Bariloche protein symposium argentine society for biochemistry and molecular biology

43
BIOCELL, 27 (Suppl. I), 2003
PL-C8.
CLONING, EXPRESSION AND BIOCHEMICAL
CHARACTERIZATION OF THE FRATAXIN HOMOLOG
FROM ARABIDOPSIS THALIANA (Athfh)
Busi, M.V.
1
; Burgos, J.L.
1
, Zabaleta, E.
1
; Araya A.
2
 and Gómez-
Casati, D.F.
1
1
Instituto de Investigaciones Biotecnológicas-Instituto Tecnológico
de Chascomús (IIB-INTECH) CONICET/UNSAM – 
2
Laboratoire
de Rèplication et Expression des Gènomes Eucaryotes et
Rètroviraux, REGER – CNRS, Universitè Bordeaux-2, France.
E-mail: [e-mail]
Frataxin is a nuclear encoded mitochondrial protein required for
maintenance of normal mitochondrial iron levels and respiration.
This protein is highly conserved from bacteria to mammals. It has
been suggested that frataxin could play the same role in all these
organisms, but its precise function remains unclear. It has been
also predicted the role of this protein in iron-sulfur cluster protein
assembly. Thus, plant frataxin homologue might be involved in
the assembly of the respiratory chain complexes at the inner
membrane in plant mitochondria. Our general working hypotesis
is that frataxim null mutants are defective in Fe-S cluster assembly
and result in abnormal iron trafficking and Fe-S protein deficiency
within plant cells. Recently, we have identified a putative plant
frataxin homologue (Athfh). The expression pattern of Athfh shows
an induction in Arabidopsis flowers. In u-atp9 Arabidopsis
transgenic plants (showing a mitochondrial dysfunction) this
protein is further induced. Purified recombinant Athfh from
Escherichia coli shows a ferroxidase activity as reported to occur
with the yeast homologue. These results, combined with
physiological studies using transgenic u-atp9 Arabidopsis lines
support a direct role of this protein in plant iron metabolism.
PL-C9.
EASTERN BLOTTING. A HIGH-THROUGHPUT
FUNCTIONAL ASSAY OF PHOSPHATASE ACTIVITY
Senn, A.M. and Wolosiuk, R.A.
Fundación Instituto Leloir, Patricias Argentinas 435, 1405 Buenos
Aires, Argentina. E-mail: [e-mail]
Phosphatases (EC 3.1.3.1) hydrolyze phosphate esters of primary,
secondary and tertiary alcohols. Most of current high-throughput
procedures to assay this ubiquitous activity rely upon artificial
substrates that yield a signal upon phosphate release; e.g. the
appearance of p-nitrophenol after the hydrolysis of p-
nitrophenylphosphate. We adapted a well-known assay for the
estimation of phosphate [Anal.Chem. 28: 1756-1758 (1956)] to
solid surfaces. As a consequence, the method became specific and
general for phosphatase reactions.
The current protocol, which does not require the lysis of bacterial
cells, was used to reveal the presence of active forms of two acid
and two alkaline phosphatases cloned in different plasmids; [acid
glucose phosphatase (agp), acid phosphatase (appA)] and
[chloroplast fructose-1,6-bisphosphatase, alkaline phosphatase
(phoA)], respectively. Moreover, it can be multiplexed to detect
these enzymes not only at a range of concentrations close to those
used in vitro but also in the presence of specific inhibitors.
Taken together these data indicate that the novel method profiles
active and inactive phosphatases, thereby can be used to screen
efficiently these enzymes in expression libraries prepared by
directed evolution or other procedures.
PL-C10.
POLYAMINE METABOLISM IN NODULES AND ROOTS
OF SOYBEAN PLANTS UNDER CADMIUM STRESS
Balestrasse, Karina B; Benavides, María P. and Tomaro María L.
Departamento de Quimica Biologica, Facultad de Farmacia y
Bioquimica, Universidad de Buenos Aires, Argentina. E-mail:
[e-mail]
Polyamines (Pas) have been reported to be involved in several
kinds of abiotic stresses in plants. However, there is no information
regarding putrescine (Put), spermidine (Spd) and spermine (Spm)
metabolism in soybean plants under cadmium stress. Both
cadmium treatments, 50 µM and 200 µM, modified Pas
metabolism in soybean, by increasing Put and Spm in nodules,
and Put and Spd in roots of treated plants. In roots and nodules,
Put formation could be attributed to the activity of both biosynthetic
enzymes, arginine decarboxylase (ADC), which was 7 times higher
than ornithine decarboxylase (ODC) in controls and 50 µM Cd-
treated nodules. ODC was responsible for Put formation in nodules
under 200 µM cadmium, where ADC activity was almost
undetectable. A clear increase in Spm and Spd content were
observed in nodules and roots respectively, after day 6 under the
metal treatments and this increase was concurrent with a drop in
ethylene formation. In nodules under cadmium stress, Spm was
higher than controls, and this effect was attributed to S-
adenosylmethionine decarboxylase (SAMDC) activity. However,
the highest Spm level was observed after SAMDC began to decline.
Our results demonstrate that the increase in Pas and the decrease
in ethylene in soybean nodules and roots did not avoid the
senescence process.
PL-C11.
FUNCTIONAL ANALYSIS  OF  THE  MAIZE
PHOTOSYNTHETIC NADP-MALIC ENZYME BY SITE
DIRECTED MUTAGENESIS
Detarsio, Enrique; Andreo, Carlos S. and Drincovich, María F.
CEFOBI. Facultad Cs Bioquímicas y Farmacéuticas (UNR).
Suipacha 531. Rosario. Argentina. E-mail:
[e-mail]
NADP-malic enzyme (NADP-ME) catalyses the oxidative
decarboxylation of L-malate to yield pyruvate, CO
2
 and NADPH.
In C
4
 plants, a plastidic isoform is located in bundle sheath
chloroplasts, which provides the CO
2
 to be fixed by RuBisCO.
Using the expression system we have previously developed for
the maize photosynthetic NADP-ME in E.Coli, four mutants were
constructed to analyse the specific function of the mutated
aminoacids. The mutants R237L and K255I produced drastic kcat
reduction, which suggests that both aminoacids can directly
participate in the catalytic mechanism. The double mutant K425/
6L showed no substantial alteration in the kcat and the Km of
malate, although the Km for NADP increased 9-fold, indicating
that one or both of these aminoacids´ positive charge are important
for the proper interaction of the enzyme with NADP. Curiously,
the Km of NAD was 4 times lower than that of the wild type,
which indicates that this mutation favours the interaction with the
alternate cofactor NAD. The mutant Y184F showed modification
only in the Km of malate, suggesting that the hydroxyl group
eliminated with this mutation is implicated in malate-binding.
Fluorescence emission and circular dichroism spectra showed no
differences between these mutants and the wild type. The
mutations introduced in the present work are also discussed in
the context of a constructed three-dimensional model of the
enzyme.

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