Bariloche protein symposium argentine society for biochemistry and molecular biology

57
BIOCELL, 27 (Suppl. I), 2003
TS-C6.
INSULIN MODULATES  PHOSPHATIDIC ACID
METABOLISM IN CEREBRAL CORTEX
SYNAPTOSOMES
Salvador, G.A.; Pasquaré, S.J.; Ilincheta de Boschero, M. and
Giusto, Norma M.
Instituto de Investigaciones Bioquímicas de Bahía Blanca
(INIBIBB), CC 857, B8000FWB Bahía Blanca, Argentina. E-mail:
[e-mail]
Many subcellular signalling events are initiated with the generation
of the biologically active lipid,  phosphatidic acid (PA). By
excluding the de novo biosynthesis, phosphatidic acid can be
generated either by phosphatidylcholine (PC) hydrolysis through
phospholipase D (PLD) or by diacylglycerol (DAG)
phosphorylation through diaglycerol kinase activity (DAGK). In
addition, PA can be further hydrolyzed by phosphatidate
phosphohydrolase (PAP) yielding DAG. We have previously
described that PLD/PAP/DAGK pathway is present in cerebral
cortex synaptosomes from adult (4 month-old) and aged (28 month-
old) rats. Our purpose was to study the effect of Insulin and of
receptor-independent tyrosine phosphorylation on PLD/PAP/
DAGK pathway. The effect of Insulin and orthovanadate on this
pathway was evaluated as a time function.  At 5 min incubation,
and in the presence of ATP and orthovanadate, Insulin stimulates
both PLD and DAGK activities (120% and 140%, respectively)
whereas PAP2 is inhibited (20%). At 30 min exposition to hormone
and orthovanadate a 10% and 60% increase was found in PLD
and DAGK respectively. Under this condition, in sinaptosomes
from aged rats, PLD activity remained unchanged and DAGK
activity only showed a 20% increase. These results demonstrate
the modulation of PA metabolism through Insulin and tyrosine
kinases and its alteration during the aging  process.
TS-C7.
1
α25(OH)
2
D
3
 AND  PTH SIGNALING IN RAT INTESTINAL
CELLS: ACTIVATION OF CYTOSOLIC PLA2
Claudia Gentili, Susana Morelli and Ana Russo de Boland.
Dept. Biologia, Bioquimica & Farmacia. Universidad Nacional
del Sur, Bahia Blanca 8000, Argentina. E-mail:
[e-mail]
 In the current study, we have probed the role of cytosolic
phospholipase A2 (cPLA2) activity in the cellular response to the
calciotropic hormones, 1
α25,dihydroxy-vitamin D
3 
 [1
α25(OH)
2
D
3
]
and PTH. Stimulation of rat enterocytes with either hormone,
increased release of arachidonic acid  [
3
H-AA] one-two fold in a
concentration and time-dependent manner. The effect of either
hormone on enterocytes was totally reduced by preincubation with
the intracellular Ca
2+ 
chelator BAPTA-AM (5 
µM), suggesting that
the release of AA following cell exposure to the calciotropic
hormones occurs mainly through a Ca
2+
-dependent mechanism
involving activation of Ca
2+
-dependent cPLA2. Calciotropic
homone stimulation of rat intestinal cells increases cPLA2
phosphorylation (3-4 fold). This effect was decreased by PD 98059
(20 
µM), a MAP kinase inhibitor, indicating that this action is, in
part, mediated through activation of the MAP kinases ERK 1 and
ERK2. Enterocytes exposure to 1
α25(OH)
2
D
3 
 (1 nM) or
 
