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Title: | 草魚胞漿內蘋果酸去氫酵素之溫度適應 Thermal Adaptation of Cytoplasmic Malate Dehydrogenases In Grass Carp , Ctenopharyngodon idella |
Authors: | Shirley MacLeod 麥莎麗 |
Publication Year : | 1994 |
Degree: | 碩士 |
Abstract: | 由於魚類體內代謝所產生的熱量經由鰓表面散失到水中的速度非常快,所以魚類無法維持恆定體溫,是屬於變溫動物,故其體溫近於環境水溫。然而魚類的地理分佈極廣,從沙漠熱泉到兩極都有魚類蹤跡。因此各種魚類如何適應於截然不同的環境水溫是值得深入探討的問題。由於魚體內的各種代謝速率及需求受環境影響很大,本研究將針對一個重要代謝酵素-蘋果酸去氫酵素(malate dehydrogenases, MDH)進行探討。MDH牽涉四個重要代謝功能,包括檸檬酸循環(TCA cycle)、醣新生作用(gluconeogenesis)、脂肪合成作用(lipogenesis)及malate-aspartate shuttle。生存於各種環境水溫中的魚類都必需維持這些代謝功能,這是我們選擇 MDH 做為溫度適應研究的第一個,也是最重要的原因。 為了選擇適當實驗魚種,首先將17種魚的白肌進行非變性膠電泳(non-denaturing gel electrophoresis),並針對MDH活性染色。結果顯示草魚(Ctenopharyngodon idella)、黑鰱(Aristichthys nobilis)、鯰魚(Parasilurus asotus)及土虱(Clarias fuscus)有較簡單的同功酵素型態,只包含兩個主要的電泳帶(electrophoretic band)。而其他魚種尤其是易於雜交的魚種(如吳郭魚,oreochromis niloticus x O. aureus和鯉魚 Cyprinus carpio)及四倍體的魚種(如虹鱒,Oncorhynchus mykiss)均有較複雜的電泳帶。由10尾魚的白肌電泳均只出現三個主要且位置相同的MDH電泳帶,顯示草魚 MDH 的同功酵素在不同的個體中並無差異。為了區分粒線體及胞漿內之MDH,先將肝臟內的粒線體分離、打破後進行非變性膠體電泳。結果發現,緊鄰移動最快的電泳帶上方之次快電泳帶是粒線體內的MDH。而將白肌萃取液加熱至45℃經30分鐘後,在電泳膠上移動最慢的電泳帶完全消失,而移動最快的電泳帶仍維持相同的量,可知前者為熱敏感型之胞漿內蘋果酸去氫酵素(thermolabile cMDH),後者為熱穩定型之胞漿內蘋果酸去氫酵素(thermostable cMDH)。進一步的針對草魚各組織中(包括腦、肝、紅肌、白肌、脾臟、心臟和腎臟)的MDH進行電泳及活性染色,也顯示這些組織均只含有二至三個明顯的電泳帶。因此證實草魚只有簡單地兩個cMDH同功酵素,易於純化分離及進一步定性。此外,草魚容易購得,方便蓄養,而且是廣溫性魚種,易於馴養於廣泛而極端之溫度中,因此本研究選擇草魚做為實驗魚種。 首先,將草魚的兩個 MDH 同功酵素經由乳酸去氫酵素親和性層析(LDH affinity chromatography)及 MDH chromatography 分離,以便進行各種結構及動力特性(structural and kinetic characterization)的定性研究。熱穩定性的cMDH首先以400mM NAD+及20mM malate洗出,而熱敏感的cMDH則必需以高梯度的基質及cofactor洗出。以分離後的cMDH同功酵素作各項定性測試後,得知兩者都有相似的(l)最適酸鹼度:由oxaloacetate到malate的反應是pH7.5,而由malate到oxaloacetate的反應則是相當高的pH10.8及11.0,(2)最適cofactor濃度:最適NADH濃度是0.2-0.3 mm,而最適NAD+濃度是2-3 mM。然而,熱穩定性的cMDH比熱敏感的cMDH有較高的最適 oxaloacetate 濃度,分別是0.1 mM及0.05mM。反之,熱穩定的cMDH則比熱敏感的cMDH有較低的最適 malate 濃度,分別是 10mM 及 100mM 。進一步比較二者對基質及 cofactor 的親和力得知在 20℃, pH7.5 的情況下測得對 NADH 的 apparent Michaelis-Menten constant(Km),在熱敏感的同功酵素是熱穩定型的二倍以上(分別是 62 mM 及 22 mM)。而對 oxaloacetate 的 apparent km 值則相反,熱穩定的 cMDH 是熱敏感 cMDH 的約 8 倍,km 值分別是 27 mM 及3.5mM。當以在42℃時的活性半衰期(half-life)來量此二者對熱的穩定性時,發現二者有極顯著的差異。熱敏感型cMDH的半衰期僅約10分鐘,而熱穩定型則長達30小時。 以上的定性研究明顯的指出這兩個 MDH 同功酵素在結構及動力特性上有許多差異。因而二者可能在代謝功能上扮演不同的角色,所以在不同的溫度環境中下有不同的重要性及其表現程度。為驗證這個假說,將草魚分別馴養在高低溫(30℃及11℃)下,經過 6 週以後測定肌肉和肝臟中 MDH 活性、蛋白質含量,並將研磨液經 45℃ 加熱 30 分鐘後,去除熱敏感 cMDH 後,測定 MDH 的剩餘活性(residual activity),以估計熱穩定 cMDH 佔總 MDH 活性之百分率。結果顯示在低溫(11℃)馴養的魚白肌內 MDH 活性明顯地高於高溫馴養的魚,分別為 75 U/g muscle (或 40 U/mg protein)及 32U/g (或 62U/mg)。這種溫度補償(temperature compensation)現像是藉由增加酵素總含量來彌補低溫會降低酵素活性的影響。此外,MDH 剩餘活性測定結果顯示,在肝臟中不論 MDH 活性或熱穩定同功酵素的比例均無明顯差異,而在白肌中熱穩定 cMDH 在高溫馴養的魚(61.6 %)遠高於低溫馴養的魚(39.6 %)。將含同量 MDH 活性的研磨液電泳後進行 MDH 活性染色,不論以目測或以 densitometer 估計兩個同功酵素含量,均同樣地顯示白肌中熱穩定 cMDH 在高溫馴養的魚含量較高(33%及13%)。 綜合各項研究結果顯示,草魚利用定量(quantitative)及定性(qualitative)兩種策略來達成對溫度的適應。