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흰쥐 말초 림프구의 분자량 22 kDa 인산화 단백의 특성

Issue Date
1993
Description
의학과/박사
Abstract
[한글] T림프구의 활성에는 적어도 4종류 이상의 protein kinase가 참여하며 이들이 서로 복합적으로 연계(crosstalk)되어 반응을 유발한다고 하나 자세한 반응기구는 아직 알려져 있지 않다. T림프구를 분열유발 물질이나 interleukin-2(IL-2) 등으로 자극시키면 인산화가 증가되며 이의 초기 생화학적 반응은 주로 phospholipase C(PLC)의 매개에 의한 세포막 인지질의 가수분해에 의해 생성된 diacyycerol(OAG)과 inositol 1,4,5-trisphosphate(IP^ ^3)에 의해 매개된다. 따라서 본 실험에서는 흰쥐 림프구를 분열 유발물질인 concanavalin A(Con A)와 phorbol 12-myristate 13-acetate(PMA)로 자극시켜 수용체 자극으로 특징적인 인산화 증가를 보이는 22 kDa단백의 인산화변동 양상 및 특성을 규명하고자 하였으며 아울러 이를 통해 T림프구 활성 초기에 일어나는 신호변환체계의 유기적 관계를 알아보고자 하였다. 실험재료로는 휜쥐(Sprague-Dawly계) 동맥혈의 림프구를 사용하였으며 nylon wool column으로 T림프구만을 분리하여 (32)**P orthophosphate로 표지시킨 다음 림프구를 균질환 한 후 원심분리하여 얻은 단백분획을 non-equilibrium pH gradient electrophoresis(NEPHGE)와 sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE )법으로 이차원 전기영동하여 단백을 분리한 후 자가방사법으로 인산화 단백을 관찰하였다. 단백의 인산화자리는 HPLC를 이용해 tryptic Peptide를 분리 후 이를 liquid scintillation counter로 방사능을 측정하여 추정하였으며 분리한 tryptic peptide를 산분해후 TLC plate위에서 전기영동하여 인산화 아미노산을 분석하였다. 실험 결과는 다음과 같다. 1. 22 kDa단백은 PKC활성제인 PMA나 분열유발 물질인 Con A처치에 의하여 인산화가 증가하였다. 2. Adenylyl cyclase 활성제인 forskolin은 22 kDa단백의 인산화 중감에 영향을 주지 못하였다. 3. 칼슘 ionophore인 A23187의 처치로 22 kDa단백의 인산화증감은 변동이 없었다. 4. 22 kDa단백의 PMA-유발 인산화 증가는 W-7 또는 H-7의 전처치로 변동이 없었으나 PKA와 PKC의 억제제인 staurosporine 전처치에 의해 용량 의존적인 억제효과를 보였다. 5. 22 kDa단백의 PMA-유발 인산화 증가는 PKC의 특이한 억제제인 calphostin C전처치에 의해 억압되었다. 6. HPLC에 의한 22 kDa단백의 tryptic peptide fractionation은 33∼36번 분획에서 대조군에 비해 인산화가 크게 증가되었다. 7. 22 bDa단백의 인산화는 아미노산 세린에서 일어났다. 이상의 실험 결과로 보아 휜쥐 말초 혈액 T림프구에서 PMA나 Con A 처치에 의해 현저한 인산화의 증가를 보이는 분자량 22 kDa의 단백은 protein kinase C에 의해서만 인산화되는 기질단백이라고 생각되며, 22 kDa의 tryptic peptide중 PKC에 의하여 인산화되는 부위는 한곳이며 세린잔기임을 알 수 있었다. The charaeteristics of 22 kDa Phosphoprotein obtained from rat Peripheral blood Iymphocytes Ilo Jou Department of Medical Sciences The Graduate School, Yonsei University (Directed by Professor Kyung Hwan Kim) Lymphocytes were found to possess all of the enzymatic machinery needed to phosphorylate and dephosphorylate proteins. At least four groups of protein kinases participate in T cell activation and interact with each other in a complex and as yet unknown manner, Increased phosphorylation in lymphocytes following stimulation with mitogens or interleukin-2, and the pivotal role of protein kinase C in the initial biochemical reaction have been reported. But the exact role of PKC in T cell activation and the substate of PKC are not well known. This study was attempted to clarify the characteristics of 22 kDa phosphoproteins obtained from rat peripheral blood Iymphocytes stimulated with Phorbol 12-myristate 13-acetate(PMA), using various kinase inhibitors as well as kinase activators. The Iymphocytes were incubated with (32)**P orthophosphate before PMA stimulation. The migration pattern of the phosphorylated proteins of PMA-treated rPBL in the two dimensional electrophoretic fields were analyzed after autoradiography. And the phosphorylation sites of 22 kDa protein were analysed by high performance liquid chromatography(HPLC) and scintilation counting. The results are as follows: 1. Increased phosphorylation of 22 kDa protein was observed with PMA or Con A. 2. Forskolin, an activator of adenylyl cyclase, cause no significant change of phosphorylation of 22kDa. 3. A 23187, a Ca**2+ ionophore, has no noticed effect on the phosphorylation of 22 kDa protein. 