鯉魚大腦嗜鈣蛋白結合蛋白之純化與免疫化學研究

Abstract

細胞外界之訊息，如荷爾蒙、生長因數、神經傳導物質及電刺激等可藉second messengers 的傳遞引起細胞內的生化反應。已知的： second messengers有Ca2+、c－AMP 、 c－GMP、 Inositol trisphosphate 及Diacylglycerol 等。本論文在探討鈣離子的結合蛋白及嗜鈣蛋白( calmodulin ，一種鈣離子結合蛋白）的結合蛋白，試圖找尋與鈣離子有關，更下游的訊號傳遞者。本論文使用 5 ％過氯酸（ perchloric acid ，簡稱 PCA ）萃取鯉魚大腦細胞質液，在蛋白質磷酸化分析中，發現 PCA 可溶蛋白可加強磷酸化反應之強度。在 SDS － PAGE 分析中，發現了 30kDa的鈣離子及嗜鈣蛋白結合蛋白。通過 HPLC C18 co1umn 後，發現此30kDa蛋白並非單純一種蛋白。以gel slicing method 製作此30kDa蛋白的抗體，發現其可專一地辨認一種 30kDa 蛋白。本論文也利用此抗體做 30kDa蛋白的免疫化學研究，發現在鯉魚大腦局部及腦下腺神經葉皆有此蛋白之分佈。有關此蛋白之確實性質尚待進一步研究。

Cells respond to a variety of external stimuli by fluctuation of concentrations of second messengers; such as calcium, cyclic adenosine monophosphate, cyclic guanosine monophosphate, inositol trisphosphate and diacylglycerol, which would then initiate a cascade of biochemical reactions within the cells. Calcium—binding proteins are believed to be the downstream regulators of calcium— initiated biochemical reactions. So far, more than twenty proteins have been identified with calcium-binding activity, but only a few of them have been demonstrated with definite physiological functions (protein kinase C, calcineurin, Ca2+/calmodulin—dependent protein kinases and calpain) . Therefore, we developed analytical methods for calcium-binding proteins and calmodulin-binding proteins in this thesis and tried to purify these binding proteins from carp brain. Using perchioric acid extraction, HPLC C18 column chromatography, Calmodulin—Sepharose affinity column chromatography, we have purified one calmodulin—binding protein of 30kDa and partially purified one calcium/calmodulin—binding protein also of 30kDa. Antiserum against this 30 k calmodulin— binding protein have been raised and used for Western blot analysis and immunocytochemistry study. Preliminary data suggest that the 30kDa calmodulin-binding protein is present only in neurohypophysis and certain areas of brain.