Bariloche protein symposium argentine society for biochemistry and molecular biology

157
BIOCELL, 27 (Suppl. I), 2003
CA-P1.
CHARACTERIZATION OF THE REACTION KINETICS
OF CATIONS WITH THE Na
+
/K
+
-ATPase MEASURING
THEIR EFFECT ON Rb
+
 OCCLUSION
Kaufman SB, González-Lebrero RM, Garrahan PJ, and Rossi RC.
IQUIFIB, Facultad de Farmacia y Bioquímica, Universidad de
Buenos Aires, Argentina. E-mail: [e-mail]
The active transport of Na
+
 and K
+
 across cell membranes is
mediated by the Na
+
/K
+
 pump, which is an intrinsic membrane
transport system, identical with the Na
+
/K
+
-ATPase. There is
evidence that during the pump cycle 2 K
+
 become bound to the
Na
+
/K
+
-ATPase in such a way that their release is slow. This type
of binding is called occlusion because it is believed that their access
to the bulk of the solvent is restricted by the protein structure. We
here show that indirect information on the reactions between the
enzyme and ligands like Na
+
, K
+
, Rb
+
, NH
4
+
, Li
+
, Tl
+
 or Mg
2+
 can
be obtained from their effects on the kinetics of Rb
+
 occlusion. To
measure the time course of 
86
Rb
+
 occlusion each cation was either
equilibrated with the enzyme before adding 20 µM 
86
Rb
+
 (condition
A) or added to the enzyme together with 
86
Rb
+
 (condition B). When
the cation was K
+
, Rb
+
, NH
4
+
 or Tl
+
 the rate of 
86
Rb
+
 occlusion was
much slower for condition A than for condition B. This result was
expected since K
+
, Rb
+
 and Tl
+
 are known to become occluded
themselves. When Na
+
, Mg
2+
 or Li
+
 were tested, no significant
difference between the time courses for both conditions were
observed. Compared to those obtained for K
+
 and its congeners
results for Na
+
, Mg
2+
 and Li
+
, strongly suggest that neither cation
forms occluded states under these conditions.
With grants from Fundación Antorchas, ANPCyT, CONICET and
Universidad de Buenos Aires.
CA-P2.
EFFECT OF LOCAL ANESTHETICS ON THE CALCIUM
ACTIVE TRANSPORT IN SARCOPLASMIC RETICULUM
MEMBRANES FROM MASTICATORY MUSCLES
Sánchez G, Takara D, Kanevsky S, Toma A, and Alonso G.
Cátedra de Biofísica. Facultad de Odontología. Universidad de
Buenos Aires. Argentina. E-mail: [e-mail]
The Ca-ATPase is a membrane intrinsic protein responsible for
active calcium transport from myoplasm to the sarcoplasmic
reticulum (SR) lumen, leading to muscle relaxation. Local
anesthetics (LA) inhibit the calcium transport in white fast muscle
(WFM) cells. The aim of this work was to compare the inhibitory
effect of lidocaine (L), bupivacaine (B), tetracaine (T), procaine
(Pr) and benzocaine (Bz) on the ATP-dependent calcium uptake
in SR membranes isolated from WFM and the masticatory muscles
(MM) masseter and medial pterygoid. SR membranes from rabbit
MBR and MM were isolated by differential centrifugation. The
ATP-dependent calcium uptake was determined at 37
o
C in the
presence of 50 mM MOPS-Tris (pH 7.2), 0.1 mM Ca-EGTA, 3
mM ATP, 3 mM MgCl
2
 and 100 mM KCl, using radioisotopic
techniques. The half-maximal LA concentration that inhibited the
calcium uptake (Ki) was significantly lower in MM for the amide
LA. Significant differences in Ki values in MM and WFM for
ester LA were not observed. The potency of the LA was: T > L >
Pr > B > Bz. A high correlation between the potency of the LA and
the partition coefficient octanol/water (taken as an index of
hydrophobicity) was found for MM (r = 0.95), masseter muscle (r
= 0.84) and medial pterygoid (r = 0.87). The results suggest that
the affinity of LA for the Ca-ATPase depends on the muscle tissue
type, the type of LA (amide or ester) and the hydrofobicity of the
drug.
CA-P3.
INFLUENCE OF CHARGED AMINO ACIDS IN THE 
α
αα
α
αM4
TRANSMEMBRANE DOMAIN ON SURFACE TARGETING
OF ACETYLCHOLINE RECEPTOR
Roccamo AM, and Barrantes FJ.
UNESCO Chair Biophys. & Molec. Neurobiol. and INIBIBB,
B8000FWB Bahía Blanca. E-mail: [e-mail]
The 
αM4 domain of the acetylcholine receptor (AChR) is flanked
by two basic amino acids (His
408
 and Arg
429
) located at the two
extremes of the transmembrane segment, at the level of the
phospholipid polar head region. A series of single- and double
αM4 mutants (H
408
A, R
429
A, H
408
A/R
429
A, R
429
E and H
408
A/R
429
E)
were produced and co-expressed with wt subunits in an
heterologous expression system (CHO-K1 cells). Surface [I
125
]
α-
BTX binding of wt cells was 60% of the total, whereas H
408
A and
R
429
A exhibited values of 40% and 5% cell-surface expression,
respectively. Mutants with reversed amino acid charge (e.g. R
429
E)
did not express measurable levels of AChR at the cell surface and
also diminished their total AChR levels. Cell-surface and total
AChR levels in the double-mutant H
408
A/R
429
A were 20% and 80%,
respectively, whereas in H
408
A/R
429
E no AChR was detected at the
surface. Basic residues in the M1 segment have been implicated
in AChR retention at the ER (Wang et al., 2002). Our study
demonstrates that in addition to such sites in M1, His
408
 and Arg
429
in 
αM4 also appear to be involved in cell-surface targeting of the
AChR.
Work supported in part by grants from UNS and FONCYT.
CA-P4.
POST GENOMIC ANALYSIS OF PUTATIVE AMINO ACID
TRANSPORTERS IN TRYPANOSOMES
Bouvier, León A.
1
; Silber, Ariel M.
2
; Canepa, Gaspar
3
; Miranda,
Mariana
3
; Manso-Alves, María J.
2
; Torres Héctor N.
1
 and Pereira
Claudio A.
3
1
INGEBI, Buenos Aires, Argentina. 
2
Instituto de Química-USP,
Brazil.
  3
IDIM Alfredo Lanari, Buenos Aires, Argentina. E-mail:
[e-mail]
Trypansoma cruzi cells use amino acids not only as a carbon source
(i.e. proline), but also in different metabolic pathways.
Phosphorilated arginine acts as an energy reservoir involved in
the renewal of ATP. It was also established that proline, aspartate
and glutamate participate in the differentiation process. However,
the molecular structure of trypanosomes amino acid transporters
remains unknown. Using the available data from the T. cruzi
genome project, we identified and characterized in silico different
putative amino acid transporter (PAT) groups, containing
paralogous sequences. More than 10,000 sequences were used to
iterative assembling of contigs and 50-100 PAT open reading
frames (ORFs) were detected and characterized. The genome
organization of these ORFs suggests that PAT genes are arranged
in tandem repeat clusters. Al last, we also identified the sequence
orthologous in other trypanosomatids such as Leishmania spp. and
Trypanosoma brucei. The amino acids transport may be considered
as the first step in their metabolic pathways, wherefore the
identification of transporter genes could be important for metabolic
research and drug design.

---