Patch clamp analysis of a H+ pump heterologously expressed in giant yeast vacuoles.

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 Biochem

journal_title

Journal of biochemistry

authors

Nakanishi Y,Yabe I,Maeshima M

doi

10.1093/jb/mvg184

keywords:

subject

Has Abstract

pub_date

2003-10-01 00:00:00

pages

615-23

issue

4

eissn

0021-924X

issn

1756-2651

journal_volume

134

pub_type

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