肝糖磷酸化酵素在吳郭魚鰓上滲透壓調節所需能量供應之角色

廣鹽性魚類在面臨外界環境鹽度改變時，鰓上相關的離子通道就必須及時且快速的作用以調節體內滲透壓恆定，而這樣的離子調節是極其耗能的。因此，在廣鹽性魚類鰓上應該具有相關的能量代謝機制以有效提供足夠的能量來源。肝糖對於能量高度消耗的組織而言，例如︰腦、肝臟、肌肉，是主要的能量儲存形式，並且扮演著當生物面臨生理壓迫時的能量即時提供者。在肝糖的代謝作用中，肝糖磷酸化酵素(GP)參與了肝糖初始分解的步驟，也就是將肝糖磷酸化分解為glucose-1-phosphate形式的代謝產物，進而使其繼續進入肝糖分解以及下游糖解等代謝途徑，以提供不同形式細胞的能量需求。在大多數的哺乳動物中，此同質雙體(homodimeric)酵素依其主要分布的組織命名，分別有肝臟型 (GPLL)，肌肉型 (GPMM)，以及大腦型 (GPBB)三種同型異構酶的存在。運用subtraction PCR，發現tGP此一基因株分別在淡水以及海水適應的吳郭魚鰓上會有基因表現量的差異。我們首次從吳郭魚鰓上選殖出tGP全長之核酸序列，並進一步利用qRT-PCR、western blot及專一的酵素活性測定，發現海水吳郭魚鰓上此酵素基因及蛋白表現量的確高於淡水；而酵素定位的影像顯示tGP的確位於吳郭魚鰓的表皮細胞上。然而，在經由與Na+-K+-ATPase蛋白的雙重染色標定影像得知，tGP與Na+-K+-ATPase並非同樣分布於MR細胞，而是主要分布於鰓上尚未定義的細胞。進一步運用GP的專一性抑制劑-caffeine卻是同樣抑制了鰓上tGP以及Na+-K+-ATPase的酵素與蛋白活性。根據這些結果，我們推測吳郭魚鰓上的肝糖分解酵素表現，會受到外界環境鹽度的刺激而改變，以提供鰓上高度耗能細胞及時的能量來源以進行滲透壓調節。並且，鰓上存在有另一類型的細胞負責能量供應的工作。

關鍵字

肝糖磷酸化酵素；鰓；滲透壓調節

並列摘要

Upon external salinity challenges, euryhaline teleosts perform rapid modulations of functions of several ion transporters or channels in gills to maintain the internal homeostasis. Ion- and osmo-regulation are highly energy consuming, therefore there must be an efficient metabolism system, which supplies immediate and sufficient energy sources for the active ion transport in gills. Glycogen is the main energy reserve in highly energy depletion tissues, such as brain, liver and muscle, and has been proposed to be an emergency fuel store during physiological stresses. In the processes of glycogen metabolism, glycogen phosphorylase (GP) catalyzes the initial glycogen degradation, i.e., the breakdown of glycogen to glucose-1-phosphate, which enters glycogenolysis or glycolysis to fulfill the energetic requirements of different cell types. The homodimeric enzyme exists as 3 isozymes named according to the tissues in which they predominate; LL (liver), MM (skeletal muscle) and BB (brain) forms in most mammalians. Using subtraction PCR, a clone of tGP was found to express differentially in the gills between freshwater (FW)-and seawater (SW)-acclimated tilapia. In present study, we have cloned and sequenced the full length cDNA of the tGP from tilapia gill. Alignment analysis showed that the tGP may be a homologue of the liver form of the mammalian GP. qRT-PCR, western blot analysis, and enzyme activity assay indicated that the tGP expression in SW tilapia gills was higher than that in FW ones. In situ hybridization result indicated that the tGP mRNA is localized in the epithelial cells of tilapia gills. However, the double labeling with Na+-K+-ATPase protein images show that tGP was not colocalized with the protein of Na+-K+-ATPase, implying that tGP mRNA was predominantly expressed in un-identified cells, but not in MR cells. In addition, tGP and Na+-K+-ATPase activity simultaneously inhibited by the GP specific inhibitor-caffeine. Taken together, the tGP gene expression is stimulated in tilapia gills upon salinity challenge, suggesting that the breakdown of glycogen may be directly providing energy for the osmoregulation mechanisms in gill highly energy consuming cells. And there are another types of cell corresponding to energy supplying.

並列關鍵字

osmoregulation ； glycogen phosphorylase ； gill

參考文獻

Andersen, B., and Westergaard, N. (2002) The effect of glucose on the potency of two distinct glycogen phosphorylase inhibitors. Biochem. J. 367 (Pt 2): 443-450.

Anderson, M.J, Cacela, D., Beltman, D., The, S.J., Okihiro, M.S., Hinton, D.E., Denslow, N., and Zelikoff, J.T. (2003) Biochemical and toxicopathic biomarkers assessed in smallmouth bass recovered from a polychlorinated biphenyl-contaminated river. Biomarkers 8 (5): 371-393.

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肝糖磷酸化酵素在吳郭魚鰓上滲透壓調節所需能量供應之角色

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