INVESTIGADORES
ROSSI juan pablo Francisco
artículos
Título:
Differential reactivity of lysine residues of red cell membranes Ca2+ pump involved in the E1-E2 conformational equilibrium. Biochem
Autor/es:
22. DONNET, C., CARIDE, A.J., FERNÁNDEZ, H.N. AND ROSSI J.P.F.C.
Revista:
BIOCHEMICAL JOURNAL
Editorial:
PORTLAND PRESS LTD
Referencias:
Lugar: Londres; Año: 1991 vol. 279 p. 121 - 127
ISSN:
0264-6021
Resumen:
Abstract
1. Modification of
Lys residues of the Ca(2+)-ATPase from human red blood cells with methyl
acetimidate (MA) inhibited up to 70% of the Ca(2+)-ATPase activity.
Furthermore, calmodulin-activated p-nitrophenyl phosphatase activity was
fully inhibited at non-limiting concentrations of MA. 2. Treatment with
MA inhibited phosphorylation of the Ca(2+)-ATPase. 3. When the enzyme
was treated with 7.2 mM-MA in the presence of 100 microM-Ca2+,
Ca(2+)-ATPase activity was decreased by 33%, whereas when the membranes
were treated with MA in the presence of 50 microM-VO4(3-), this activity
was decreased by only 8%. 4. When membranes were either proteolysed or
preincubated with 1 mM-Ca2+, MA quickly inactivated the Ca(2+)-ATPase (k
= 1.2 min-1). On the other hand, inactivation of membranes preincubated
in the absence of Ca2+ and Mg2+ was slow (k = 0.08 min-1). 5. When the
activity was measured in the absence of calmodulin, MA decreased to the
same extent the values of KCa (the apparent dissociation constant for
Ca2+) and Vmax, but in the presence of calmodulin the treatment
decreased Vmax. only. 6. The results are consistent with the idea that
MA reacts readily with the Ca(2+)-ATPase when the enzyme is in an E1
conformation, but not an E2 conformation, and that, reciprocally,
treatment of the enzyme with MA shifts the enzyme to E1. 7. Provided
that Ca2+ is present, ATP, with low apparent affinity (K0.5 = 195
microM), protected against inactivation by MA. However, MA treatment did
not change the Km values of either the high-affinity or the
low-affinity site for ATP, suggesting that protection results from a
shift to a conformation in which the Lys residues are inaccessible to
MA.