Bariloche protein symposium argentine society for biochemistry and molecular biology

153
BIOCELL, 27 (Suppl. I), 2003
LI-P22.
RESCUE OF GANGLIOSIDE-DEFICIENT CHO-K1 CELLS
AND ACETYLCHOLINE RECEPTOR DOMAINS
Bonini I, Picardi MV, Bermudez V, de los Santos B, Roccamo AM,
and Barrantes FJ.
UNESCO Chair Biophys. & Molec. Neurobiol. and INIBIBB,
B8000FWB Bahía Blanca. E-mail: [e-mail]
In order to investigate the possible association of the nicotinic
acetylcholine receptor (AChR) with lipid microdomains (“rafts”)
we have exploited the intrinsic GM2 synthase intrinsic deficiency
of the CHO-K1/A5 cell line that stably expresses adult muscle -
type AChR, to produce a double-transfectant new clone (coined
CHO-K1/GM2) rescued from such deficiency by expression of
UDP-GalNAc:lactosylceramide/ GM3/GD3 
β-1,4-N-
acetylgalactosaminyl transferase, a med-Golgi localized enzyme.
The resistance to Triton X-100 and other mild detergent (CHAPS,
sulfobetain and octyl-POE) solubilization was used as one of the
assays for microdomain occurrence. Similar solubilization profiles
were observed for both cell lines. Density gradients and Western
blots of detergent-treated cells labelled with [
125
I]
α-bungarotoxin
showed the preferential AChR distribution in high density
fractions. In view of the importance of cholesterol in AChR
function, we modified its content in both cell lines using
cyclodextrins. CHO-K1/A5 cells treated with 15 mM methyl-
β-
cyclodextrin (M
βCD) showed a decrease in [
125
I]
α-bungarotoxin
label of up to 50%. A similar decrease in label occurred in Triton
X-100 soluble fractions of both cell lines treated with 0-15 mM
M
βCD.
Work supported in part by grants from UNS, FONCYT, and FIRCA
1-RO3-TWO 1225-01 NIH.
LI-P23.
StarD7 IS SURFACE ACTIVE AND INTERACTS
DIFFERENTIALLY WITH PHOSPHOLIPID
MONOLAYERS
Angeletti S
1
, Maggio B
2
, and Genti-Raimondi S
1
1
Dpto. de Bioquímica Clínica. 
2
CIQUIBIC, Dpto. de Química
Biológica. Fac. Ciencias Químicas. U.N.C. Haya de la Torre y
Medina Allende. Ciudad Universitaria. 5000 Córdoba. Argentina.
E-mail:[e-mail]
Previously, we described the cloning and characterization of a new
gene up-regulated in the choriocarcinoma JEG-3 cell line,
denominated GTT1 (Gestational  Trophoblastic  Tumor 1).
Nucleotide sequence analysis of the cDNA and computer-assisted
homology search of the deduced amino acid sequence showed
approximately 25% identity and 49% similarity with human,
bovine or mouse PCTP. In addition, GTT1 shares a conserved
extended central region -from amino acids 66 to 250- with the
START domain proteins proposed to bind lipids and interact with
membranes. For this reason GTT1 was renamed StarD7 (START
domain containing 7). In the present report, we demonstrate that
StarD7 protein forms stable Gibbs and Langmuir monolayers at
the air-buffer interface showing marked surface activity. The latter
is enhanced by penetration into phospholipids films at an initial
surface pressure above the protein´s own equilibrium adsorption
surface pressure to a lipid-free interface. The protein-phospholipid
stabilizing interactions at the interface depend on the lipid, with
preference for phosphatidylserine, cholesterol and
phosphatidylglycerol, and the increases of lateral surface pressure
generated are comparable to those of other membrane-active
proteins. The surface activity of StarD7 is strong enough to
thermodynamically drive and retain StarD7 at the lipid membrane
interface where it may undergo lipid-dependent reorganization as
indicated by changes of surface pressure and electrostatics.
LI-P24.
INTESTINAL FATTY ACID BINDING PROTEINS:
IMPORTANCE OF 
αI-HELIX IN FATTY ACID TRANSFER
MECHANISM TO PHOSPHOLIPID MEMBRANES
Gisela R. Franchini
1
, Betina Corsico
1
,
 
