Bariloche protein symposium argentine society for biochemistry and molecular biology

40
BIOCELL, 27 (Suppl. I), 2003
CA-C3.
ACTIVATION OF PLANT PLASMA MEMBRANE H
+
-
ATPase (AHA2) BY 14-3-3 PROTEIN MEDIATED
OLIGOMERIZATION
G. Yudowski
1
, G. Berberian
1
, M. Palmgren
2
, L. Beaugé
1
 and G.
Roberts
1
.
1
INIMEC, Córdoba, Argentina. 
2
The Royal Veterinary and
Agricultural University, Denmark. E-mail: [e-mail]
Plant plasma membrane H
+
-ATPase is regulated by an
autoinhibitory C-terminal domain that serves as binding site for
scaffolding protein 14-3-3. The 14-3-3 protein releases the
constraint exerted by this domain. We had established that
activation of the catalysis by C-terminal blocking or deletion
produces a strong negative cooperativity (or double michaelian)
on the substrate kinetic. In this work we have employed fluorescent
resonance energy transfer (FRET) between a donor-acceptor
fluorescent pair to follow self-association between H
+
-ATPase
monomers in response to treatments related with activation of the
pump. We used recombinant AHA2 y aha
∆92 (C-terminal truncated
form). The results show that loss of cooperativity occurs in parallel
with disruption of protein-protein interactions. The activation of
H
+
-ATPase by 14-3-3 protein changes the substrate kinetics towards
a negative cooperativity and produces oligomerization. Also,
oligomerization was observed when lipids restore the negative
cooperativity (lost after solubilization) in C-terminal truncated
enzyme. These results suggest that there are specific
supramolecular arrangements involved in the optimal performance
of this pump and the role of the C-terminal domain of H
+
-ATPase
might be related to prevent self-association and further interaction.
Supported by FONCYT-Argentina (PICT99 05-05158) and Agencia
Córdoba Ciencia (181/01).
CA-C4.
SITES INVOLVED IN THE SPONTANEOUS OCCLUSION
OF K
+
 IN THE Na,K-ATPase
González-Lebrero Rm, Kaufman Sb, Garrahan Pj, Rossi Rc
IQUIFIB, Facultad de Farmacia Y Bioquímica, Universidad De
Buenos Aires, Argentina. E-mail: [e-mail]
The Na
+
/K
+
-ATPase is a protein of the cell membrane that exports
3 Na
+
 and imports 2 K
+
 ions in each transport cycle, during which
these ions become temporarily occluded (trapped within the
protein). Occluded K
+
 can also be formed by mixing the enzyme
with the cation in media lacking Na
+
, Mg
2+
 and ATP (“direct
route”). While K
+
 occlusion in physiological conditions occurs
through extracellular sites of the enzyme, it is believed that
occlusion via the direct route takes place through intracellular
sites, but this is still unclear. We here investigated whether the
sites of the enzyme implicated in the occlusion of K
+
 via the direct
route are extracelullar or intracellular. Under these conditions,
K
+
 occlusion and deocclusion occur through ordered processes with
a fast and a slow components of similar sizes. This behaviour
resembles that found for K
+
 deocclusion in the presence of Mg
2+
and orthophosphate (MgPi) where the sites involved are
extracellular. Using 
86
Rb
+
 as a K
+
 congener, and a double incubation
sequence (with 
86
Rb
+
 or Rb
+
) we labelled the slow- or fast-exchange
pools. Depending on whether the deocclusion velocity of 
86
Rb
+
 in
the presence of MgPi was slow or fast after the double incubation
sequence, we could infer if the occlusion via the direct route
occurred through extracelullar or intracellular sites of the enzyme.
Results suggest that the release of Rb
+
 in media with MgPi take
place through the same sites as those from which the cation entered
the enzyme, i.e. the external ones.
With grants from Fundación Antorchas, ANPCyT and CONICET.
CA-C5.
FORSKOLINE ACTIVATED CURRENT IN WILD TYPE
AND VINCRISTINE RESISTANT K562 CELL LINE
Assef, Yanina A.; Cavarra, Soledad; Damiano, Alicia; Zotta, Elsa;
Ibarra, Cristina; Kotsias, Basilio A.
Inst. Inv. Médicas A. Lanari, UBA and Fac. Medicina, UBA,
Argentina. E-mail: [e-mail]
The cell line K562 obtained from a chronic myeloid leukemia
express integral membrane proteins such as CFTR (cystic fibrosis
transmembrane regulator) and MDR1 (multidrug resistance). We
studied with patch clamp and RT-PCR techniques, the relationship
between ion currents and the pattern of expression of these
proteins, in wild type (K562WT) and vincristine resistant
(K562vinc) cell lines. The amplified products in K562WT showed
two bands of 300 bp and 170 bp corresponding to CFTR and
MDR1, respectively. These two bands were of smaller and greater
intensity in K562vinc cells. In the whole cell configuration,
forskoline (20 ì M), an activator of adenylate cyclase, added to
the extracelular side turned on an outward current in both cell
lines, 8 out of 10 experiments in K562WT and 5 out of 9 in
K562vinc. The cAMP-activated current was blocked by
diphenylamine-2-carboxylate (DPC, 0.5 mM) added to the bath.
Normalized current (I/Imax) at 80 mV were: 3.92±0.95 and
2.69±0.56 for forskoline and DPC in K562WT, respectively. In
K562vinc the currents were: 9.62±4.81 and 4.56±2.14 for
forskoline and DPC, respectively. The presence of an outward
cAMP-activated current blocked by DPC in both cell lines suggest
that these currents are not altered in spite of the different pattern
of expression of CFTR and MDR1.
CA-C6.
ARABIDOPSIS THALIANA PLANT PLASMA MEMBRANE
AQUAPORINS SHUT DOWN AT LOW CYTOPLASMIC pH
Moira Sutka
1
, Karina Alleva
1
, Colette Tournaire-Roux
2
, Mario
Parisi
1
, Christophe Maurel
2
 and Gabriela Amodeo
1
.
1
Biomembranas, Fisiología, Fac. Medicina, UBA, Argentina.
2
Biochim. et Physiol. Moléc. des Plantes, CNRS/INRA/Agro-M,
Montpellier, France. E-mail: [e-mail]
Mechanisms that regulate water permeability (Pf) in plants are
basic to understand plant responses under limiting factors like
abiotic stress. One of the proposed candidates involved in
aquaporin modulation is pH (J. Memb. Biol., 2002 187:175-184;
Plant. J., 2002 30:71-81). In order to understand its action we
performed the following approaches: 1) At the cell level, isolated
leaf protoplasts showed very low Pf values (10-20 
µm.s
-1
) in the
presence of an acidic bath medium (pH 5.5) when employing a
developed micropipette technique combined with video
microscopy. 2) At the membrane level, when plasma membrane
vesicles were purified and studied using stopped flow
spectrophotometry, we found that Pf was blocked by protons when
medium pH was lowered on both sides of the membrane. In these
experiments, when vesicles were equilibrated at different pH (final
concentration 10 mM of buffer) Pf showed a half-inhibition at pH
values of 7.2-7.5. 3) At the molecular level, PIPs were expressed
in Xenopus oocytes. In osmotic swelling assays, Pf was markedly
sensitive (85%) to a 10 min pre-treatment by sodium acetate (pH
6.0) which induced a marked cytosolic acidification.
These findings clearly demonstrate not only that pH allows short-
term regulation of aquaporin activity but also that these proteins
can be directly blocked by an intracellular acidification.

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