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    Please use this identifier to cite or link to this item: https://ir.fy.edu.tw:8080/ir/handle/987654321/7019


    Title: Aspartyl residue 10 is essential for ATPase activity of rat hsc70.
    Authors: Shiao-ping Huang;Ming-Ying Tsai;Ywh-Min Tzou;Wen-gueyWu;Chung Wang
    Contributors: 輔英科技大學 醫學檢驗生物技術系
    Date: 1993-01-01
    Issue Date: 2010-10-15 11:16:03 (UTC+8)
    Abstract: Three mutants of rat hsc70 were constructed, overexpressed in Escherichia coli, purified, and characterized. First, site-directed mutation was utilized to substitute Asn for Asp-10. The recombinant protein, hsc70(D10N), loses not only its peptide-stimulated ATPase activity but also its basal ATPase activity. The measured dissociation constants of ATP (0.3 microM) and S-peptide (5 microM) for hsc70(D10N), however, are virtually identical to those of hsc70. The intrinsic fluorescence spectra of hsc70(D10N) also remain largely unchanged. Therefore, the overall structure of the hsc70 protein is most likely intact after mutation. Second, the entire C-terminal peptide-binding domain was deleted and the resultant mutant contains only the N-terminal ATPase domain of hsc70. This recombinant protein, Nt-hsc70, is a peptide-independent ATPase. The ATPase activity at 37 degrees C of the Nt-hsc70, 270 pmol/h/micrograms of protein, is comparable to that of maximally peptide-activated hsc70. Third, the Asp-10 of Nt-hsc70 was replaced by Asn. Despite that this mutant, Nt-hsc70(D10N), is capable of binding ATP and it loses the capability to hydrolyze ATP. Taken together, these results indicate that aspartyl residue 10 of hsc70 is essential for ATP hydrolysis. Purified hsc70 and its mutants autophosphorylate in vitro at a substoichiometric level. On average, less than 1% of the hsc70 and Nt-hsc70 proteins are phosphorylated. Although the amount of phosphate incorporated into hsc70(D10N) and Nt-hsc70(D10) is reduced, a significant level of phosphorylation can still be achieved in these two site-directed mutants. Hence, autophosphorylation of hsc70 and its mutants is not correlated with their ability to hydrolyze ATP.
    Relation: J. Biol. Chem., 268(3),2063-2068
    Appears in Collections:[醫學檢驗生物技術系] 期刊論文

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