臺灣博碩士論文加值系統

在酵母菌Saccharomyces cerevisiae中的ARR4蛋白與細菌ArsA為同源蛋白。生長曲線的實驗中突變蛋白ARR4[G30R]展現negative dominance的現象，表示ARR4蛋白在生物體內可形成雙聚體的結構來行使它的功能。在非變性膠體電泳顯示純化的ARR4蛋白呈現單體、雙聚體或是多聚體的構形狀態。另外以chemical cross-linkers可以確認在非變性膠體上的ARR4雙聚體的所在位置。重金屬離子Co2+, Sb3+, As3+,As5+對ARR4的ATPase活性與雙聚體的形成皆無影響。ARR4水解ATP的程度以測量磷酸根釋放與NADH吸光值變化，分別為50-70與100-120nmol/min/mg低的活性，當ATP與Mg2+為2:1比例時有最大活性。突變蛋白ARR4[G30R]大約只有ARR4蛋白一半的ATP水解速度。當ARR4蛋白分別地加入ATP或Mg2+，在非變性膠體上單體與雙聚體比例無變化，而ARR4[G30R]蛋白則不受任處理何影響。但同時加入ATP與Mg2+時，ARR4蛋白雙聚體的形成就有明顯增加的趨勢。以不同的molar比例(1:1, 1:2及1:4)混合ARR4與突變蛋白ARR4[G30R]但維持相同的蛋白濃度，發現ATPase活性與ARR4的含量成線性關係，因此推測ARR4胺基酸結合位在雙聚體中可以獨立地執行水解ATP作用。另外在非變性膠體上ARR4與ARR4[G30R]形成雙聚體的程度也與ARR4蛋白的含量成比例，而ATP與Mg2+同時與ARR4作用會使ARR4構形改變有利雙聚體的形成。

The ARR4 gene from Saccharomyces cerevisiae was highly homological with E. coli arsA. The defective nucleotide-binding domain of ARR4[G30R] exhibited negative dominance from the growth curve experiment, showing that ARR4 protein might act as a dimer in vivo. On the nonreducing gel, the purified 40 kDa ARR4 presented as monomer, dimer and oligomer conformational states. Besides, the chemical cross-linking experiments confirmed the positional size of dimeric ARR4. The metal ions of Co2+, Sb3+, As3+ and As5+ have not effect on the ATPase activity and ARR4 oligomerization. ARR4 exhibit a low level of Mg2+-ATPase activity in the range of 50-70 and 100-120 nmol/min/mg respectively as estimated by phosphate released and NADH-coupled assay. The maximal activity was obtained at a ratio of ATP:Mg2+ of 2:1 and the ATPase activity of ARR4[G30R] was about 50% of that of the ARR4 from both ATPase assays. In the presence of ATP or Mg2+, ARR4 has similar ratios of dimer and monomer. When incubated with Mg2+-ATP, ARR4 tended to form more dimer than with ATP and Mg2+ individually. ARR4[G30R] exhibited less dimer formation. Mixing ARR4 and ARR4[G30R] at a constant total protein concentration with different molar ratios (1:1, 1:2 and 1:4), a near linear relationship of activity versus amount of the ARR4 protein was observed. This observation suggests that ATP hydrolysis takes places in one ATP binding site independent of the other site of NBD. The proportional ATPase activities were consistent with the ratios of ARR4-ARR4[G30R] dimerization on the nonreducing gels. In conclusion, the conformational changes induced by Mg2+-ATP is related to the dimerization of ARR4 and both its NBDs would hydrolyze ATP independently.

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