Patch clamp analysis of a H+ pump heterologously expressed in giant yeast vacuoles.
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.
Nakanishi
Yoichi
Y
Laboratory of Cell Dynamics, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601.
Yabe
Isamu
I
Maeshima
Masayoshi
M
D001669:Biochemical Phenomena; D001671:Biochemistry; D001692:Biological Transport; D018076:DNA, Complementary / Q000378:metabolism; D005822:Genetic Vectors; D006859:Hydrogen / Q000378:metabolism; D007477:Ions; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D009154:Mutation; D018408:Patch-Clamp Techniques; D010957:Plasmids / Q000378:metabolism; D017494:Proton Pumps / Q000737:chemistry*; D011522:Protons; D011755:Pyrophosphatases / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry; D014617:Vacuoles / Q000378:metabolism
D018076:DNA, Complementary; D007477:Ions; D017494:Proton Pumps; D011522:Protons; D006859:Hydrogen; D011755:Pyrophosphatases; D025262:Vacuolar Proton-Translocating ATPases; D008274:Magnesium