Bariloche protein symposium argentine society for biochemistry and molecular biology

163
BIOCELL, 27 (Suppl. I), 2003
BG-P3.
CHARACTERIZATION OF RAT LIVER AND KIDNEY
MITOCHONDRIA NITRIC OXIDE SYNTHASE
Zaobornyj T, Valdez LB, and Boveris A.
Laboratory of Free Radical Biology, School of Pharmacy and
Biochemistry, UBA. Junín 956, 1113, Buenos Aires, Argentina.
E-mail: [e-mail]
Mitochondrial nitric oxide synthase (mtNOS) catalyses NO
production in the mitochondrial matrix. The aim of this work was
to study the factors that have influence on mtNOS biochemical
activity. The highest activity in liver and kidney submitochondrial
membranes was obtained at pH 7.4, with a NO production of
1.3
±0.2 and 0.59±0.02 nmol/min.mg protein, respectively. Thus,
the mitochondrial matrix pH offers an optimal environment for
NO formation. At the same pH, NOS activity in the cytosol was
51% lower than in mitochondrial fraction. Values for the apparent
K
m
 (
µM) and V
max
 (nmol/min.mg protein) for O
2
 were 40 and 0.51
for liver, 37 and 0.42 for kidney. The apparent K
m
 (
µM) and V
max
(nmol/min.mg protein) for L-arginine were 70 and 1.7 for liver; 4
and 0.62 for kidney. Enzymatic activity was modulated by Ca
2+
concentration. Given the apparent K
m 
values for L-arginine and
NADPH, the regulation of activity by these substrates seems
unlikely under physiological conditions. NO production was
influenced by the metabolic state of intact mitochondria: the rates
at state 3 were 40-50% lower than those at state 4 suggesting a
novel kind of regulation for this enzyme. Addition of Ca
2+
 to the
reaction medium produced a 200% increase in O
2 
consumption
and a 40% increase in mtNOS activity. Characterization of mtNOS
activity provides understanding of the regulation of mitochondrial
function by NO.
BG-P4.
FERRITIN RADICALS GENERATED BY IRON UPTAKE
Galleano, Monica; Estévez, Maria Susana; Alcalde, Myriam;
Augusto, Ohara and Puntarulo, Susana.
Physical Chemistry-PRALIB. School of Pharmacy and
Biochemistry. UBA-CONICET. E-mail: [e-mail].
Ferritin-radicals produced during aerobic iron (II) uptake by ferritin
was studied by electronic paramagnetic resonance (EPR) in the
presence of the trapping agents N-tert-butyl-
α-phenyl nitrone
(PBN) or 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The
spectrum in the presence of PBN was a superposition of two types
of signals, an anisotropic signal from a slowly tumbling adduct
(2a
N
zz
 = 68.1 G) and an isotropic signal from a rapidly tumbling
adduct (a
N
=15.9 G and a
H
=3.75 G). In the presence of DMPO, the
spectrum obtained showed the dominant triplet pattern expected
for a slowly tumbling nitroxide (a
H
α=22.3±1.1 G). In order to
identify the detected radicals, chemical modifications were carried
out on the native protein. By blocking thiol groups did not affect
significantly the signal, however, by blocking tyrosine residues
the signal was decreased in approximately 50%. Treatment with a
strong oxidant (peroxynitrite) produced a significant decrease in
tryptophan fluorescence associated to the same EPR signal,
suggesting a role for these chemical species of similar nature in
both processes. These results showed evidence of the presence of,
at least, two radicals during iron uptake by ferritin: a tyrosyl-
centered radical, that could act as an intermediate by transferring
the radical character to another specie, tentatively identify as a
tryptophan-centered radical.
Supported by ANPCyT, IUPAB and the University of Buenos Aires.
MG and SP are career investigators from CONICET.
NT-P1.
DYNAMIC CHARACTERIZATION OF THE GLOBIN LIKE
FAMILY
Maguid S, Ferrelli L, Fernandez-Alberti S, and Echave J.
Centro de Estudios e Investigaciones, Universidad Nacional de
Quilmes, Argentina. E-mail: [e-mail]
The equilibrium dynamics of proteins in the folded state is directly
related to their structures. In that sense, we can attempt to identify
the common essential feature of equilibrium dynamics for a given
protein fold.
In spite of the large number of biological functions that are achieved
by a small number of protein structural families, a common
dynamics behavior associated to each protein fold might be
expected. Thus, a typical dynamic fluctuation behavior could be
identified as an invariant of the evolution of each family.
In the present work we propose to explore the global dynamics of
structurally similar proteins but sequential and functionally
different. We seek the identification of common collective
coordinates that were conserved during the evolution. The finding
of invariants of evolution can be a first step in the development of
simulation models for protein evolution.
We have studied the vibrational dynamics of the globin-like family
by normal modes analysis (NMA). The low frequency normal
modes describe collective movements that are closely related to
the protein biological function. Representative structures of the
globin family were considered.
NT-P2.
TOLERANCE OF Escherichia coli CELLS TRANSFORMED
WITH CHLOROPLAST 2-CYS PEROXIREDOXIN TO
OXIDATIVE STRESS
Aran, Martin; Senn, Alejandro M.; Caporaletti, D. and Wolosiuk,
Ricardo A.
Fundación Instituto Leloir, Patricias Argentinas 435, 1405 Buenos
Aires, Argentina. E-mail: [e-mail]
2-Cys peroxiredoxin (2-Cys Prx) is a ubiquitous family of
peroxidases that are devoid of the prosthetic heme group. Two
highly conserved cysteines play an important role in modulating
the concentration of hydrogen peroxide, thereby control signal
transduction and impart tolerance to oxidative stress. To
characterize the contribution of 2-Cys Prx to chloroplast
metabolism, we cloned and expressed the mature form from
rapeseed leaves in pET 22b(+) and subsequently studied the
functional characteristics. Next, we analyzed, as a functional assay
for undertaking studies of directed evolution, whether this
recombinant protein bestows the capacity to tolerate oxidative
stress in heterologous systems. Therefore, E.coli cells were
transformed with the abovementioned plasmid and subsequently
grew in media containing a variety of oxidants. Relative to cells
transformed with the vector bearing chloroplast fructose-1,6-
bisphosphatase (control), counterparts carrying the plasmid that
codes for 2-Cys Prx tolerated higher concentrations of
hydroperoxides as well as chemical oxidants. At variance, both
strains elicited similar response to chemicals that deprive the
intracellular concentrations of thiols, diamide and diethylmaleate.
These results not only suggested that chloroplast 2-Cys Prx
functions in the defense system against oxidation in E.coli cells,
likely as electron donor to hydrogen peroxide, but also provided a
protocol to estimate the functional capacity of chloroplast 2-Cys
Prx.

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