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Patch Clamp Analysis of a H+ Pump Heterologously Expressed in Giant Yeast Vacuoles.
- Source :
-
Journal of Biochemistry . Oct2003, Vol. 134 Issue 4, p615-623. 9p. - Publication Year :
- 2003
-
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/µm2) using the patch-clamp technique in the whole-vacuole recording mode. We determined the inhibitor sensitivity and affinity for substrate (Km, 4.6 µM). The enzyme number in a giant vacuole (4.2 × 106) 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 Km. The high accuracy, effectiveness and applicability of the method for exogenously expressed ion transporters were also discussed. [ABSTRACT FROM PUBLISHER]
Details
- Language :
- English
- ISSN :
- 0021924X
- Volume :
- 134
- Issue :
- 4
- Database :
- Academic Search Index
- Journal :
- Journal of Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 44543487
- Full Text :
- https://doi.org/10.1093/jb/mvg184