4. Neither W-7 nor H-7 inhibit the increase in phosphorylation of 22 kDa by PMA. 5. Staurosporine, a potent PKC inhibitor, showed inhibitory action on PMA-stimulated phosphorylation of 22 kDa in a dose-dependent manner. 6. Calphostin C, specific PKC inhibitor, inhibited the PMA-stimulated phosphorylntion of 22 kDa. 7. Among tryptic peptide fractions of 22 kDa by HPLC, one (32)**P phosphopeptide peak was observed at about 20% acetonitrile. From the above results, it could be suggested that the 22 kDa phosphoprotein of rat Peripheral bloodl ymphocytes would be a substrate of PKC, and has one phosphorylation on serine residue.
[영문] Lymphocytes were found to possess all of the enzymatic machinery needed to phosphorylate and dephosphorylate proteins. At least four groups of protein kinases participate in T cell activation and interact with each other in a complex and as yet unknown manner, Increased phosphorylation in lymphocytes following stimulation with mitogens or interleukin-2, and the pivotal role of protein kinase C in the initial biochemical reaction have been reported. But the exact role of PKC in T cell activation and the substate of PKC are not well known. This study was attempted to clarify the characteristics of 22 kDa phosphoproteins obtained from rat peripheral blood Iymphocytes stimulated with Phorbol 12-myristate 13-acetate(PMA), using various kinase inhibitors as well as kinase activators. The Iymphocytes were incubated with (32)**P orthophosphate before PMA stimulation. The migration pattern of the phosphorylated proteins of PMA-treated rPBL in the two dimensional electrophoretic fields were analyzed after autoradiography. And the phosphorylation sites of 22 kDa protein were analysed by high performance liquid chromatography(HPLC) and scintilation counting. The results are as follows: 1. Increased phosphorylation of 22 kDa protein was observed with PMA or Con A. 2. Forskolin, an activator of adenylyl cyclase, cause no significant change of phosphorylation of 22kDa. 3. A 23187, a Ca**2+ ionophore, has no noticed effect on the phosphorylation of 22 kDa protein. 4. Neither W-7 nor H-7 inhibit the increase in phosphorylation of 22 kDa by PMA. 5. Staurosporine, a potent PKC inhibitor, showed inhibitory action on PMA-stimulated phosphorylation of 22 kDa in a dose-dependent manner. 6. Calphostin C, specific PKC inhibitor, inhibited the PMA-stimulated phosphorylntion of 22 kDa. 7. Among tryptic peptide fractions of 22 kDa by HPLC, one (32)**P phosphopeptide peak was observed at about 20% acetonitrile. From the above results, it could be suggested that the 22 kDa phosphoprotein of rat Peripheral bloodl ymphocytes would be a substrate of PKC, and has one phosphorylation on serine residue.
URI
http://ir.ymlib.yonsei.ac.kr/handle/22282913/117089
Appears in Collections:
2. 학위논문 > 1. College of Medicine (의과대학) > 박사
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