Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes
A quantitative description of transmembrane water transport requires specification of osmotic (Pf) and diffusional (Pd) water permeability coefficients. Methodology has been developed to measure Pf and Pd simultaneously on the basis of the sensitivity and rapid response of the fluorophore aminonaphthalenetrisulfonic acid (ANTS) to solution H2O/D2O content. Cells loaded with ANTS in an H2O buffer were subjected to an inward osmotic gradient with a D2O buffer in a stopped-flow apparatus. The time courses of cell volume (giving Pf) and H2O/D2O content (giving Pd) were recorded with dual photomultiplier detection of scattered light intensity and ANTS fluorescence, respectively. The method was validated by using sealed red cell ghosts and artificial liposomes reconstituted with the pore-forming agent gramicidin D. At 25 degrees C, red cell ghost Pf was 0.021 cm/s with Pd 0.005 cm/s (H2O/D2O exchange time 7.9 ms). Pf and Pd were inhibited by 90% and 45% upon addition of 0.5 mM HgCl2. The activation energy for Pd increased from 5.1 kcal/mol to 10 kcal/mol with addition of HgCl2 (18-35 degrees C). In 90% phosphatidylcholine (PC)/10% cholesterol liposomes prepared by bath sonication and exclusion chromatography, Pf and Pd were 5.1 X 10(-4) and 6.3 X 10(-4) cm/s, respectively (23 degrees C). Addition of gramicidin D (0.1 micrograms/mg of PC) resulted in a further increment in Pf and Pd of 7 X 10(-4) and 3 X 10(-4) cm/s, respectively. These results validate the new methodology and demonstrate its utility for rapid determination of Pf/Pd in biological membranes and in liposomes reconstituted with water channels.
- Research Organization:
- Univ. of California, San Franciso (USA)
- OSTI ID:
- 6030675
- Journal Information:
- Biochemistry; (United States), Vol. 28:2
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
CELL MEMBRANES
PERMEABILITY
WATER
MEMBRANE TRANSPORT
CHOLESTEROL
CHROMATOGRAPHY
DEUTERIUM
DIFFUSION
ERYTHROCYTES
FLUORESCENCE
LIPOSOMES
OSMOSIS
TRACER TECHNIQUES
BIOLOGICAL MATERIALS
BLOOD
BLOOD CELLS
BODY FLUIDS
CELL CONSTITUENTS
HYDROGEN COMPOUNDS
HYDROGEN ISOTOPES
HYDROXY COMPOUNDS
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LUMINESCENCE
MATERIALS
MEMBRANES
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
OXYGEN COMPOUNDS
SEPARATION PROCESSES
STABLE ISOTOPES
STEROIDS
STEROLS
550201* - Biochemistry- Tracer Techniques