Bariloche protein symposium argentine society for biochemistry and molecular biology

162
BIOCELL, 27 (Suppl. I), 2003
CA-P21.
AMINO ACID  TRANSPORT ALTERATION  IN
SACCHAROMYCES CEREVISIAE MUTANTS DERIVED
FROM A L-THREONINE AUXOTROPH (thr1) STRAIN
Stella, Carlos A.; Burgos, Hilda I.; Alonso, Manuel; Ramos,
Eugenia H. and Mattoon, James R.
Departamento de Bioquímica Humana, Facultad de Medicina,
UBA. E-mail: [e-mail]
In the yeast S.cerevisiae mutations in five amino acid permease
genes (AGP1, DIP5, GNP1, MUP3 and LET2) have been shown
to decrease L-threonine transport activity. In addition disruption
of the SSY1 gene which encodes an amino acid sensor localized
on the plasma membrane, reduces L-threonine transport activity.
Considering this multiplicity of potential threonine transporters
we decided to study: a) which permease operates as the primary,
high affinity threonine permease in wild type strains and b)
physiological conditions where one or more of the other permeases
transport L-threonine. To this end a L-threonine auxotroph bearing
a  thr1 gene was mutagenized with EMS and plated on minimal
medium containing ammonium ion as nitrogen source and 0.3 mM
L-threonine. This concentration of L-threonine is near the growth-
limiting threshold. Small-colony mutants were selected as potential
mutants with L-threonine transport defects. Studies with these
mutants, including direct transport measurements, inhibitor
resistance and genetic complementation indicated that: 1) There
are at least three phenotypic categories among the four mutants 2)
Individual mutants respond differently to growth medium
composition 3) In minimal medium, higher L-threonine
concentration reduces growth rate in the parent and all but one
mutant [T2] 4) One mutant [T4] produces very high L-threonine
transport activity which decays rapidly in L-threonine-less medium
and 5) All mutants except T2 show enhanced L-leucine uptake.
CA-P22.
APPARENT ACTIVATION OF Ca
2+
-ATPase ACTIVITY BY
DILUTION
Vanagas L, Rossi RC, and Rossi JPFC.
IQUIFIB. Facultad de Farmacia y Bioquímica, Junín 956. Buenos
Aires, Argentina. E-mail: [e-mail]
Ca
2+
 pumps (PMCAs) are widely distributed among different
tissues. Generically the pump is a single polypeptide chain of
127,000 to 137,000 daltons. PMCA is a calmodulin-regulated P-
type ATPase that is encoded by a multigene family. To characterize
the mechanism of PMCA under different conditions, it is necessary
to measure Ca
2+
-ATPase activity, Ca
2+
 transport and the partial
reactions like, phospho and dephosphorylation and occlusion of
Ca
2+
.
We have found that specific activity of Ca
2+
-ATPase of erythrocyte
membranes, measured at concentrations below 50 
µg/ml of protein,
increases steeply up to 3-5 times at concentrations of 1 
µg/ml of
total membrane protein. Dilution of the protein did not modify
the activation by ATP, Ca
2+
 or Ca
2+
-calmodulin. This apparent
activation of the enzyme is not affected by ionic strength and is
independent of the method of Pi measurement (colorimetric or
determination of release of 
γ
32
P-ATP). The apparent activation on
the enzyme is mimicked by 0.0025-0.01% of detergent C
12
E
10
 and
is enhanced when membranes were slowly frozen instead of frozen
by immersion in liquid nitrogen. This behavior is also observed in
microsomal preparations of h4b PMCA expressed in insect Sf9
cells. These results seem to be accounted for by two reasons: a)
dilution reveals sites hidden by high lipid-protein concentrations
or b) dilution reveals the action of a modulator like FXYD proteins
in Na,K-ATPase.
BG-P1.
INHIBITION OF THE RENIN-ANGIOTENSIN SYSTEM
IMPROVES MITOCHONDRIAL FUNCTION
Bárbara Piotrkowski, Elena MV de Cavanagh, César G. Fraga.
Physical Chemistry-Pralib, School of Pharmacy and Biochemistry,
UBA. E-mail: [e-mail]
It has been shown that nitric oxide (NO) can act as a signal that
modulate the number of mitochondria in different cell types. In a
recent work we found that long term inhibition (9 months)of the
renin-angotensin system (RAS) attenuates structural and functional
changes in mitochondria, and the decrease in the number of
mitochondria that occurs with aging. Here we investigated whether
short term inhibition of RAS with enalapril, could modify kidney
mitochondrial function, and the role of NO in such effect. Thirty-
two 3 mo-old male Wistar rats were divided into 4 groups that
received enalapril (E,10mg/kg.day), enalapril+Lname (E+L,1mg/
kg.day), L-Name(L-N), or water without additions (Control),
during 14 days. Blood pressure was significantly lower (96±5 vs.
113 ± 1 mm Hg,p<0.05), and total mitochondrial protein per gram
of wet tissue significantly higher in E vs. Control. Mitochodrial
membrane potential was significantly higher in Enal and E+L vs.
L-N and Control (154±10, 149±11, 114±7 and 117±5mV,
respectively). Mitochondrial H
2
O
2
 production was lower in E, E+L-
N and L-N vs. Control (28.7 ± 5.1, 31.2 ± 4.0, 44.8 ± 10.2, and
93.6 ± 13.5 nmol H
2
O
2
/min.mg protein, respectively).
Mitochondrial uncoupling protein-2 and eNOS protein were
significantly higher in E vs. E+L-N, L-N and Control. In conclusion,
short-term inhibition of the RAS with E modifies mitochondrial
function in rat kidney, independently of the NOS activity. The
present results are consistent with an interaction between E and
NO, in the regulation of the number of mitochondria.
BG-P2.
FLAVAN-3-OL AND PROCYANIDINS AS INHIBITORS OF
ANGIOTENSIN CONVERTING ENZYME (ACE)
Lucas Actis-Goretta, Javier I. Ottaviani, and Cesar G. Fraga.
Physical Chemistry-PRALIB, School of Pharmacy and
Biochemistry, UBA. E-mail: [e-mail]
Flavonoids have been mainly claimed to act as free radical
scavengers. We have observed that after regular consumption of a
flavan-3-ol and procyanidin rich-diet during 14 days, there was a
significant decrease in systolic and mean blood pressure in a group
of young people. The aim of the present work was to determine if
this effect on blood pressure could be ascribed to an inhibition of
the angiotensin converting enzyme (ACE). We determined if
purified (-)-epicatechin (EC) and its related oligomers (dimers to
hexamers) have an inhibitory effect on ACE activity. Determination
of IC
50
 using N-hippuryl-L-histidyl-L-leucine (HHL) as substrate,
showed that tetramers and hexamers present the lowest values of
IC
50 
(12 and 10 µM, respectively). The same inhibition was
confirmed using other synthetic substrate (FAPGG). A group of
flavonoids belonging to other families, quercetin, miricetin,
kaempherol and rutin, showed lower or negligible inhibitory effect.
EC, dimers and hexamers presented a competitive inhibition
respect to the HHL and FAPGG on ACE activity. Lower Ki values
were, 4 and 12 µM for hexamer, for HHL and FAPGG. ACE
inhibitory activity was confirmed in different systems, such as rat
organ membranes. Procyanidins-rich beverages as red wine and
tea showed inhibitory effect on ACE activity depending the
polyphenols content. These effects of flavan-3-ols and procyanidins
on ACE could contribute to explain the health beneficial effects
observed in populations that consume high amounts of foods rich
in flavonoids.

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