The characterisation of fluoresceinphosphatidylethanolamine (FPE) as a real-time indicator of the electrostatic nature of the cell membrane surface is described. The conditions appropriate for the labelling of cell membranes and the implementation of FPE as a tool to monitor the interactions of various proteins and peptides with membranes are outlined. Some complications attributed to the erythrocyte glycocalyx are examined. In addition it is shown using neuraminidase as an example, that some types of enzyme-catalysed reactions on the cell surface may be monitored in real time. It is also shown that information concerning the binding of several proteins such as serum albumin and monoclonal antibodies are accessible with this technique. The albumin in particular is shown to exhibit a saturation of binding, the analysis of which indicates that the dissociation constant for erythrocytes was determined to be 8 microM and for lymphocytes to be almost 3 microM. On the basis of this comparison together with artificial membranes, the membrane protein components of the lymphocyte surface are implicated in the binding of albumin or the erythrocyte membrane proteins reduce the affinity of the cell surface for albumin.

REFERENCES

Antohe
F.
,
Heltianu
C.
,
Simionescu
M.
(
1991
).
Albumin-binding proteins of endothelial cells: immunocytochemical detection of the 18 kDa peptide.
Eur. J. Cell Biol
56
,
34
42
Arcangeli
A.
,
Carla
M.
,
Del Bene
M.
,
Becchetti
A.
,
Wanke
E.
,
Olivotto
M.
(
1993
).
Polar/apolar compounds induce differentiation by modulating cell-surface potential.
Proc. Nat. Acad. Sci. USA
90
,
5858
5862
Cevc
G.
(
1990
).
Membrane electrostatics.
Biochim. Biophys. Acta
1031
,
311
382
Chen
P.
,
Cambier
J. C.
(
1989
).
Measurement of phosphoinositide hydrolysis in isolated cell membrane preparations.
Meth. Enzymol
168
,
338
347
Dewald
B.
,
Dulaney
J. T.
,
Touster
O.
(
1974
).
Solubilisation and polyacrylamide gel electrophoresis of membrane enzymes with detergents.
Meth. Enzymol
32
,
82
91
Dobrilla
L.
,
Antohe
F.
,
Heltianu
C.
,
Simionescu
M.
(
1992
).
Albumin binding proteins of endothelial cells identified by anti-idiotypic antibodies.
Int. Immunol
4
,
789
796
Dodge
J. T.
,
Mitchell
C.
,
Hanahan
D. J.
(
1979
).
The preparation and chemical characteristics of haemoglobin-free ghosts in human erythrocytes.
Arch. Biochem. Biophys
100
,
119
130
Donath
E.
,
Voigt
A.
(
1986
).
Electrophoretic mobility of human erythrocytes. On the applicability of the charged layer model.
Biophys. J
49
,
493
499
Eylar
E. H.
,
Madoff
M. A.
,
Brody
O. V.
,
Oncley
J. L.
(
1962
).
The contribution of sialic acid to the surface charge of the erythrocyte.
J. Biol. Chem
237
,
1992
2000
Fernandez
N.
,
Labeta
M.
,
Kurpisz
M.
,
Nakatsuji
T.
,
Sachs
J.
,
Festenstein
H.
(
1990
).
Major histocompatibility complex (MHC) protein analysis by optimised two-dimensional electrophoretic methods.
Electrophoresis
11
,
167
174
Fernandez
N.
,
Kurpisz
M.
,
Labeta
M.
,
Sachs
J.
,
Pawalec
G.
(
1992
).
A ligand-epitope in vitro analysis of major histocompatibility determinants expressed on B and T lymphocytes.
Immunology
77
,
116
122
Grynkiewicz
G.
,
Poenie
M.
,
Tsien
R.
(
1985
).
A new generation of Ca2+indicators with greatly improved fluorescence properties.
J. Biol. Chem
260
,
3440
3450
Gurdon
J.
(
1987
).
Embryonic induction- molecular prospects.
Development
99
,
285
306
Gutierrez
C.
,
Bernabe
R. R.
,
Vega
J.
(
1979
).
Purification of human T and B cells by a discontinuous density gradient of Percoll.
J. Immunol. Meth
29
,
57
63
Izumida
Y.
,
Seiyama
A.
,
Maeda
N.
(
1991
).
Erythrocyte aggregation: bridging by macromolecules and electrostatic repulsion by sialic acid.
Biochim. Biophys. Acta
1067
,
221
226
Jones
L.
,
O'Shea
P.
(
1994
).
The electrostatic nature of the cell surface of Candida albicans: a role in adhesion.
Exp. Mycol
18
,
110
120
Kim
J.
,
Mosior
M.
,
Chung
L. A.
,
Wu
H.
,
McLaughlin
S.
(
1991
).
Binding of peptides with basic residues to membranes containing acidic phospholipids.
Biophys. J
60
,
135
148
Levine
S.
,
Levine
M.
,
Sharp
K. A.
,
Brooks
D. E.
(
1983
).
Theory of the electrokinetic behaviour of human erythrocytes.
Biophys. J
42
,
127
13
Maeda
N.
,
Imaizumi
K.
,
Sekiya
M.
,
Shiga
T.
(
1984
).
Rheological characterisation of desialylated erythrocytes in relation to fibrinogen-induced aggregation.
Biochim. Biophys. Acta
776
,
151
158
Martin
M. M.
,
Lindqvist
L.
(
1975
).
The pH dependence of fluorescein fluorescence.
J. Lumin
10
,
381
390
McLaughlin
S.
,
Szabo
G.
,
Eisenmann
G.
(
1971
).
Divalent ions and the surface potential of charged phospholipid membranes.
J. Gen Physiol
58
,
667
687
McLaughlin
S.
(
1989
).
The electrostatic properties of membranes.
Annu. Rev. Biophys. Biophys. Chem
18
,
113
136
O'Shea
P.
(
1988
).
Physical fields and cellular organisation.
Experientia
44
,
684
694
Schwarz
G.
,
Beschiaschvili
G.
(
1989
).
Thermodynamic and kinetic studies on the association of melittin with a phospholipid bilayer.
Biochim. Biophys. Acta
979
,
82
90
Schwoch
G.
,
Passow
H.
(
1973
).
Preparation and properties of human erythrocyte ghosts.
Mol. Cell. Biochem
2
,
197
218
Sharp
K. A.
,
Brooks
D. E.
(
1985
).
Calculation of the electrophoretic mobility of a particle bearing bound electrolyte using the non-linear Poisson-Boltzmann equation.
Biophys. J
47
,
563
566
Thelen
M.
,
Petrone
G.
,
O'Shea
P. S.
,
Azzi
A.
(
1984
).
The use of fluoresceindipalmitoylphosphatidylethanolamine for measuring pH-changes in the internal compartment of phospholipid vesicles.
Biochim. Biophys. Acta
766
,
161
168
Thelen
M.
,
O'Shea
P. S.
,
Petrone
G.
,
Azzi
A.
(
1985
).
Proton translocation by a native and subunit III-depleted cytochrome c oxidase reconstituted into phospholipid vesicles.
J. Biol. Chem
260
,
3626
3631
Wall
J. S.
,
Golding
C.
,
van Veen
M.
,
O'Shea
P. S.
(
1995
).
The use of fluoresceinphosphatidylethanolamine as a real-time probe for peptide-membrane interactions.
Mol. Membr. Biol
12
,
183
192
This content is only available via PDF.