Electrogenic plasma membrane H+-ATPase activity using voltage sensitive dyes.
Fast responding voltage sensitive dyes, RH421 and di-4-ASPBS, were used to study the electrogenic properties of plant plasma membrane proton pumps on sealed plasma membrane vesicles extracted by two-phase partitioning from Beta vulgaris and Avena sativa cv Swan root material. Fluorescence spectroscopy in the presence of the dye RH421 (10.8 nM) was sufficiently sensitive to detect electrogenic activity of the extracted plant vesicles. The dye detection system could detect inhibition of electrogenic activity of vesicles by vanadate (75 μM) and stimulation by nigericin (0.5 μM). The newly developed dye di-4-ASPBS was less sensitive to detecting the electrogenic proton pump activity. This study represents an important innovation in plant biophysics as this class of fast responding voltage sensitive dyes have never to our knowledge been used to study electrogenic proton pump activity derived from plant membranes and represents a novel approach for carrying out such studies.
Amoroso
Steve
S
The School of Biological Sciences, The University of Sydney, Macleay Building (A12), Sydney, NSW, Australia 2006.
Clarke
Ronald J
RJ
Larkum
Anthony
A
Quinnell
Rosanne
R
D018554:Avena / Q000201:enzymology* / Q000502:physiology; D027461:Beta vulgaris / Q000201:enzymology* / Q000502:physiology; D001703:Biophysics; D002462:Cell Membrane / Q000378:metabolism*; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology*; D015394:Molecular Structure; D009550:Nigericin / Q000494:pharmacology; D010710:Phosphates / Q000378:metabolism; D018517:Plant Roots / Q000201:enzymology* / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011726:Pyridinium Compounds / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013343:Styrenes / Q000737:chemistry; D014638:Vanadates / Q000494:pharmacology; D056969:Voltage-Sensitive Dye Imaging
D010710:Phosphates; D011726:Pyridinium Compounds; D013343:Styrenes; C070534:RH 421; D014638:Vanadates; D006180:Proton-Translocating ATPases; D009550:Nigericin