PTH (10
nM) also resulted in P-cPLA2 translocation from cytosol to nuclei
and membrane fractions, where phospholipase subtrates reside.
Collectively, these data suggest that PTH and 1
α25(OH)
2
D
3
activate in duodenal cells, a Ca
2+
-dependent cytosolic PLA2 and
attendant arachidonic acid release and that this activation requieres
prior stimulation of intracellular ERK1/2. 1
α,25(OH)
2
D
3
  and PTH
modulation of cPLA2 activity may change membrane fluidity and
permeability and thereby affecting intestinal cell membrane
function.
TS-C8.
NITRIC OXIDE, cGMP, CDPKs AND MAPKs ARE
INVOLVED IN THE IAA-INDUCED ADVENTITIOUS
ROOT FORMATION IN CUCUMBER
Lanteri, M. Luciana; Pagnussat, Gabriela C. and Lamattina,
Lorenzo.
IIB, FCEyN, UNMdP, CC 1245, 7600, Mar del Plata, Argentina.
E-mail: [e-mail]
Adventitious root formation (ARF) is a key step in vegetative
propagation. We have previously found that nitric oxide (NO) and
cGMP act downstream auxins in the signal transduction pathway
leading to ARF in cucumber (Pagnussat et al., Plant Physiol. 2002,
129:954-956 and Plant Physiol. 2003, 132:1241-1248). Since ARF
is partially dependent on the entrance of calcium from the
extracellular space and on the release from intracellular stores,
we further tested how auxins and NO affect the calcium dependent
protein kinase (CDPK) activity when ARF is induced. In vitro
CDPK activity increased in explants treated either with the auxin
indole-3-acetic acid (IAA) or with the NO-donor sodium-
nitroprusside, and was partially prevented by the specific NO-
scavenger cPTIO. The activation of a CDPK of 45 kDa was detected
by in-gel assays after 1 day of IAA- or NO-treatments. We also
investigated the involvement of a mitogen-activated protein kinase
(MAPK) signaling cascade during ARF. Our results indicated that
MAPK activity is induced by IAA in a NO-mediated pathway.
Collectively, these results are strong evidence indicating that ARF
is regulated by a complex and intricate set of cellular messengers
involving auxins, NO, cGMP, calcium, CDPKs and MAPKs.
Further investigations will be directed to establish what type of
relationship exists between them.
Supported by CONICET, ANPCyT, Fundación Antorchas and
UNMdP.
BT-C1.
SELECTIVE AND COVALENT IMMOBILIZATION OF
PROTEINS ONTO POLYMERIC SURFACES.
BIOTECHNOLOGYCAL APPLICATIONS
Carbajal, M.L.; Santos, J.; Ermacora, M.R. and Grasselli, M.
Universidad Nacional de Quilmes. R. Sáenz Peña 180, Bernal
(B1876BXD) Argentina. E-mail: [e-mail]
Covalent chemical immobilization is an important field of
biochemistry and physic chemistry. Also, it is a useful tool in
modern biotechnology to develop heterogeneous systems for
diagnostic kits. Carbodiimide coupling reaction is one of the most
used as a consequence of its very well known mechanism and
reaction conditions. However, the high abundance of carboxylic
acids and amino groups in protein structures renders this reaction
unspecific. In order to study less explored chemical reactions for
protein immobilization, sulphur and arsenic containing compounds
were reinvestigated. The chemistry of these compounds has
convenient features: they form covalent bonds that are reversible
under mild conditions and involve short reaction times. In this
work, reactions of aminophenyl arsine oxide (APA3), aminophenyl
arsine chloride (APA3Cl2), and aminoarsalinic acid (APA5) with
biological sulfhidryl compounds were studied in solution. Reaction
products were analyzed by UV, HPLC and MALDI-MS. As
expected, APA3 reacted readily with mercaptoethanol and
dithiotreitol. Interestingly, APA3Cl2 showed higher solubility than
APA3 and similar rates of reaction with the thiol compounds.
Natural or mutant proteins showing Cys-X-X-Cys motive in their
primary structure could be immobilized onto polymeric surfaces
containing APA3. Additionally it was possible to selectively
immobilize these proteins from a crude extract without previous
purification.

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