IQUIFIB   02644
INSTITUTO DE QUIMICA Y FISICOQUIMICA BIOLOGICAS "PROF. ALEJANDRO C. PALADINI"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Is the Plasma Membrane Calcium Pump regulated by the cytoskeleton?
Autor/es:
L. VANAGAS; I. C. MANGIALAVORI; A. J. CARIDE; R. C. ROSSI; J. P. F. C. ROSSI
Lugar:
Rosario, Argentina
Reunión:
Congreso; XXXV Reunión Anual de la Sociedad Argentina de Biofísica; 2006
Institución organizadora:
Sociedad Argentina de Biofísica
Resumen:
Plasma membrane calcium pump (PMCA) is an integral
membrane protein that actively transports Ca2+ towards the extra
cellular milieu, maintaining low intracellular Ca2+ concentrations
which are necessary for efficient signaling. PMCA is activated with acidic
phospholipids and regulated by protein kinases, but the principal regulatory
mechanism of enzymatic activity is mediated by the binding of calmodulin (CaM) to
a domain located near the C-terminus of PMCA, which increase the maximum
velocity and the apparent affinity for calcium of the pump. We have previously
shown that specific activity of Ca2+-ATPase of erythrocyte
membranes, measured at concentrations below 50 mg/ml of protein, increases steeply
up to 3-5 times when the membrane protein concentration decrease from 40 µg/ml
to 1 µg/ml. The activation by dilution was also observed during ATP-dependent
Ca2+ uptake measurements into inside-out vesicles both from erythrocyte
membranes and from Sf9 cells which over-express the enzyme, confirming that it
is a property of PMCA. Dilution of the protein did not modify the activation by
ATP, Ca2+ or Ca2+-calmodulin.
Present results show that the phenomenon can be
described as an activation-inhibition behavior as a function of protein
concentration which is dependent on the degree of PMCA expression. The dilution
effect is present in different PMCA isoforms and in the truncated variant CT120
which lacks the segment of 15 kDa pertaining to the calmodulin binding site. These
results indicate that the effect is dependent of the ratio PMCA/cytoskeletal
proteins. Further, a solubilized highly-purified micellar PMCA preparation show
that specific activity is constant alongside the protein concentration tested,
provided that enough concentration of phosphatidylcholine was added. This fact
indicates that the activation-inhibition behavior does not occur in the absence
of cytoskeletal proteins. On the other hand, it is known that Cytochalasin D
could promote or inhibit the polymerization of actin depending on the
concentration of Mg2+ and K+ ions present in the media. Our
results show that 1 mg/ml Cytochalasin D on erythrocyte membranes inhibits the
activation effect at low concentration of protein in the presence of 120 mM K+.
Conversely, in the presence of 0.5 mM Mg2+, Cytochalasin D activates
the enzyme at high protein concentration.
Taken together, these results strongly suggest that
the activation-inhibition behavior is a consequence of interaction of
cytoskeletal proteins with PMCA.
With grants of ANPCYT, CONICET, NIH and UBACYT.