Abstract:
:Despite the usefulness of the patch-clamp technique, its application to ion pumps and transporters in biomembranes is limited. We developed a novel method for determining the activity of a proton-pumping pyrophosphatase (H(+)-PPase) made of a single protein. We heterologously highly expressed the enzyme in Saccharomyces cerevisiae, prepared giant vacuoles from the cells, and measured a PPi-dependent electrical current of 18 pA (10.5 fA/ micro m(2)) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (K(m), 4.6 micro M). The enzyme number in a giant vacuole (4.2 x 10(6)) and the molecular activity of the expressed H(+)-PPase (14 s(-1)) were determined. An uncoupling-type H(+)-PPase mutant, of which the 263rd glutamate residue was replaced by aspartate, and of which H(+) pump activity was not detected with the fluorescence quenching method, showed a weak current with a high K(m). The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Nakanishi Y,Yabe I,Maeshima Mdoi
10.1093/jb/mvg184keywords:
subject
Has Abstractpub_date
2003-10-01 00:00:00pages
615-23issue
4eissn
0021-924Xissn
1756-2651journal_volume
134pub_type
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journal_title:Journal of biochemistry
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doi:10.1093/oxfordjournals.jbchem.a002977
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pub_type: 杂志文章
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pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:2009-01-01 00:00:00
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pub_type: 杂志文章
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更新日期:2009-07-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a122939
更新日期:1989-11-01 00:00:00
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journal_title:Journal of biochemistry
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更新日期:2013-03-01 00:00:00
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a132883
更新日期:1980-05-01 00:00:00
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a124275
更新日期:1993-12-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:2010-05-01 00:00:00
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pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:2016-04-01 00:00:00
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pub_type: 杂志文章
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更新日期:1990-05-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:1996-01-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:1995-10-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a132601
更新日期:1979-10-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:1992-09-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:1989-02-01 00:00:00