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Direct
visualization of the membrane pool ATP.
The figure shows that it is possible to visualize
directly by confocal microscopy the membrane pool when it is filled
with the fluorescent analog of ATP, 2',3'-0-(2,4,6-trinitrocyclohexadienylidine)
adenosine-5-triphosphate (TNP-ATP). (The confocal micrograph depicts
porous, hemoglobin-free red blood cell ghosts loaded by preincubation
with fluorescent TNP-ATP and subsequently washed prior to visualization.)
TNP-ATP is not a substrate for the Na/K pump but is known to act as
a competitive inhibitor. The ghosts so labeled present a punctate appearance
in the distribution of the fluorescent loci contained in the boundaries
of the ghost membranes (the ghost interior being empty). Because TNP-ATP
(as well as its diphosphate form TNP-ADP) is known to be a substrate
for membrane bound glycolytic enzymes, it is possible to follow the
sequential changes in the fluorescent appearance of TNP-ATP in ghosts
during their loading or unloading, when provided with the appropriate
forward and backward substrates for the GAPDH/PGK reactions. It is too
early to know the extent to which analysis of the punctate loci will
provide topographical information on the distribution of ATP pools (and
Na/K pumps) or information concerning the membrane elements that provide
for the evident fluorescent enhancement of TNP-ATP. Even so, it is tempting
to speculate that the cytoskeletal proteins dematin and pallidin represent
interesting candidates.
Recent publications:
Hoffman,
J.F., Joiner, W., Nehrke, K., Potapova, O., Foye, K. and Wickrema, A.
The
hSK4 (K NN4) isoform is Ca2+ activated K+ channel (Gardos channel) in
human red blood cells. Proc. Natl. Acad. Sci. USA 100:7366-7371,
2003.
Hoffman,
J.F., Wickrema, A., Potapova, O., Milanick, M. and Yingst, D.R. Na
pump isoforms in human erythroid progenitor cells and mature erythrocytes.
Proc. Natl. Acad. Sci. USA 99: 14572-14577, 2002.
Hoffman,
J.F. A
Brief History of the Red Blood Cell Club Blood Cells Mol Dis.
27:54-56, 2001.
Hoffman,
J.F. Questions
for red blood cell physiologists to ponder in this millenium. Blood
Cells Mol Dis. 2001 Jan-Feb;27(1):57-61.
Mairbaurl,
H., Schulz, S., Hoffman, J.F. Cation
transport and cell volume changes in maturing rat reticulocytes.
Am J Physiol Cell Physiol. 2000 Nov;279(5):C1621-30.
Stengelin,
M.K. and Hoffman, J.F. Na,
K-ATPase Subunit Isoforms in Human Reticulocytes: Evidence from RT-PCR
for the Presence of alpha1, alpha3, Beta2, Beta3, and gamma. Proc.
Natl. Acad. Sci. USA 94:5943-5948, 1997
joseph.hoffman@yale.edu
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