在定量策略上,草魚白肌藉由增加總 MDH 含量來維持需有的酵素活性;而在定性策略上,藉由表現不同比例的具有不同結構及動力特性的同功酵素來維持酵素作用的效率。 A survey of 17 different fish species showed that white muscle malate dehydrogenases of grass carp (Ctenopharyngodon idella), bighead carp (Aristichthys nobilis), catfish (Parasilurus asotus)as well as air-breathing catfish (Clarias fuscus) have a fairly simple electrophoretic banding pattern made up of two clear bands, while many of the other species (particularly the highly hybrid species such as tilapia and goldfish)exhibited very complex patterns. After heat treatment of white muscle extract of grass carp at 45℃ for 30 minutes, the slow-migrating (thermolabile) cytoplasmic malate dehydrogenase (cMDH) band totally disappeared while the fast-migrating (thermostable) band remained at the same intensity. Structural and kinetic characteristics of the thermostable and thermolabile cMDH partially purified from grass carp are further investigated. Both isozylmes had similar optimum pH for oxaloacetate (OAA) reduction, and optimum cofactor concentrations for both NAD+ and NADH. The thermostable cMDHs had a higher optimal OAA concentration as compared to that of the thermolabile cMDHs. However, the thermostable cMDHs had a lower optimal malate concentration than that of the thermolabile cMDHs. Both the thermostable and thermolabile cMDHs had a very high optimal pH for the oxidation of malate. Half–life for thermolabile cMDH at 42℃was 10 min in comparison to 24 hr for the thermostabile one. The apparent Km value of NADH measured at 20℃ (PH 7.5) for thermolabile cMDHs was more than twice that of the thermostable cMDHs. In contrast, the Km of OAA for thermolabile cMDHs was about one third that of thermostable cMDHs. Temperature acclimation of grass carp showed that thermolabile cMDH appeared in higher concentrations in white muscle and liver of cold-acclimated fish, while the thermostable form was more abundant in the warm-acclimated fish. In muscle, the residual activity after 30 minutes heat treatment at 45℃ was significantly lower in 11℃ acclimated fish as compared to that of the 30℃ acclimated group .After scanning the activity-stained non-denaturing gel by densitometer, it was revealed that the ratio of the band intensity of thermostable to thermolabile in 30℃ acclimated fish was more than twice that in 11℃ acclimated fish. Moreover, in white muscle(but not in liver), total MDH activity in 11℃ acclimated fish was about twice that of the 30℃ acclimated group. These results suggested that temperature acclimation could induce temperature compensation in MDH activity and differential expression of thermostable and thermolabile cMDH isozymes. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76084 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 漁業科學研究所 |
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