Quantitation of PMCA Phosphorylated Intermediates by Electrophoresis"

ECHARTE, MM; LEVI, V; VILLAMIL GIRALDO, A. M.; ROSSI, R.C.; ROSSI, J.P.F.C.

ANALYTICAL BIOCHEMISTRY

Academic Press

Lugar: New York; Año: 2001 p. 267 - 273

0003-2697

P-ATPases are characterized by the formation ofacid-stable phosphorylated intermediates (EP) duringtheir reaction cycle. We have developed a microscalemethod to determine EP that involves the phosphorylationof the enzyme using [g-32P]ATP and precipitationwith TCA; separation of the sample by SDS-PAGE,and measurement of the enzyme protein and 32PlabeledEP by digital analysis of both the stained geland its autoradiogram, respectively. The principal advantagesof this method over typical procedures (filtrationand centrifugation) are the low amount of enzymerequired and the substantial decrease in theblank values and data scattering produced by unspecificphosphorylation and nonquantitative recoveringof the enzyme. Application of this new method to apurified preparation of the plasma membrane calciumATPase (PMCA) results in overcoming the difficultiesof measuring EP at high ATP concentrations. A biphasicbehavior of the substrate curve for EP was observedwhen the study was extended to ATP levelswithin the physiological range. Since, in principle, themethod does not require the use of highly purifiedpreparations, it could be helpful for the study of phosphorylatedintermediates especially under conditionsin which small amounts of protein are available, e.g.,mutated variants of P-ATPases. © 2001 Academic PressKey Words: calcium pump; phosphorylation; m