Proteoliposomes carrying reconstituted yeast plasma membrane H(+)-ATPase in their lipid membrane or plasma membrane vesicles are model systems convenient for studying basic electrochemical processes involved in formation of the proton electrochemical gradient (Deltamicro(H) (+)) across the microbial or plant cell membrane. Deltapsi- and pH-sensitive fluorescent probes were used to monitor the gradients formed between inner and outer volume of the reconstituted vesicles. The Deltapsi-sensitive fluorescent ratiometric probe oxonol VI is suitable for quantitative measurements of inside-positive Deltapsi generated by the reconstituted H(+)-ATPase. Its Deltapsi response can be calibrated by the K(+)/valinomycin method and ratiometric mode of fluorescence measurements reduces undesirable artefacts. In situ pH-sensitive fluorescent probe pyranine was used for quantitative measurements of pH inside the proteoliposomes. Calibration of pH-sensitive fluorescence response of pyranine entrapped inside proteoliposomes was performed with several ionophores combined in order to deplete the gradients passively formed across the membrane. Presented model system offers a suitable tool for simultaneous monitoring of both components of the proton electrochemical gradient, Deltapsi and DeltapH. This approach should help in further understanding how their formation is interconnected on biomembranes and even how transport of other ions is combined to it.

Institute of Physical Biology University of South Bohemia Nové Hrady Czech Republic

Zobrazit více v PubMed

Biochim Biophys Acta. 1984 Aug 31;766(2):375-85 PubMed

Biophys J. 1990 Apr;57(4):835-49 PubMed

Biochim Biophys Acta. 1991 Jan 30;1061(2):297-303 PubMed

Biochemistry. 1980 Jun 24;19(13):2925-31 PubMed

Biochim Biophys Acta. 1993 Feb 23;1146(1):87-96 PubMed

J Biol Chem. 1982 Aug 25;257(16):9365-71 PubMed

J Biol Chem. 1981 Dec 10;256(23):12081-7 PubMed

Photochem Photobiol. 2001 Jul;74(1):8-13 PubMed

J Membr Biol. 1977 May 6;33(1-2):109-40 PubMed

Eur J Biochem. 1982 Apr 1;123(2):447-53 PubMed

Methods Enzymol. 1988;157:513-28 PubMed

J Photochem Photobiol B. 1996 Apr;33(2):101-24 PubMed

Biochim Biophys Acta. 2000 Mar 10;1469(1):1-30 PubMed

J Biol Chem. 1990 Nov 15;265(32):19524-34 PubMed

Biochim Biophys Acta. 1987 Oct 16;903(3):480-94 PubMed

Proc Natl Acad Sci U S A. 1976 May;73(5):1485-8 PubMed

Biochim Biophys Acta. 1983 Jan 13;722(1):107-15 PubMed

Biochim Biophys Acta. 1994 Nov 1;1188(1-2):131-8 PubMed

Arch Biochem Biophys. 1972 Dec;153(2):413-25 PubMed

Biophys J. 1996 Jan;70(1):339-48 PubMed

J Membr Biol. 1973;14(4):305-38 PubMed

Biochemistry. 1990 Apr 24;29(16):3859-65 PubMed

Biochemistry. 1981 Mar 17;20(6):1534-8 PubMed

Biochemistry. 1990 Apr 24;29(16):3865-71 PubMed

Biochim Biophys Acta. 2003 Jan 10;1609(1):71-9 PubMed

Biophys J. 1979 Jan;25(1):63-85 PubMed

Biochim Biophys Acta. 1981 Dec 30;639(3-4):197-223 PubMed

Biochim Biophys Acta. 1978 May 18;509(2):289-99 PubMed

J Biol Chem. 1984 Jun 25;259(12):7884-92 PubMed

Biochim Biophys Acta. 1996 Dec 4;1285(2):175-82 PubMed

Anal Biochem. 1976 May 7;72:248-54 PubMed

Biochemistry. 1976 Nov 16;15(23):5094-105 PubMed

J Biol Chem. 1978 Oct 10;253(19):7026-32 PubMed

Methods Enzymol. 1989;172:63-84 PubMed

Biochim Biophys Acta. 1987 Oct 7;893(3):499-507 PubMed

Biochim Biophys Acta. 1997 Apr 26;1325(2):155-64 PubMed

Biochim Biophys Acta. 1969 May;180(1):1-8 PubMed