 Horacio A. Garda
1
 and
Judith Storch
2
.
1
Instituto de Investigaciones Bioquímicas de La Plata (INIBIOLP),
CONICET-UNLP, Facultad de Ciencias Médicas, calles 60 y 120,
1900 La Plata, Argentina. 
2
Department of Nutritional Sciences,
Rutgers University, New Brunswick NJ 08901-8525. E-mail:
[e-mail]
Intestinal fatty acid binding protein (I-FABP) and liver FABP (L-FABP) are
both coexpressed in intestinal entherocytes. These proteins, as well as the
whole family, have a well conserved terciary structure consisting of a 
β-barrel
domain with a hydrophobic pocket and a portal domain with two short 
α-
helixes (
αI y αII). Fatty acid transfer from I-FABP to membranes occurs by
direct collisional interaction with lipid bilayer while L-FABP shows an aqueous
diffusion mediated process. In addition we have shown the importance of the
portal region in determining the FA transfer mechanism to artificial membranes.
Based on previous evidence and considering the existence of an amphipatic
helix (
αI) in I-FABP, we decided to construct two chimeric proteins by
exchanging the 
αI region between I and L-FABP.  We obtained a chimeric
protein with the ligand binding domain of L-FABP and the 
αI-helix of IFABP
(
α
II-
β
LFABP) and the corresponding reversal chimeric (
α
IL-
β
IFABP).
Integrity of the binding cavity assays (Kd and Kp determination) showed no
drastic differences between the chimeras and wild-type proteins. A fluorescence
resonance energy transfer assay was used to monitor the rate and mechanism
of transfer of FA to phospholipid membranes. Results showed important
modifications in rate and mechanism of transfer for 
α
IL-
β
IFABP chimeric
protein compared to wild-type I-FABP and no drastic change for 
α
II-
β
LFABP
chimera compared to wild-type LFABP.
LI-P25.
STUDIES OF EFFECTS OF FLUNITRAZEPAM ON THE
LAMELLAR-HEXAGONAL II PHASE TRANSITION OF
PHOSPHATIDYL ETHANOLAMINE. USE OF
MEROCYANINE 540 AS A FLUORESCENT INDICATOR
Daniel A. García and María A. Perillo.
Biofísica-Química. Depto de Química. FCEFYN. Universidad
Nacional de Córdoba. E-mail: [e-mail].
Flunitrazepam (FNT) is an extensively used benzodiazepine (BZD)
due to its potent anxiolytic and hypnotic action. Previous studies
of our group demonstrated that BZD-membrane interaction could
be explained by a partition equilibrium model. We showed that
BZD partitioning in the membrane increased in quantity and depth
as the membrane structural order decreased. Moreover, the
localization of BZDs at the phospholipid polar head region could
explain the decrease in the size of dpPC vesicles, through a
mechanism that involves the increment in the relative volume of
this polar head region inducing an increase in the vesicle’s surface
curvature. In the present work we studied if FNT can affect the
lamellar-hexagonal II phase transition of phosphatidyl
ethanolamine through a similar mechanism. This study was
approached by using the fluorescent dye merocyanine 540 which
is sensitive to the molecular packing of membrane lipids. A detailed
analysis of the effects of FNT on merocyanine absorption,
fluorescence emission and excitation spectra, was performed. The
results indicated that merocyanine behaved as a good indicator of
this phase transition as was previously described. FNT did not
affect the transition temperature but showed a tendency to diminish
the dye fluorescence emission intensity which could involve
topological changes leading to modifications in the polarity of the
region sensed by merocyanine.
Supported by SeCyT-UNC and ACC.

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