ATPase and phosphatase activities are differentially inhibited by photo-oxidation of the sarcoplasmic reticulum Ca(2+)-ATPase.
We have already described that photo-oxidation of the sarcoplasmic reticulum Ca(2+)-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 Ca(2+)-dependent and Ca(2+)-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 (t1/2 20-30 min). Here we have focused on photo-inhibition of the Ca(2+)-independent pNPPase activity, and the counteracting effects of ATP and FITC. Following photo-oxidation, the Ca(2+)-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.
Mignaco
J A
JA
Departamento de Bioquímica Médica, ICB/CCS, Universidade Federal do Rio de Janeiro, Cidade Universitária, Rio de Janeiro, Brazil. jmignaco@server.bioqmed.ufrj.br
Barrabin
H
H
Scofano
H M
HM
D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002474:Cell-Free System; D004923:Erythrosine / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010777:Photochemistry; D011817:Rabbits; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D010744:Phosphoric Monoester Hydrolases; D000251:Adenosine Triphosphatases; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate; D004923:Erythrosine