초록 ▼

1) Protein Kinase A가 calmodulin sensitive한 adenylate cyclase의 desensitization에 어떻게 관여하는 지를 밝히기 위하여 G-protein이 없는 catalytic subunit를 calmodulin affinity column으로 분리하였다. 분리한 adenylate cyclase는 protein kinase A에 의해 활성이 떨어졌으며, 특히 calmidulin에 의한 활성화가 forskolin에 의한 활성화 보다 급격히 저해되었다. 이 결과는 calmodulin sensi

1) Protein Kinase A가 calmodulin sensitive한 adenylate cyclase의 desensitization에 어떻게 관여하는 지를 밝히기 위하여 G-protein이 없는 catalytic subunit를 calmodulin affinity column으로 분리하였다. 분리한 adenylate cyclase는 protein kinase A에 의해 활성이 떨어졌으며, 특히 calmidulin에 의한 활성화가 forskolin에 의한 활성화 보다 급격히 저해되었다. 이 결과는 calmodulin sensitive adenylate cyclase는 protein kinase A에 의한 phosphorylation에 의해 desensitization이 일어나며, 이 phosphorylation이 calmodulin에 의한 활성화를 저해시킴을 보여준다. 2) Protein kinase A에 의한 lens membrane substrate의 phosphorylation을 cell homogenate(in vivo like) 이용하여 추적하였다. cAMP를 넣어주지 않으면 Ca??, forskolin에 의해 phosphorylation이 증가되지만, 충분한 cAMP를 넣어주지 않으면 Ca??이 physophorylation을 감소시켰다. Ca??, 에 의한 phosphorylation의 감소는 calmodulin이 protein kinase A의 substrate에 붙어서 일어나는 것임을 calmodulin inhibitor와 calmodulin을 이용하여 확인하였다. 이 결과는 lens cell communcation 과정에서 calmodulin과 cAMP가 서로 반대로 작용할 경우, 그 기작의 이해에 도움을 줄 것이다.

Abstract ▼

1) Proloned treatment of cells with hormones can lead to a subsequent loss of reponsivenss. This phenomenon is called desensitization. To fiqure out possible roles of protein kinase A in the process of adenylaste cyclasedesensitization, Ca??/ calmodulin sensitive adenylate cyclase was isolated from

1) Proloned treatment of cells with hormones can lead to a subsequent loss of reponsivenss. This phenomenon is called desensitization. To fiqure out possible roles of protein kinase A in the process of adenylaste cyclasedesensitization, Ca??/ calmodulin sensitive adenylate cyclase was isolated from lens fiber cell membranes using calmodulin affinity column. The isolated adenylate cyclase which is free from GTP binding proteins, was gradually activated by various calmodulin concentration. However, the adenylate cyclase activity was dramatically decreased in the presence of protein kinase A. Especially, the stimulation of adenylate cyclase activity by calmodulin was almost completely abolished in the presence of protein kinase A. These results suggest that protein kinase A phosphorylates catalytic subunit of adenylate cyclase, then inactivate enzyme activity. 2) Protein kinase A mediated phosphorylation of the lens membrane substrates was performed using lens homogenates in the presence of lens adenylate cyclase activators. All three(26, 28, 18KD) lens membrane substrates were phosphorylated by endogenous protein kinase A. However, in the presence of sufficient amount of cAMP Ca?? inhibits ??P-incorporation into the major substrates. When calmodulin inhibitor, calmidazolium was added to lens homogenates phosphorylation system marked increase of ??P-incorporation into the major substrates was observed. Moreover, phosphorylation of the buffer- washed membranes using exogenous protein kinase A was gradually decreased by addition of exogenous calmodulin. These results demonstrate that calmodulin inhibits protein kinase A mediated phosphorylation of the lens membrane substrates.

목차 Contents

• 서론...8
• 실험재료및 방법...10
• 결과및 고찰...12
• 참고문헌...23