裂殖性酵母菌ATCC 2476鎘結合物質之研究

Abstract

裂殖性酵母菌Schizosaccharomyces pombe ATCC 2476對鎘具有耐受性。加鎘培養後，發現ATCC 2476不產生金屬硫蛋白質 (metallothionein, MT)，也無植物螯合素 (phytochelatin, PC)。ATCC 2476產生一類鎘誘生物質，可與鎘結合，命名為胞內鎘結合物質 (intracellular cadmium binding complex, ICBC)。ICBC不含硫醇基 (-SH)，原態分子量約為9.6 kDa，經有機溶劑萃取、蛋白酶處理、核酸脢處理，皆不影響其原態分子量及鎘結合能力；利用Dubois’s法測定總醣，發現ICBC亦非醣類；其熱穩定性高，經100oC處理後，仍保有結合的鎘。觀察紅外線光譜與pH值對ICBC鎘結合能力的影響，推測ICBC有別於MT及PC利用Cys與鎘結合之特性，可能是以-COOH和鎘結合。ATCC 2476以鎘濾紙擴散培養，在特定區域菌體表面出現金屬光澤，推測可能是細胞外之鎘累積物所造成，收集並純化此結合物質，稱為胞外鎘結合物質 (extracellular cadmium binding complex, ECBC)。實驗結果顯示，ECBC與ICBC有相同特性，例如：不含硫醇基、相同原態分子量、其鎘結合能力不受有機萃取、蛋白酶、核酸酶處理的影響。ATCC 2476菌體內累積的鎘，隨著鎘誘導時間增加而上升，在無鎘環境下，會將菌體內的鎘排出；ICBC亦隨著此趨勢消長。經實驗證明，當細胞內鎘向外排出時，細胞表面的ECBC上升，且和ICBC下降成正相關。而細胞內機制，目前仍不清楚。

Schizosaccharomyces pombe ATCC 2476 is tolerant to environmental cadmium. ATCC 2476 produced neither metallothionein nor phytochelatin for the sequestration of invading cadmium. However, ATCC 2476 did produce an intracellular cadmium binding complex (ICBC) as the chelating agent responding to the invading cadmium. Molecular weight of ICBC was determined by gel filtration chromatography as 9.6 kDa. No thiol group can be detected by Ellman’s reagent, and there was no effect on the molecular weight or its cadmium binding ability after lipid extraction or protease and nuclease treatment. There was no detectable glucan in ICBC by Dubois’s method. By infrared spectroscopy and effect of the pH on its cadmium binding ability, we proposed that carboxylic group might play a central role in chelating cadmium ions. This mechanism was different essentially from that of metallothionein and phytochelatin. When ATCC 2476 was cultured on a square agar plate, then a narrow strip of filter paper soaked with high concentration of CdCl2 was placed in the center of the plate. The surface of the cell near the Cd strip produced metal luster after several days of incubation. The metal luster was supposed to be caused by the accumulation of cadmium binding complex. The cells with luster were collected, and the surface cadmium binding complex was isolated and denoted as the extracellular cadmium binding complex (ECBC). ECBC shared similar properties with ICBC, e.g. having no thiol group, no change in molecular weight and cadmium binding ability after enzyme treatments. The concentration of cadmium accumulated in the cell increased when ATCC 2476 exposed to cadmium. If the environmental cadmium were then relieved, ATCC 2476 rejected cadmium from cells. The amount of cadmium chelated by ECBC increased corresponding to the cadmium released by ICBC. These results suggested a new cadmium chelating complex which sequestered cadmium ions by carboxylic groups in the cells, and then excluded the complex out of the cells by an unknown mechanism.