INVESTIGADORES
ROSSI Rolando Carlos
artículos
Título:
Quantitation of Plasma Membrane Calcium Pump Phosphorylated Intermediates by Electrophoresis
Autor/es:
M. M. ECHARTE; V. LEVI; A. M. VILLAMIL; R. C. ROSSI; J.P.F.C. ROSSI
Revista:
ANALYTICAL BIOCHEMISTRY
Editorial:
Academic Press
Referencias:
Año: 2001 vol. 289 p. 267 - 273
ISSN:
0003-2697
Resumen:
P-ATPases are characterized by the formation of acid-stable
phosphorylated intermediates (EP) during their reaction cycle. We have
developed a microscale method to determine EP that involves the
phosphorylation of the enzyme using [gamma-(32)P]ATP and precipitation
with TCA; separation of the sample by SDS-PAGE, and measurement of the
enzyme protein and (32)P-labeled EP by digital analysis of both the
stained gel and its autoradiogram, respectively. The principal
advantages of this method over typical procedures (filtration and
centrifugation) are the low amount of enzyme required and the
substantial decrease in the blank values and data scattering produced
by unspecific phosphorylation and nonquantitative recovering of the
enzyme. Application of this new method to a purified preparation of the
plasma membrane calcium ATPase (PMCA) results in overcoming the
difficulties of measuring EP at high ATP concentrations. A biphasic
behavior of the substrate curve for EP was observed when the study was
extended to ATP levels within the physiological range. Since, in
principle, the method does not require the use of highly purified
preparations, it could be helpful for the study of phosphorylated
intermediates especially under conditions in which small amounts of
protein are available, e.g., mutated variants of P-ATPases.