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.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0810106-115918 |
| Date | 10 August 2006 |
| Creators | Chang, Chih-kang |
| Contributors | Jong-kang Liu, Ming-hong Tai, Ching-mei Hsu |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0810106-115918 |
| Rights | unrestricted, Copyright information available at source archive |
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