|Abstract: ||摘要 鯉魚消化道組織之鋅濃度遠高於其它種類的魚，本研究室且已了解此與鋅結合之物質為膜蛋白質。鯉魚全血中之鋅主要在其紅血球；而紅血球外膜鋅濃度高時，紅血球全鋅濃度即高。因此鯉魚紅血球外膜上與鋅結合的物質極可能係位於紅血球細胞膜上之膜蛋白質，本研究擬嘗試分離鯉魚紅血球之細胞膜，並了解鋅在紅血球之分布；分離與純化此細胞膜之「與鋅結合蛋白質」。 將經滲透壓破裂之鯉魚RBC，以100,000 g, 4 hr離心，分別測定其上層液與沉澱之鋅、Na+/K+-ATPase和蛋白質的濃度，結果得知可將鯉魚RBC之「細胞膜+細胞核」與其細胞質分離。再以0.25 M及2.3 M sucrose solution之蔗糖梯度，經超高速離心後，可以將細胞膜 (以Na+/K+-ATPase為指標)與細胞核 (以DNA為指標) 分離。鯉魚血液RBC的鋅濃度在不同個體間變化甚大，其範圍為0.8至38.2 μg/ml whole blood。將鯉魚RBC分劃成3個部份，細胞質、細胞膜和細胞核後，分別測定其中蛋白質的含量。不同鯉魚RBC個體間之蛋白質濃度變化很大，但RBC的蛋白質主要來自其細胞質。鯉魚細胞膜蛋白質的濃度，不會因為RBC蛋白質濃度上升而有太大的變化，均維持在20 mg/ml whole blood左右。鯉魚細胞核蛋白質濃度的變化和細胞膜相同，也均維持在一定的濃度，亦在20 mg/ml whole blood上下。鯉魚血液中，以單位蛋白質鋅之含量來看，以RBC 細胞膜之含鋅量最為特殊，其單位蛋白質中，平均鋅含量為233 μg/g protein，但是個體差別極大，最少者為97 μg/g protein，最高者為1,590 μg/g protein，相差達16倍 因此為了解鯉魚RBC細胞膜上是否有與鯉魚消化道組織相同之「與鋅結合蛋白質」 (Zn binding protein, ZnBP)，故利用清潔劑Lubrol抽取RBC膜上之蛋白質。Lubrol Extract經IMAC分離後，IMAC adsorbed protein的部分有許多的band，分子量45,000左右僅有幾條模糊的band (ZnBP所在位置)，其中那一條band是ZnBP，需要進一步研究。|
Abstract Zn concentration in the digestive tract tissue of common carp is higher then other fish species. It is known that this high Zn might come form a Zn-binding protein. The Zn in the whole blood of common carp mainly comes from its RBC; the higher the Zn concentration in the outer plasma membrane of RBC, the higher the whole Zn concentration in RBC will be. The Zn-binding substance in the common carp’s outer plasma membrane of RBC might be the membrane protein in its plasma membrane. This study plans to purify the plasma membrane of RBC, understand the Zn distribution in RBC, and separate the Zn-binding protein from plasma membrane of common carp’s RBC. Common carp’s RBC was osmotic hemolyzed, and subjected to centrifugation at 100,000 xg, for 4 h and the concentrations of Zn, Na+/K+-ATPase and protein in different fractions were measured. It was found that cytosols could be separated from plasma membrane (and nuclei) by this method. Sucrose density gradient ultracentrifugation with 0.25 M and 2.3 M sucrose solution was further applied to separate plasma membrane from nuclei. Zn concentration in common carp’s RBC varies greatly among different individuals ranging from 0.8 to 38.2 μg/(ml whole blood). RBC of common carp was divided into three parts: cytosols, plasma membrane and nuclei, and the protein concentrations in these parts were measured. It was found that the protein concentration in RBC varied greatly among different individuals, but the protein in RBC mainly comes from cytosols. The protein concentration in plasma membrane of common carp RBC remained constantly in different individual, which had a value of 20 μg/(ml whole blood). The protein concentration in the nuclei of common carp RBC also remained constantly in different fish which was 20 μg/(ml whole blood). In terms of Zn amounts per protein, the Zn concentration in plasma membrane of common carp RBC is very unique. The average Zn concentration was 233 μg/(g protein), and the range was 97-1,590 μg/(g protein), the highest one was 16 times higher than the lowest one. In order to understand whether the common carp’s RBC plasma membrane has the same “Zn-binding protein” as in its digestive tract tissue, detergent Lubrol was used to extract the Zn-binding protein from plasma membrane of RBC. After separating by Zn2+ IMAC, there were a lot of bands in IMAC adsorbed protein, but there were only several blurred band located on MW 45,000 where ZnBP located. Further researches are needed to study which band is Zn-binding protein.