Caracterización del estado de acoplamiento de las preparaciones de retículo sarcoplásmico e interacción de la Ca²⁺ -ATPasa con vanadato

Abstract

The coupling (Ca²⁺/ATP ratio) of sarcoplasmic reticulum vesicles can be calcülated from measurements of ATPase activity by using a pHmeter. Such ratio has been established in about 2 mol Ca²⁺/mol ATP, in agreement with other authors.The effect of calcium ionophore Lasalocid (X537A) and of non ionic detergents (C₁₂E₉) on sarcoplasmic reticulum vesicles is studied. The action of these agents is to give an increase of the calcium dependent ATPase activity, as a consecuence of the greatly increasing of the permeability of the membrane to Ca²⁺, but they do not affect the basal ATPase (calcium independent activity). A method to uncouple the sarcoplasmic reticulum vesicles is described, by treatment with EGTA at 37° C. Such uncoupling produces an enhacement of the calcium dependent activity no affecting the basal ATPase. Preparations of native ATPase, ATPase uncoupled by treatment with EGTA and ATPase purified with detergents have been used to study the interaction of orthovanadate ions with the enzyme. The dissociation constant of the enzyme-vanadate complex was found to depend on the previous history of the enzyme and therefore it was found to be different in native ATPase, ATPase uncoupled by treatment with EGTA or in ATPase purified with detergents. The interaction of vanadate ions with the Ca²⁺-ATPase - is a slow process. A histeretic process is observed when ATPase activity of sarcoplasmic reticulum vesicles, preincubated with vanadate, is assayed in the presence of the substrates of the enzyme, Ca⁺² and ATP.