Bariloche protein symposium argentine society for biochemistry and molecular biology

41
BIOCELL, 27 (Suppl. I), 2003
CA-C7.
LIPID PHASE DISTRIBUTION OF NICOTINIC
ACETYLCHOLINE RECEPTOR PROTEIN
Wenz, Jorge J. and Barrantes, F.J.
UNESCO Chair Biophys. & Molec. Neurobiol. and INIBIBB,
B8000FWB Bahía Blanca, Argentina. E-mail:  [e-mail]
Fluorescence quenching, fluorescence polarization, and Förster
resonance energy transfer (FRET) techniques in combination with
a detergent insolubility assay were used to investigate the ability
of purified nicotinic acetylcholine receptor (AChR) to partition in
lipid environments having defined phase characteristics.
Fluorescence data indicate that in reconstituted bilayers composed
of DPPC, DOPC, the fluorescent probe DPH, and the quenching
lipid 12-SLPC, which form a distinct two-phase system, the AChR
favours the saturated lipid (DPPC)-enriched phase. This
segregation becomes more evident in the presence of cholesterol.
In addition, the presence of AChR or cholesterol enhances Triton
X-100 insolubility of the phospholipid systems, supporting the
hypothesis that the protein is preferentially distributed in the
saturated DPPC-rich, Triton-insoluble, ordered lipid phase.
Work supported in part by grants from UNS, FONCYT, and FIRCA
1-RO3-TW01225-01 (NIH).
CA-C8.
A MODEL ACCOUNTING FOR SARCOPLASMIC
RETICULUM Ca-ATPase MAXIMAL ACTIVATION AT
HIGH ATP AND METAL CONCENTRATIONS
González Débora A, Ostuni Mariano A. and Alonso Guillermo L.
Cátedra de Biofísica, Facultad de Odontología, Universidad de
Buenos Aires, Argentina. E-mail: [e-mail]
The sarcoplasmic reticulum Ca-ATPase uses Mg
2+
 as ATP
cosubstrate. Results already presented strongly suggest the
participation of 2 Me
2+
 and 2 ATP for maximal enzyme activation.
Previously, we showed that 2 Mn
2+
 (Mg
2+
 analogue) are involved
in maximal phosphorylation with Pi. To evaluate the stoichiometry
for the binding of Me
2+
 and ATP, we studied, in the absence of
enough Ca
2+
 to activate the enzyme, the ATP effect on micromolar
(
54
Mn)Mn binding and the Mn
2+
 effect on the intrinsic fluorescence
changes induced by ATP binding. We also measured ATPase activity
in an extended range of [Mg
2+
] and [Mn
2+
], in the presence of
different [ATP]. The explanation of all these results required the
participation of 2 Me
2+
 and 2 ATP. Here we present a model for
the ATPase cycle that includes species with 2 Me
2+
 and 2 ATP
simultaneously bound. We assigned the kinetic constants values
and the initial concentrations of the species in the model to obtain,
by numerical simulation, the steady state concentrations of the
species at a given time. Pi production is the index of the ATPase
activity. The simulated curves for activity as a function of [Mn
2+
]
satisfactorily reproduce the experimental results.
PL-C1.
POTASSIUM UPTAKE -KINETICS AND GENE
EXPRESSION ALONG BARLEY ROOT AXIS
Vallejo A.J., Peralta M.L., Danna C.H. and Santa-María G.E.
Instituto de Investigaciones Biotecnológicas, Universidad
Nacional de San Martín. Bs.As., Argentina. E-mail:
[e-mail]
Potassium acquisition by higher plants requires the involvement
of outer root cell layers. It has been postulated that the AKT1
inward rectifier K
+
 channel and HAK1 transporters play a major
role in this process. While evidence supporting this assertion has
been already provided for AKT1 in Arabidopsis thaliana, it remains
scant for HAK1. In situ studies performed with barley roots shown
that HvHAK1 is preferentially expressed at the root epidermis. A
detailed examination of the longitudinal AKT1 and HAK1
expression patterns showed that both genes are expressed at lower
levels in the first centimeter from the apex relative to mature zones.
A similar expression pattern was observed for the putative K
+
transporters HvHAK1b, HvHAK2 and HvHAK3. If the contribution
of different transport entities differs along the root, and if these
entities differ in their kinetic properties, a change in Rb
+
 transport
characteristics along the root could be expected. We observed that
all root zones exhibit a saturable high-affinity component and a
linear “non-saturable” one, being the Vmax minimal in the growing
region. No differences were observed for the Rb
+
 Km and the K
+
Ki among root segments. Cs
+
 and NH
4
+
 inhibited high-affinity
rubidium uptake in all the segments, while Ba
2+
 and TEA did not.
However, 10 mM Na
+
 exerted a differential inhibition along the
root that matches the HvHAK1b/HvHAK1 expression quotient.
These results indicate that high-affinity K
+
 transport in all root
zones is mediated by several HAK1-like transporters, which share
most, but not all, kinetic attributes.
PL-C2.
CELL WALL DEGRADING ENZYMES DURING RIPENING
OF STRAWBERRY FRUIT
Rosli, Hernán Guillermo; Civello, Pedro Marcos; Martínez
Gustavo Adolfo.
IIB-INTECH. Camino Circunvalación Laguna Km.6. Chascomús,
Buenos Aires, Argentina. E-mail: [e-mail]
The primary cell wall of fruits undergoes structural changes during
ripening which leads to softening. Cell wall modification is
mediated by enzymes like polygalacturonase (PG), pectin methyl
esterase (PME), endoglucanase (Egase), ß-xylosidase (ß-Xyl) and
ß-galactosidase (ß-Gal). We studied the activity of these enzymes
during ripening of three strawberry cultivars with different
softening extent (Camarosa was the firmest, Toyonaka the softest
and Pajaro intermediate). Fruits were classified in large green
(LG), white (W), 50% red (50%R) and 100% red (100%R). PG
activity was detected from W in Toyonaka and Pajaro, being the
first higher than the second. Toyonaka maintained this level
throughout ripening and Pajaro increased reaching values similar
to those of Toyonaka in 100%R. PG activity started from 50%R in
Camarosa and reached higher values than the other varieties in
100%R. PME activity increased during ripening until 50%R,
without significant differences among varieties. From 50%R to
100%R, only Pajaro increased its Egase activity, having Toyonaka
the highest values, with almost no differences between Camarosa
and Pajaro. ß-Gal activity increased in the three cultivars. Toyonaka
presented the highest values, Camarosa the lowest and Pajaro
intermediate. ß-Xil presented two peaks of activity, one at W and
other at 100%R. For all stages Toyonaka showed higher activities,
Camarosa and Pajaro had similar levels except in 100%R. We
conclude that the higher activity levels presented by the softer
cultivar might be one of the reasons for differences in firmness.

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