Bariloche protein symposium argentine society for biochemistry and molecular biology

149
BIOCELL, 27 (Suppl. I), 2003
LI-P6.
CHARACTERIZATION OF ERYTHROCYTE MEMBRANE
DOMAINS RESISTENT TO EXTRACTION WITH THE
ZWITTERIONIC DETERGENT CHAPS
Rodi PM
1
, Velázquez MM
1
, Gennaro AM
1,2
1
Departamento de Física, Facultad de Bioquímica y Ciencias
Biológicas, Universidad Nacional del Litoral. Paraje El Pozo S/
N, 3000 Santa Fe, Argentina. E-mail: [e-mail]
2
INTEC (CONICET - UNL). Guemes 3450, 3000 Santa Fe.
The usual approach to study membrane rafts, which are laterally
segregated domains enriched in sphingolipids, cholesterol and
specific proteins, involves isolation of detergent resistant
membrane domains (DRM). In previous works we have obtained
erythrocyte DRM using the non-ionic detergent Triton X-100, and
characterized their protein pattern and lipid ordering. We showed
that DRM could also be obtained from cholesterol depleted
erythrocytes (Rivas and Gennaro, Chem. Phys. Lipids 122:165-9,
2003). In the present work, we investigate the effects of treating
erythrocytes with the zwitterionic detergent CHAPS. We obtained
DRM both from intact and from cholesterol-depleted erythrocytes.
However, a detergent/lipid rate larger than that used with Triton
was needed in order to attain complete hemolysis. The insoluble
material was characterized in lipid and protein composition by
TLC and SDS-PAGE electrophoresis, and in lipid ordering by spin
label EPR spectroscopy. The observed differences between these
DRM and those extracted with Triton X-100 reinforce the
hypothesis that DRM and rafts should not be considered as
identical entities, as DRM characteristics depend on the
methodology used in the extraction procedure.
Work supported by Universidad Nacional del Litoral.
LI-P7.
EPR STUDY OF MIXED MEMBRANES OF DMPC AND
C8CERAMIDE
Dolores Carrer
1
, Shirley Schreier
2
 and Bruno Maggio
1
.
1
Departamento de Química Biológica-CIQUIBIC, Facultad de
Ciencias Químicas, Univ. Nac. de Córdoba, Córdoba, Argentina
,
and 
2
Instituto de Química, Universidad de Sao Paulo, Sao Paulo,
Brasil. E-mail: [e-mail]
In mixtures of dmPC and C8Cer, calorimetry shows the presence,
at 25-30 mole% C8Cer, of a single phase which behaves as a
complex. Up to 20 mole% C8Cer and in the gel phase, a part of
the membrane is laterally segregated in domains of pure or almost
pure dmPC, while C8Cer becomes incorporated in a second phase
of composition equal to the complex. EPR experiments show that
the rise in C8Cer concentration does not change the mobility of
the mixed membrane at neither of the three positions measured at
10
°C. When temperature rises to Tm-4°C, the rise in C8Cer
concentration produces a change in the mobility of the membrane
in the region close to the interface. In the complex samples, the
mobility gradient is lost at this temperature, with the interface
region as mobile and disordered as the C-12 region. In all samples,
in gel phase as well as in fluid phase, the results indicate there is
no immobilisation of the terminal part of the chains, which is
diagnostic of a non-interdigitated or a partially interdigitaded
membrane and rules out the possibility of mixed-interdigitated
organisation.
LI-P8.
THE GLUCOCORTICOID-INDUCED FACTOR (FDx)
REGULATING FATTY ACID DESATURASE ACTIVITIES
PROVED TO BE A LIVER- FABP
Marra, Carlos Alberto; Rimoldi, Omar and Alaniz, María J.T. de
INIBIOLP (Instituto de Investigaciones Bioquímicas de La Plata)
CONICET-UNLP. Calles 60 y 120, 1900 La PLata, Argentina.
E-mail: [e-mail]
Previous studies from our laboratory demonstrated that either
treatment of isolated liver cells with glucocorticoids or the injection
of these steroids to rats were able to produce an inhibitory effect
on both 
∆5 and ∆6 fatty acid desaturase activities concomitantly
with the stimulation of the 
∆9 desaturase. These modulatory actions
were dose-dependent and required specific steroid receptor
occupancy, although the 11-
β-OH group in the steroid molecule
was not an essential requirement for biological activity. The
regulatory effects were mediated by a soluble protein (FDx)
recovered from the cytosolic fraction of treated cells or injected
rats. In this work we reported the isolation and purification of the
FDx factor through a protocol that included fractionated saline
precipitation, HPLC gel-permeation and ion-exchange
chromatography. FDx was purified ca. 6,200 times up to
homogeneity as judged by native and SDS-PAGE. Protein
sequencing of six olygopeptides obtained after trypsin digestion
of native FDx demonstrated a complete homology with rat L-FABP.
In vitro experiments testing the regulatory capacity of FDx vs.
purified L-FABP confirmed this finding. Thus, the main
contribution of this work was to reveal a novel regulatory function
of  L-FABP on the hormonal control of the unsaturated fatty acid
biosynthesis.
LI-P9.
TOPOGRAPHY OF MYELIN LIPIDS MONOLAYERS
WITH AND  WITHOUT  PLP  PROTEIN AND
DISTRIBUTION OF GANGLIOSIDE GM1
Rosetti Carla Mariana and Maggio Bruno.
Departamento de Química Biologica-CIQUIBIC. Facultad de
Ciencias Químicas. Universidad Nacional de Córdoba, Argentina.
E-mail: carla dqb.fcq.unc.edu.ar
Myelin membranes extracted with a mixture of non-polar solvents
contains the majority of lipids of the original membrane and
proteolipid (PLP) protein (13.6 weight %). Besides, we isolated
an extract of the lipid fraction without protein.  Monolayer films
were spread from both fractions and observed by Epifluorescence
and Brewster Angle microscopy (BAM).  The lipid mixture shows
domain segregation with coexistence of liquid expanded (LE) and
liquid ordered (LO) phases that merge at low surface pressure (
≈
5mN/m). By contrast,  as shown before (Oliveira R, Maggio B.
2002), the system constituted by the lipids and PLP, exhibit domain
coexistence up to collapse on a Tris Ca++ subphase. The
distribution of some components in LE and LO phases was analysed
in monolayers with and without PLP protein. In particular,
ganglioside GM1 that partitions into LE domains in mixtures
containing PLP,  is also enriched in LE phases in absence of the
protein. By contrast, in  defined mixtures with LE/LO domains,
ganglioside GM1 was found in LO phases. BAM showed a marked
reorganization of the LE phase containing the PLP protein during
compression up to around 20mN/m, where the domain shape also
changes. The domains lacking the protein show only slight
variations of the optical thickness with surface pressure increases.

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