Bariloche protein symposium argentine society for biochemistry and molecular biology

39
BIOCELL, 27 (Suppl. I), 2003
LI-C7.
INTERACTION OF AN ASPARTYL PROTEASE WITH
LIPID INTERFACES CONTAINING
PHOSPHATIDYLGLYCEROL OR PHOSPHATIDYL
ETHANOLAMINES
M. Florencia Martini and E. Anibal Disalvo.
Laboratorio de Fisicoquímica de Membranas Lipídicas y
Liposomas, Cátedra de Química General e Inorgánica, Facultad
de Farmacia y Bioquímica, Universidad de Buenos Aires. E-mail:
[e-mail]
Aspartyl proteases are proteolytic enzymes with two aspartyl
groups in the active site that are involved in various human
diseases.
Previous papers of our laboratory have shown that an aspartyl
protease has a significant adsorption on lipid interfaces depending
on the net charges and the lipid composition.
The penetration of the protein is higher in phosphatidylcholine
than in phosphatidylethanolamine  monolayers at the same initial
surface pressure. The penetration in PC monolayers was correlated
with a slight decrease in the enzyme activity at 25
°C with respect
to its activity in water. In contrast, the activity increased when the
lipid interface was composed by phosphatidylethanolamine in
which the penetration was much lower. A comparable result was
obtained when the activity was measured in the presence of
membranes composed by phosphatidylglycerol.
For these reasons, our interest was to explore the changes in the
structural properties of both the protein and the membrane when
they are composed by phosphatidylethanolamine or
phosphatidylglycerol.
LI-C8.
COLLISIONAL TRANSFER OF FATTY ACIDS FROM
IFABP TO MEMBRANES: IMPORTANCE OF THE LYSINE
RESIDUES IN THE 
α-HELICAL DOMAIN
Lisandro Laborde
1
, Lisandro Falomir
1
, Betina Corsico
1
, Horacio
A. Garda
1
 and Judith Storch
2
.
1
Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP),
CONICET-UNLP, Fac. de Cs Médicas, calles 60 y 120, 1900-La
Plata, Argentina. 
2
Dpt. of Nutritional Sciences, Rutgers University,
New Brunswick NJ 08901-8525.
Fatty acid (FA) transfer from intestinal FABP (IFABP) to PL
membranes occurs by a collisional mechanism. Electrostatic
interactions are crucial in this process, so lysines residues of the
IFABP surface could be interacting with acidic groups in
membrane. We have shown that the helical domain of IFABP is
critical in determining the FA transfer mechanism.  To analyze the
role of the Lys residues of the 
α-helical domain on the transfer
mechanism, we eliminated and reverted the charge of these
residues (K16I, K16E, K20I, K20E, K27I, K27E, K29I and K29E)
by site directed mutagenesis. Analysis of the structural integrity
of  point mutants by CD spectra and quantum yield showed no
major differences compared to the wild-type protein. Binding to
the native ligand and anthroyloxy-oleate (12AO) were also
unaltered.Transfer rates for 12AO to zwiterionic membranes
revealed a collisional transfer mechanism  for all the mutants
except for K27I. Positions 27 and 29 showed the largest decrease
in transfer rate. More drastic changes were observed for vesicles
containing anionic phospholipids; the highest decrease was shown
by K27E and K29E mutants, with a 78% and 90% decrease in
transfer rate to cardiolipin-containing vesicles respectively. These
results indicate that lysines 27 and 29 have a  important role in
this process.
CA-C1.
MEASUREMENT OF CELL VOLUME CHANGES IN WILD
TYPE AND AQUAPORIN TRANSFECTED RENAL CELLS
Ford, Paula; Rivarola, Valeria; Chara, Osvaldo; Parisi, Mario
and Capurro, Claudia.
Laboratorio de Biomembranas, Departamento de Fisiología y
Biofísica, Facultad de Medicina, Universidad de Buenos Aires,
Argentina. E-mail: [e-mail].
Measurement of water transport across cell membranes, with high
temporal resolution and sensitivity, is essential for determining
osmotic water permeability and changes in cell volume associated
with stimulation or inhibition of ion transport mechanisms under
isotonic conditions. Various techniques have been employed to
study cellular volume changes. The use of fluorescent dye-dilution,
based on an inversely proportional relationship between relative
fluorescence intensity and relative cell water volume, provides
excellent sensitivity for measuring volume changes in single cells.
However the application of this technique in confluent monolayers
of epithelial cells results on a directly proportional relationship,
opposited to what was expected. These results were interpreted
by other authors as a consequence of fluorescence self-quenching.
In the present study, we investigated in detail whether this
relationship depends on the region of the cell studied. To do this
we used a cellular line established from rat cortical collecting
duct (RCCD
1
), which maintains the cellular types and the transport
characteristics of the native duct. We found that the relationship
between relative fluorescence intensity and relative cell water
volume does change with the region of the cell studied.
Comparative studies between wild type and water channel
(aquaporin-2 transfected) cells were made using this technique.
CA-C2.
NICOTINIC RECEPTOR M3 TRANSMEMBRANE
DOMAIN: ROLE  IN CHANNEL ACTIVATION
De Rosa M.J. and Bouzat C.
Instituto de Investigaciones Bioquímicas, UNS-CONICET, Bahía
Blanca, Argentina. E-mail: [e-mail]
The nicotinic acetylcholine receptor (AChR) is a pentamer of
homologous subunits. The primordial AChR presumably contained
only one type of 
α subunit and evolution led to subunit diversity
resulting in a wide spectrum of structurally and functionally
different AChRs. AChRs in the adult muscle have a composition
of 
α
2
βεδ.  Each subunit contains 4 transmembrane domains (M1-
M4). The role of M3 in channel gating is not well understood. To
determine its contribution to AChR activation, we constructed
muscle 
α chimeric subunits containing M3 segments of the
neuronal 
α7 and the ε subunit. The chimeric subunits were
coexpressed with non-
α subunits and the functional changes were
evaluated at the single channel and macroscopic current level.
Single-channel recordings of AChRs in which 
αM3 was replaced
by 
α7M3 showed briefer open durations and prolonged closing
intervals within clusters. Kinetic analysis revealed a 6-fold
decrease in the channel opening rate and a 2-fold increase in the
closing rate. Qualitatively different changes were observed in the
AChR containing the chimeric 
αεM3α subunit. These channels
showed significatively prolonged durations, indicating a ~ 20-fold
decrease in the closing rate, and brief mean closed times within
clusters. The open probability is about 1 at ACh concentrations as
low as 1 µM. Taken together our results add new experimental
evidence that gives support to the involvement of the M3 domain
in channel gating and gives new insights into the structural
functional relationships of its contribution to channel gating.

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