Abstract:
:Vacuolar H(+)-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton-translocase; it contains a single type of polypeptide of approximately 81kDa. A line of evidence demonstrated that the carboxyl terminus of V-PPase is relatively conserved in various plant V-PPases and presumably locates in the vicinity of the catalytic site. In this study, we attempt to identify the roles of the C-terminus of V-PPase by generating a series of C-terminal deletion mutants over-expressed in Saccharomyces cerevisiae, and determining their enzymatic and proton translocating reactions. Our results showed that the deletion mutation at last 5 amino acids in the C-terminus (DeltaC5) induced a dramatic decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase; but the mutant lacking last 10 amino acids (DeltaC10) retained about 60-70% of the enzymatic activity of wild-type. Truncation of the C-terminus by more than 10 amino acids completely abolished the enzymatic activity and proton translocation of V-PPase. Furthermore, the DeltaC10 mutant displayed a shift in T(1/2) (pretreatment temperature at which half enzymatic activity is observed) but not the optimal pH for PP(i) hydrolytic activity. The deletion of the C-terminus substantially modified apparent K(+) binding constant, but exert no significant changes in the Na(+)-, F(-)-, and Ca(2+)-inhibition of the enzymatic activity of V-PPase. Taken together, we speculate that the C-terminus of V-PPase may play a crucial role in sustaining enzymatic activity and is likely involved in the K(+)-regulation of the enzyme in an indirect manner.
journal_name
Arch Biochem Biophysjournal_title
Archives of biochemistry and biophysicsauthors
Lin HH,Pan YJ,Hsu SH,Van RC,Hsiao YY,Chen JH,Pan RLdoi
10.1016/j.abb.2005.08.010subject
Has Abstractpub_date
2005-10-15 00:00:00pages
206-13issue
2eissn
0003-9861issn
1096-0384pii
S0003-9861(05)00352-8journal_volume
442pub_type
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journal_title:Archives of biochemistry and biophysics
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1016/j.abb.2004.02.024
更新日期:2004-05-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:2015-06-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:2005-06-15 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:1996-08-01 00:00:00
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pub_type: 杂志文章
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
doi:10.1006/abbi.1994.1300
更新日期:1994-07-01 00:00:00
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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journal_title:Archives of biochemistry and biophysics
pub_type: 杂志文章
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更新日期:2008-02-15 00:00:00
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pub_type: 杂志文章
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