ATPase and phosphatase activities are differentially inhibited by photo-oxidation of the sarcoplasmic reticulum Ca2+-ATPase

Abstract

We have already described that photo-oxidation of the sarcoplasmic reticulum Ca2+-ATPase with the halogenated dye erythrosin B produces inhibition of the ATPase activity (J.A. Mignaco et al., Biochemistry 35 (1996) 3886-3891). We now show that the Ca2+-dependent and Ca2+-independent p-nitrophenylphosphatase activities are also inhibited by this treatment. Modification of rapidly (< 10 min) oxidized residue(s) is responsible for the major loss of ATPase activity, whereas photo-inhibition of the phosphatase activities occurs more slowly (t( 1/4 ) 20-30 min). Here we have focused on photo-inhibition of the Ca2+-independent pNPPase activity, and the counteracting effects of ATP and FITC. Following photo-oxidation, the Ca2+-independent pNPPase activity decreases monotonically. ATP partially protects against the inactivation of the pNPPase, whereas labeling the enzyme with FITC does not. However, the protective effect of ATP is completely abolished by the attached FITC. These data are interpreted in terms of two different sites that are susceptible to photo-oxidation and are involved in different events related to substrate hydrolysis.