사, 세포증식 반응, 세포막에서의 이온운반과 면역반응등 다양한 세포기능을 매개한다고 생각된다. T 림프구를 칼슘 ionophore와 phorbol 12-myristate 13-acetate (PMA)로 자극하면 분열이 유발되며 세포막 인지질 대사산물인 diacylglycerol에 의해 활성화된 protein kinase C(PKC)가 세포 분열의 초기 단계에서 조절 물질로 작용함이 밝혀졌다. 따라서 본 실험에서는 세포내 전달과정에 대한 연구의 기초작업의 일부로 흰쥐로부터 채취한 림프구를 분열유발물질인 concanavalin A(Con A)와 PMA로 자극시켜 수용체 자극의 전후에 일어나는 단백의 인산화 과정을 전기 영동을 시행하여 관찰하고 calcium/calmodulin dependent pro tein kinase(CaM kinase)억제제인 W-7과 PKC억제제인 H-7을 이용하여 이들 효소에 특이적으로 반응하여 인산화되는 기질단백을 찾고자 시도하였다. 한편 세포활성에 따를 신호전달 체재에서의 인산화 효소들의 유기적 관계를 보기위해 시간에 따른 인산화 반응의 변화 를 보았다. 실험 재료로는 흰쥐(Sprague-Dawley계) 동맥혈의 림프구를 사용, nylon wool column을 이용하여 T림프구 만을 분리한 후 분리된 림프구를 (32)**P orthophosphate로 표지시킨 후 림프구를 균질화 한 후 원심 분리하여 단백 분획을 얻고, 이를 non-equilibrium pH gradient electrophoresis(NEPHGE)와 sodium dodecyl sulfate-poiyacrylamide gel electrophoresis(SDS-PAGE)를 이용한 이차원 전기 영동법을 이용하여 단백을 분리한 후 건조시킨 gel을 자가 방사기록법으로 X-ray film에 감광시켜 인산화 단백을 관찰하였다. 실험 결과는 다음과 같다. 1. 횐쥐 T 림프구를 PMA로 자극하면 5개의 인산화 단백이 새로이 나타나고 7개 단백의 인산화가 증가되었으며, Con A 자극으로는 1개의 단백이 새로이 인산화 되고 7개 단백의 인산화가 증가되었다. 2. PMA 및 Con A 자극으로 인산화 되는 13개 단백은 kinase 억제제 전처치에 의하여 세군으로 구분되며, H-7 전처치로 24kDa/pⅠ 7.1, 24/7.2, 26/6.1, 74/6.2 단백의, W-7 전처치로 14kDa/pⅠ 5.9, 28/6.8, 29/6.9, 28/7.0, 44/6.8, 58/6.2 단백의 인산화가 현저히 감소되었으며, 18kDa/pⅠ 5.4, 25/7.3 및 54/5.2 단백은 두 억제제에 의해 영향을 받지 않았다. 3. 이들 인산화 단백은 대부분 세포의 Soluble fraction에서 확인되며 자극후 반응 초기에 인산화 된 후 인산화가 감소하나, 침전물에서 관찰되는 소수의 인산화 단백은 지속적인 인산화를 보였다. 4. Kinase 억제제 처치에 의하여 구분된 3군에 속하는 단백들의 시간에 따른 인산화 양상을 관찰한 결과 각 군에 따른 인산화 양상에 상호 연관성이 없었다. 이상의 실험결과로 보아 림프구 활성의 초기 단계에서 인산화 되는 단백에는 PKC, CaM kinase 및 다른 kinase에 의해 인산화 되는 3종류의 단백이 존재하며, 세종류의 kinase의 활성은 단계적인 활성이 아니라 독립적 또는 상호 협동적으로 작용하여 림프구 활성을 유발시키는 것으로 생각된다.
[영문]The reversible phosphorylation of specific cellular proteins is an important biochemical reaction for the functional regulation, which is conserved to all of the mammalian cells. Mitogens trigger reorganigation of phospholipid molecules in the cytoplasmic membrane, which activate the protein kinases. The activated kinases phospherylate certain proteins to generate physiologically diverse responses on the various cellular stimuli. The nature of the substrate proteins to be phosphorylated in this pathway remains unknown. This study was attempted to classify the proteins involved in the specific phosphorylation using the rat peripheral blood lymphocytes(rPBL) stimulated with mitogens, phorbol 12-myristate 13-acetate(PMA) and concanavalin A(Con A). The lymphocytes were incubated with (32)**P -orthophosphate before PMA or Con A stimulation. The migration patterns of the phosphorylated proteins of mitogen-treated rPBL in two dimensional electrophoretic fields were analyzed after autoradiography. The stimulation of the lymphocytes with PMA and Con A increased the phosphorylation of thirteen protein fractions. The phosphorylating intensities of the protein spots were different to the treatments of the cells with specific kinase inhibitors, H-7 and W-7. These protein fractions were grouped into 3 classes, those are PKC-mediated, CaM kinase-mediated, and other kinase mediated proteins. Effect of the duration of the stimulation on the phosphorylated behaviors of the proteins were also examined. The phosphorylation of 3 classes of protein fractions occurred concurrently, not sequentially, although individual protein fraction had different time Point for the peak phosphorylation during the stimulation period up to 30 min. The phosphoproteins found in the cytosolic soluble fraction were phosphorylated earlier than those in the pellet, whose phosphorylations were sustained high level over 10 min. The above results suggest that the early events in lymphocyte activation involves 3 different sets of proteins which are phosphorylated by CaM kinase, PKC and other kinase, and these kinases do not work sequentially, but indepeThe reversible phosphorylation of specific cellular proteins is an important biochemical reaction for the functional regulation, which is conserved to all of the mammalian cells. Mitogens trigger reorganigation of phospholipid molecules in the cytoplasmic membrane, which activate the protein kinases. The activated kinases phospherylate certain proteins to generate physiologically diverse responses on the various cellular stimuli. The nature of the substrate proteins to be phosphorylated in this pathway remains unknown. This study was attempted to classify the proteins involved in the specific phosphorylation using the rat peripheral blood lymphocytes(rPBL) stimulated with mitogens, phorbol 12-myristate 13-acetate(PMA) and concanavalin A(Con A). The lymphocytes were incubated with (32)**P -orthophosphate before P MA or Con Astimulation. The migration patterns of the phosphorylated proteins of mitogen-treated rPBL in two dimensional electrophoretic fields were analyzed after autoradiography. The stimulation of the lymphocytes with PMA and Con A increased the phosphorylation of thirteen protein fractions. The phosphorylating intensities of the protein spots were different to the treatments of the cells with specific kinase inhibitors, H-7 and W-7. These protein fractions were grouped into 3 classes, those are PKC-mediated, CaM kinase-mediated, and other kinase mediated proteins. Effect of the duration of the stimulation on the phosphorylated behaviors of the proteins were also examined. The phosphorylation of 3 classes of protein fractions occurred concurrently, not sequentially, although individual protein fraction had different time Point for the peak phosphorylation during the stimulation period up to 30 min. The phosphoproteins found in the cytosolic soluble fraction were phosphorylated earlier than those in the pellet, whose phosphorylations were sustained high level over 10 min. The above results suggest that the early events in lymphocyte activation involves 3 different sets of proteins which are phosphorylated by CaM kinase, PKC and other kinase, and these kinases do not work sequentially, but independently or cooperatively.
URI
http://ir.ymlib.yonsei.ac.kr/handle/22282913/115704
Appears in Collections:
2. 학위논문 > 1. College of Medicine (의과대학) > 박사
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